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Home , Gordon Covell, Human red cell antigens, Plasmodium schwetzi

SERIES EDITORS D. ROLLINSON S. I. HAY Department of Zoology, Spatial Epidemiology and Ecology Group, The Natural History Museum, London, UK Tinbergen Building, Department of [email protected] Zoology, University of Oxford, South Parks Road, Oxford, UK [email protected]

EDITORIAL BOARD M. G. BASÁÑEZ R. E. SINDEN Reader in Parasite Epidemiology, Immunology and Infection Section, Department of Infectious Disease Department of Biological Sciences, Epidemiology Faculty of Medicine Sir Alexander Fleming Building, Imperial (St Mary’s campus), Imperial College, College of Science, Technology and London, London, UK Medicine, London, UK

S. BROOKER D. L. SMITH Wellcome Trust Research Fellow and Johns Hopkins Malaria Research Professor, London School of Hygiene and Institute & Department of Epidemiology, Tropical Medicine, Faculty of Infectious Johns Hopkins Bloomberg School of Public and Tropical, Diseases, London, UK Health, Baltimore, MD, USA

R. B. GASSER R. C. A. THOMPSON Department of Veterinary Science, Head, WHO Collaborating Centre for The University of Melbourne, Parkville, the Molecular Epidemiology of Parasitic Victoria, Australia Infections, Principal Investigator, Environmental Biotechnology CRC N. HALL (EBCRC), School of Veterinary and School of Biological Sciences, Biomedical Sciences, Murdoch University, Biosciences Building, University of Murdoch, WA, Australia Liverpool, Liverpool, UK X. N. ZHOU R. C. OLIVEIRA Professor, Director, National Institute of Centro de Pesquisas Rene Rachou/ Parasitic Diseases, Chinese Center for CPqRR - A FIOCRUZ em Minas Gerais, Disease Control and Prevention, Shanghai, Rene Rachou Research Center/CPqRR - People’s Republic of China The Oswaldo Cruz Foundation in the State of Minas Gerais-Brazil, Brazil VOLUME EIGHTY ONE

Advances in PARASITOLOGY

Edited by S. I. HAY Spatial Epidemiology and Ecology Group Tinbergen Building, Department of Zoology, University of Oxford, South Parks Road, Oxford, UK RIC PRICE Centre of Tropical Medicine, University of Oxford, Oxford, UK J. KEVIN BAIRD Eijkman-Oxford Clinical Research Unit Jalan Diponegoro No. 69 Jakarta, Indonesia

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Printed and bound in UK 13 14 15 10 9 8 7 6 5 4 3 2 1 CONTRIBUTORS

Myriam Arevalo-Herrera Caucaseco Research Center, Cali, Colombia J. Kevin Baird Eijkman-Oxford Clinical Research Unit, Jakarta, Indonesia; Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK John W. Barnwell Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Emory University, Atlanta, Georgia, USA; Centers for Disease Control and Prevention, Malaria Branch, Division of Parasitic Diseases and Malaria, Atlanta, Georgia, USA Katherine E. Battle Department of Zoology, University of Oxford, Oxford, UK Jane M. Carlton Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA William E. Collins Institutional Association: Malaria Branch, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia Aparup Das Evolutionary Genomics and Bioinformatics Laboratory, Division of Genomics and Bioinformatics, National Institute of Malaria Research (ICMR), Dwarka, New Delhi, India Ananias A. Escalante Center for Evolutionary Medicine and Informatics, The Biodesign Institute, Arizona State University, Tempe, AZ, USA Marcelo U. Ferreira Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo (SP), Brasil Mary R. Galinski Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Emory University, Atlanta, Georgia, USA; Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia, USA Simon I. Hay Department of Zoology, University of Oxford, Oxford, UK Rosalind E. Howes Department of Zoology, University of Oxford, Oxford, UK

ix x Contributors

Christopher L. King Center of Global Health & Diseases (CGHD), Case Western Reserve University, V eterans Affairs Medical Center, Cleveland, OH, USA Odile Mercereau-Puijalon Institut Pasteur, Centre National de la Recherche Scientifique Unité de Recherche Associée, Unité d’Immunologie Moléculaire des Parasites, Paris, France Esmeralda V.S. Meyer Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia, USA Ivo Mueller Walter + Eliza Hall Institute, Infection & Immunity Division, Parkville, Victoria, Australia; Barcelona Centre for International Health Research (CRESIB, Hospital Clínic-Universitat de Barcelona), Barcelona, Spain Jean-Louis Pérignon Inserm, UMR-S 945, Paris, France; Faculté de Médecine Pitié-Salpêtrière, Université Pierre et Marie Curie-Paris 6, CHU Pitié-Salpêtrière, Paris, France; Faculté de Médecine Paris 5, Université René Descartes-Paris 5, CHU Necker-Enfants Malades, Paris, France Ari W. Satyagraha Eijkman Institute for Molecular Biology, Jakarta, Indonesia Georges Snounou Inserm, UMR-S 945, Paris, France; Faculté de Médecine Pitié-Salpêtrière, Université Pierre et Marie Curie-Paris 6, CHU Pitié-Salpêtrière, Paris, France Takafumi Tsuboi Cell-Free Science and Technology Research Center and Venture Business Laboratory, Ehime University, Matsuyama, Ehime, Japan Peter A. Zimmerman Center for Global Health & Diseases, Case Western Reserve University, Cleveland, Ohio, USA PREFACE

The epidemiology of Plasmodium vivax: history, hiatus and hubris forms is a two volume special issue of Advances in Parasitology on the epidemiology of P. vivax. The aim of the review collection is to present a contemporary summary of what is known about P. vivax, with the challenge set to the authors to (1) retrieve what has been ‘lost’ from ‘history’, (2) summarize objectively the current state of knowledge including the reasons for the ‘hiatus’ in interest and; (3) identify research gaps/directions/priorities to gently temper the prevailing ‘hubris’ with respect to control and elimination. Part A (volume 80) was published in December 2012. It was composed of six chapters and dealt principally with the most practical dimensions of vivax malaria, describing the epidemiology, clinical consequences, treatment, and control strategies shaped by the biology of the parasite. Part B (volume 81) is published here and brings together a further six chapters that investigate more fully aspects of the parasite life cycle, innate and inherited aspects that confer host resistance to P. vivax infection, the epidemiological importance of G6PD deficiency, what is known about the genome of P. vivax and finally the lessons that can still be learned from the interpretation of the old neurosyphilis literature. Chapter 1 by Mary R. Galinski and colleagues looks in detail at the parasite’s life cycle, how it is adapted to its life history challenges and how this differentiates it from P. falciparum. They also explore the research challenges that remain in combining non-human primate models with new technologies to potentially provide further insights and epidemiological understanding of the biology of this parasite. Chapter 2, by Peter A. Zimmerman and colleagues, reviews fascinating new complexities to what is canonically taught about red blood cell polymorphism (predominantly of the Duffy gene) and susceptibility to P. vivax infection at the individual and population levels. Chapter 3 led by Ivo Mueller reviews the natural acquisition of immunity to P. vivax. Epidemiological observations are synthesised and used to support the premise that a multi-stage P. vivax vaccine may be feasible. Chapter 4 by Rosalind E. Howes reviews the geography of G6PD deficiency. The global distribution of its prevalence and genetic variants are discussed along with the implications that this has for the potential risk of haemolysis triggered by primaquine therapy in different parts of the world. Chapter 5, written by Jane M. Carlton and colleagues, considers the genomics, population genetics

xi xii Preface and evolutionary history of P. vivax. New insights from detailed genomic investigations of P. vivax across India are synthesised, as well as, research avenues opening as a result of next generation sequencing technologies, discussed. In the final Chapter 6, Georges Snounou and Jean-Louis Péri- gnon conclude these volumes by reviewing the epidemiological insights gained from a thorough analysis of the malaria therapy for neurosyphilis literature. They outline further some of the insights that might be gained in relation to the current challenges of P. vivax epidemiology, immunology and pathology by a deeper engagement with this literature. It is perhaps worth noting that the enormous literature summarized in these two volumes evidences a rich history that we are unwise to forget. Moreover, that the authorship have perhaps helped turn the corner on the research hiatus of P. vivax. Conversely, and perhaps predictably, it also reveals there is much still to learn and therefore that we must approach with some hubris the immediate challenges of P. vivax control and future challenges of its elimination. Finally we take this opportunity to thank all the authors for their considerable time and energy devoted to putting these chapters together. We hope that these two volumes “lower the bar” for a new cohort of malariologists inspired by future challenges in the epidemiology and control of P. vivax.

Simon I. Hay, Ric N. Price and J. Kevin Baird CHAPTER ONE

Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle

Mary R. Galinski*,†,1, Esmeralda V. S. Meyer†, John W. Barnwell*,‡ *Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Emory University, Atlanta, Georgia, USA †Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia, USA ‡Centers for Disease Control and Prevention, Malaria Branch, Division of Parasitic Diseases and Malaria, Atlanta, Georgia, USA 1Corresponding author: E-mail: [email protected]

Contents 1. Introduction 2 2. The General Life Cycle of Plasmodium vivax and Other Primate Malaria Parasite Species 3 2.1. The Hypnozoite: An Alternative Life Style for Liver-stage Development 6 2.2. The Reticulocyte as a Host Cell: An Environmental Safety Program for P. vivax 8 2.3. Fast and Furious: The Sexual Life Strategies of P. vivax 9 3. In Vitro and Ex Vivo Models for Examining P. vivax Biology 11 4. Neotropical Non-Human Primate Models (New World Monkeys) for Investigating the Varied Biology of vivax Malaria 14 5. The Relapsing Malaria Parasites of Southern Asian Macaque Monkeys as Models for P. vivax Biology 15 6. From Genomics to Systems Biology: The Bigger Picture Puzzles 17 7. Conclusions 20 Acknowledgements 20 References 20

Abstract Plasmodium vivax has unique attributes to support its survival in varying ecologies and climates. These include hypnozoite forms in the liver, an invasion preference for reticulocytes, caveola–vesicle complex structures in the infected erythrocyte membrane and rapidly forming and circulating gametocytes. These characteristics make this species very different from P. falciparum. Plasmodium cynomolgi and other related simian species have identical biology and can serve as informative models of P. vivax infections. Plasmodium vivax and its model parasites can be grown in non-human primates (NHP), and in short-term ex vivo cultures.

For P. vivax, in the absence of in vitro culture systems, these models remain highly relevant side by side with human clinical studies. While post-genomic technologies allow for greater exploration of P. vivax-infected blood samples from humans, these come with restrictions. Two advantages of NHP models are that infections can be experimentally tailored to address hypotheses, including genetic manipulation. Also, systems biology approaches can capitalise on computational biology combined with set experimental infection periods and protocols, which may include multiple sampling times, different types of samples, and the broad use of “omics” technologies. Opportunities for research on vivax malaria are increasing with the use of existing and new methodological strategies in combination with modern technologies.

1. INTRODUCTION Plasmodium vivax has been neglected as a disease of major global importance. Recently, expanded efforts have been made to bring more widespread attention to this disease and to overcome perceptions that there are insurmountable barriers to advancing research and basic knowledge on vivax malaria (Carlton et al., 2011; Galinski and Barn- well, 2008; Lacerda et al., 2012; Mueller et al., 2009; Price et al., 2011). In fact, research and methodological strategies are in place to move forward using the most modern technologies available, and advances are being made. Basic vivax malaria research is benefiting from the incor- poration of ex vivo samples from humans, non-human primate (NHP) experimentation and in vitro analyses. In addition, an increased focus on the epidemiology of P. vivax, with an increased attention to interactions with other species, and greater consideration of the ecological factors that affect this parasite’s range is apparent (Gething et al., 2012). Various clinical, epidemiological and biological attributes associated with vivax malaria have also gained the attention of mathematical modellers and computational biologists who wish to apply currently available knowledge on the host and vector interactions with this parasite to understand transmission and the influence of current control interventions and drug treatments on those dynamics (Aguas et al., 2012; Chamchod and Beier, 2012; Gething et al., 2011; Mueller et al., 2009; Price et al., 2011; White, 2011). However, to improve modelling efforts and control strategies, interventions or drug therapies will benefit from a better understanding of the biological attributes that afford P. vivax and its sibling species life strategies that enable it to persist when confronted with control methods implemented by its human host. Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle 3

2. THE GENERAL LIFE CYCLE OF PLASMODIUM VIVAX AND OTHER PRIMATE MALARIA PARASITE SPECIES In general terms, the life cycle of P. vivax is like that of all of the other primate malaria species in that it requires an invertebrate and a vertebrate host for survival and perpetuation; a female mosquito of a susceptible Anopheles species and a primate, whether human or NHP (Fig. 1.1). When the female mosquito bites, or more precisely, probes the dermis with her proboscis looking for a vessel to obtain her blood meal, she releases salivary fluid and along with it a few sporozoites from her salivary glands. In the dermal tissues, the sporozoites are motile and capable of penetrating small blood vessels, and beginning to stimulate a host immune response (Guilbride et al., 2012; Sinnis and Zavala, 2008). In the circulating blood, they are swept into the liver sinusoid vessels where they penetrate through the professional phagocytes known as Küpffer cells into the Space of Disse to begin the exoerythrocytic or liver-stage cycle of growth (reviewed in Baer et al., 2007b; Frevert et al., 2008; Meis et al., 1983; Pradel and Frevert, 2001). Once there, the sporozoite then penetrates a hepatocyte, rounds up and differentiates into a small trophozoite (∼4 µ in diameter) growing in size over the next few days eventually differentiating into a multinucleated schizont in 5 days. By 6 or 7 days, of primary growth and development, a fully mature schizont 40–60 µ in diameter has differentiated into thousands of individual invasive single nucleated merozoites surrounded by a parasitophorous membrane capsule. As reported for rodent malaria experimental model systems (not yet investigated in primate malarias), the plasma membrane of an infected hepatocyte breaks down, and blebs of the parasitophorous membrane full of merozoites called merosomes break off and flow into the circulation of the liver sinusoid vessels (Prudencio et al., 2006; Thiberge et al., 2007). These merosomes are carried into the faster flowing general blood circulation and break apart releasing the imprisoned merozoites (Baer et al., 2007a), which then attach to and invade red blood cells (RBCs) to start the erythrocytic cycle of infection, also known as the blood-stage cycle. The newly invaded merozoite immediately differentiates into an erythrocytic trophozoite and begins remodelling the anucleate RBC to provide a suitable environment for it to grow larger over a period of 48 hours feeding upon the haemoglobin of the parasitised RBC. Thirty-eight or 40 h into this cycle of growth, the nucleus divides in two to create a schizont and over the next 8 or so hours continues to divide by schizogony to form 4 Mary R. Galinski et al.

Figure 1.1 Schematic of the life cycle of Plasmodium vivax and comparable sibling simian species, depicted to represent the unique biological features of these species in the life cycle of primate malaria species and the importance of clinical and experimental interventions. The monkey figure represents neotropical NHP models of P. vivax or macaque NHP models of P. cynomolgi and other simian parasite species that serve as surrogates for P. vivax. The purple and green icons indicate where natural events and experimental manipulations can take place. The green mosquito icons refer to the natural inoculation of sporozoites through biting and the purple mosquito icons refer to the natural biting and infection of Anopheles ssp. mosquitoes by drawing in gametocyte-infected blood. The green medical symbol and syringe denoting the inoculation of sporozoites into the human and NHP hosts, respectively, refer to the possibility of challenging these hosts after immunisation with a vaccine candidate to determine if protection can be induced. The purple medical symbol and syringe denote the collection of blood for testing involving human and NHPs, respectively. The purple syringe also signifies the specific collection of blood containing gametocytes from NHP to artificially feed and infect Anopheles mosquitoes, to support experiments on the transmission of sporozoites or transmission blocking vaccines. The unique biological features of P. vivax and comparable species depicted are the hypnozoite, the preferential invasion of merozoite into reticulocytes, the production of CVCs, represented as a mottled appearance of the infected RBCs, and the early and rapid development and circulation of gametocytes. Red arrows refer to processes relating to these features, in need of special research emphasis: 1) what is the make-up of hypnozoites and how are they activated, 2) what are the similarities and differences in primary and relapsing liver-stage schizonts and is their biology with merosome release in the blood stream comparable to other Plas- modium species, 3) what critical factors are required for reticulocyte host cell selection, invasion and growth in these cells, and 4) what factors determine the development and circulation of gametocytes, potentially permitting transmission from the early stages of a blood-stage infection. (For interpretation of the references to colour in this figure legend, the reader is referred to the online version of this book). Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle 5

12–16 or more differentiated merozoites in the case of P. vivax. When the host cell membrane ruptures after 48 h of parasite intracellular growth, the merozoites are released and invade new RBCs to begin the entire asexual blood cycle again. The merozoites in some schizonts are preprogrammed to differentiate into sexual-stage male or female micro- and macro-gametocytes, respectively, and not into asexual trophozoites upon entry into a new RBC (Bruce et al., 1990; Reininger et al., 2012; Silvestrini et al., 2000). The insect or sexual stage of the life cycle begins when a feeding female Anopheles mosquito takes up some of these circulating male and female gametocytes in her blood meal. In the midgut of the blood-engorged mosquito, the gametocytes lose their RBC membrane outer cover and become sexually stimulated. The nucleus of the microgamete fractures into eight nuclei and eight flagellating bodies are formed (Carter and Nijhout, 1977; Prasad et al., 2011). When one penetrates a macrogamete, a diploid zygote is formed that over a period of 24–36 h metamorphoses into an ookinete, another tissue invasive parasite stage. The ookinete penetrates through the mosquito midgut lining to lodge under the basal membrane where it transforms into an oocyst that undergoes multiple nuclear divisions to form a capsule of several thousand elongated sporozoites. At maturity, the capsule breaks open, releasing the thousands of sporozoites into the haemocoel of the mosquito, which then migrate to and penetrate the salivary glands to lodge in the glandular spaces waiting until the mosquito probes dermal tissue seeking a blood meal. As the mosquito probes, she releases salivary fluid and along with it several dozen up to a few hundred sporozoites into the dermis of the skin, basically coming full circle from where we started this story and completing the life cycle of a primate malaria parasite (reviewed in Ejigiri and Sinnis, 2009). Plasmodium vivax and its three sibling species in Southeast Asian macaque monkeys, P. cynomolgi, P. simiovale and P. fieldi, form a group of malaria parasites that have evolved a set of distinctive biological features in their life cycles that set them apart from the other most often studied of the human malaria parasite species, P. falciparum (reviewed in Carlton et al., 2008b; Galinski and Barnwell, 2012, 2008). While there is much more to be understood regarding the evolutionary advantages of these life cycle features for the parasite, both our present knowledge about their biology and intuitive reasoning suggest that these parasites have evolved these characteristics as life strategies to evade and persist in hostile environments whether it be within their hosts or ecological. In particular, these parasites have dormant hypnozoite forms in the liver that are unique to the P. vivax clade of relapsing malaria parasites (Cogswell, 1992; 6 Mary R. Galinski et al.

Cogswell et al., 1991; Krotoski et al., 1982b, 1986). The distantly related human malaria parasite, P. ovale, has also been known to produce relapse infections and thus suspected of forming hypnozoites, which may be a good example of convergent evolution. However, this species and its possible relapses are much less studied (Richter et al., 2010). The adjunctive liver cycle of the hypnozoite, as explained further below, enables recurring blood-stage infections, termed relapses, to occur weeks or months after a primary infection in the blood has taken place (reviewed in White, 2011). A second life strategy that P. vivax and the other relapsing malaria parasite species as well have evolved is to preferentially select reticulocytes as host cells (Galinski et al., 1992; Kitchen, 1938; Kosaisavee et al., 2011; Li and Han, 2012), which has been hypothesised to be the source of the benign status of P. vivax because it limits parasite levels in the blood. However, as explained below, this probably is not the only reason this young RBC population provides a selective advantage for P. vivax and its cousin species. Another distinctive characteristic for P. vivax and other relapsing malarias, which perhaps is also correlated to the invasion of reticulocytes, is that in Giemsa-stained blood smears the infected erythrocytes are recognised by a multitude of reddish dots known as Schüffner’s stippling. These dots actually represent numerous elaborate flask-shaped and tubular membranous structures, known as caveola-vesicle complexes (CVCs), which are created by the parasite and positioned all along the inside of the host cell membrane and open to the exterior (Aikawa et al., 1975; Akinyi et al., 2012; Barnwell et al., 1990). A fourth life cycle difference from P. falciparum and likely the other species of the Laverania subgenus is that P. vivax gametocytes develop quickly and circulate early in an acute infection, a condition that would allow transmission prior to patients feeling severely ill and seeking treatment (Bousema and Drakeley, 2011). Each of these biological features is discussed in more detail in the context of Fig. 1.1, depicting the life cycle of P. vivax, sum- marising key points that are relevant for understanding and designing experiments using available technologies, human clinical samples or NHP models.

2.1. The Hypnozoite: An Alternative Life Style for Liver-stage Development When a sporozoite of a relapsing malaria parasite enters the liver, it will differentiate into an early small liver trophozoite of about 4.0 or 5.0 µ in size and then it may enter either of two very different developmental pathways. Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle 7

First there can be immediate growth in the host liver cell all the way through to schizogony and the production of thousands of merozoites that will initiate the erythrocytic cycle. This is the primary pathway for primate Plasmo- dium species including most strains of P. vivax detailed above. Alternatively, the small trophozoites become dormant and may remain in this quiescent metabolic state for weeks, months or up to 2 years in a hepatocyte. Whether the route taken is a predetermined genetic or epigenetic trait programmed in the mosquito, a factor of the hepatocyte environment or a combination of both is unknown as is almost everything else about P. vivax liver-stage parasites and their relationship with the hepatic host cell. The small dormant forms of P. vivax (and P. ovale, and the simian species P. cynomolgi, P. simiovale and P. fieldi), termed hypnozoites, remain a black box for researchers to decipher. At about 4–5 µ, the hypnozoite is a fraction of the size of a growing primary liver-stage schizont (40–60 µ) (Krotoski et al., 1982b, 1986), and its make-up and what reactivates it for growth is entirely unknown. What is known is that P. vivax hypnozoites remain dormant in the liver for a varied range of times depending to some extent on geographical distribution but more so on what appears to be different “strains” of the parasite (Garnham et al., 1975; White, 2011). In the Northern latitudes the so-called “temperate” strains, also known as hibernans types, were characterised early on by their delayed relapse patterns of blood-stage infections. These strains exhibited a pattern of a first relapse that would occur 8, 9 or 12 months after being infected with sporozoites. In these cases, there may or may not be an initial primary parasitaemia following within 2 or 3 weeks after sporozoite infection, which results from some liver-stage trophozoites going directly through the primary cycle of development. The lack of an early primary cycle of exo-erythrocytic development indicates that the sporozoites from the temperate strains mostly differentiate into hypnozoites. The other basic type that is designated as “tropical” strains have early and frequent relapse patterns at regular intervals of a few weeks initially over 18–24 months and almost always with an initial primary blood-stage infection. It is clear that the temperate form is a life-cycle strategy suited to climates with long winters and exists to infect mosquitoes emerging in late spring and early summer (reviewed in Galinski and Barnwell, 2012; White, 2011). It is not clear, though, what the selective advantage is for the early and frequent relapse of tropical strains is except perhaps to thwart an emerging host immune response. Most relapse parasites in individual patients with tropical strains, even in low transmission conditions, have been shown to be genetically heterogeneous reflecting past and present infections with 8 Mary R. Galinski et al. different genotypes of P. vivax (Chen et al., 2007; Imwong et al., 2007). Much work remains to be done, to fully understand the origins and mechanisms of dormancy and activation of hypnozoites.

2.2. The Reticulocyte as a Host Cell: An Environmental Safety Program for P. vivax For P. vivax and the few species of primate Plasmodium dependent on reticulocyte host cells, an efficient process must ensue, enabling these organisms with the ability to effectively find and latch onto the limited number of young RBCs amidst a virtual sea of more mature erythrocytes. Generally, reticulocytes only make up 0.5–2.0% of the erythrocytes in circulation. A few proteins have so far been identified and characterised as being required during the invasion process of the reticulocyte by the merozoite. The reticulocyte-binding proteins have been defined as critical proteins that select these host cells in the circulation (Galinski and Barnwell, 1996; Galinski et al., 1992, 2000). The parasite’s Duffy Binding Protein has also been recognised for several decades as a critical RBC adhesin (Chitnis and Miller, 1994; Wertheimer and Barnwell, 1989); only recently have P. vivax infections been associated with individuals with Duffy-negative RBC phenotypes (Cavasini et al., 2007; Menard et al., 2010; Ryan et al., 2006). Other proteins in common with P. falciparum and other Plasmodium species are believed to have comparable roles across species, as elaborated in Mueller et al. (Chapter 3). But, critically, the reticulocyte must be targeted prior to the release of other internally localised adhesins in the merozoite apical organelles, which would otherwise permit the abortive binding of merozoites to more mature cells, which likely do not support the continued growth and propagation of P. vivax (Galinski and Barnwell, 1996; Galinski et al., 1992). As introduced above, P. vivax trophozoites (and related simian model parasites, like P. cynomolgi ) grow in the reticulocyte and early during this growth phase the parasitised erythrocyte develops elaborate CVCs all along the surface of the infected host cell (Aikawa et al., 1975), each appearing in 3D analyses to have its own signature with a different number and length of tubules and associated vesicles (Akinyi et al., 2012); these structures and isolated vesicles in the cytoplasm suggest a biogenesis mechanism that may involve the incremental fusion of such vesicles. These structures include dozens of parasite-encoded proteins (Barnwell et al., 1990) Udagama et al., 1988. What metabolic functions may be served by the CVC are only speculative at this time but based on early experiments (Aikawa et al., 1975) and 3D images of hollow openings to the surface (Akinyi et al., 2012) uptake and perhaps release of metabolites is suggested. The need for these structures Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle 9 in P. vivax and in each of the other relapsing, reticulocyte-preferring species and the apparent interconnectedness with the host cell and extracellular environment of the host may explain why the field has faced challenges growing these parasites in long-term in vitro culture. We would argue that the parasite does not only require reticulocytes but also other unknown critical factors for its development in the reticulocyte that are not currently supplied by or adequate in current culture media. Ongoing research shows that the CVCs can release vesicles akin to exosomes as an active mechanism as well as upon rupture of the infected RBC (Dluzewski et al, unpublished data). Restricting an infection to these young RBCs, which typically represent 0.5–2% of the circulating RBCs, may limit the rise in parasitaemia, but this strategy may also enable P. vivax to alter the infected RBCs (iRBCs) to remain or become more flexible and able to circulate through small capil- lary vessels and more likely to survive passage through the sinusoidal vascu- lature of the spleen (Handayani et al., 2009). This is the opposite strategy of P. falciparum that invades mature erythrocytes and increases the rigidity of its host cell such that it must display cytoadherence ligands, which are variant antigens, at the surface of infected erythrocytes. This allows the parasitised erythrocyte to sequester in small vessels by adhesion to endothelium and thus avoid passage through and destruction in the spleen when the intracellular parasite matures. Plasmodium vivax does not have var genes encoding variant antigens like P. falciparum (Baruch et al., 1995; del Portillo et al., 2001; Smith et al., 1995; Su et al., 1995) and because of the fluidity/flexibility of its host cell does not need to sequester to avoid spleen passage. To what degree the evolution of the CVC structures and their placement all along the iRBC membrane (Aikawa et al., 1975; Akinyi et al., 2012; Barn- well et al., 1990) impacts this cellular flexibility remains to be investigated. The recently reported but limited adhesive potential of P. vivax iRBCs (Carvalho et al., 2010) compared to the strong cytoadherence and deep vascular sequestration of P. falciparum iRBCs does not impact the hypothesis that selecting and further altering reticulocytes by P. vivax and cousin species also permit the parasitised RBC to circulate through the spleen and survive.

2.3. Fast and Furious: The Sexual Life Strategies of P. vivax Gametocyte development for P. falciparum compared to P. vivax gametocyte development is slow and complex occurring over a period of about 10–11 days (Carter and Miller, 1979). The first stage recognisable as gametocytes in P. falciparum are small round compact forms with hemozoin pigment granules in infected erythrocytes, which soon disappear from the circulation. 10 Mary R. Galinski et al.

These sexual-stage parasites are sequestered in the spleen and bone marrow sinusoids as they mature towards the typical stage V sausage-shaped crescents that reappear in the blood and are infective for mosquitoes (Smalley et al., 1980). In sharp contrast, while not studied nearly as much as P. falciparum gametocytes, especially in the more recent time period, P. vivax gametocytes at maturity are large, round and fill an enlarged RBC with prominent Schüff- ner’s stippling and a large nucleus in pink (male) and blue (female) staining cytoplasm. P. vivax gametocytes are known to develop early in an acute primary infection, within 5 days of the clinical onset. In fact, gametocytes are known to be produced earlier, perhaps, within 8 days after mosquito inoculation before they can be seen by light microscopy (30–60 gametocytes per microliter) as mosquitoes can become infected at this time (Boyd et al., 1936; Boyd and Andstratman-Thomaws, 1934). Although not well studied, P. vivax gametocyte development requires probably about 48 h and they do not remain more than 3 days after differentiation towards sexual maturity. However, gametocyte densities become greater as blood-stage infections progress, seeming to come in waves at 5-day intervals and the production of gametocytes continues as the infection progresses on into chronic- ity becoming asymptomatic or more mildly symptomatic. Plasmodium vivax gametocytes, which can be efficiently infective to mosquitoes throughout this time, comparatively seem to be more infective towards susceptible mosquito species than P. falciparum, likely attributable to intrinsic attributes of both the parasite and the mosquito species (Bousema and Drakeley, 2011; Boyd et al., 1935). Nevertheless, this ability to form infective gametocytes early and continuously, in addition to the periodic renewal of blood infections (and gametocyte propagation) by reactivated hypnozoites, makes P. vivax transmission fast, effective and persistent. Like the asexual stages, the P. vivax-gametocyte-infected erythrocyte, which continues to circulate in the blood and not become immobilised in a tissue site as P. falciparum gametocytes do, remains highly flexible and capable of passing through small cap- illaries or splenic sinusoidal vessels despite containing a large parasite body. Although we have only discussed P. vivax gametocytes above, the gametocyte biology of P. cynomolgi, or any of the other relapsing malaria parasite species, parallels that of P. vivax. There are many Anopheline mosquito species in the tropical, subtropical and, most illustratively, in the temperate latitudes quite far north that can act as efficient and effective transmitters of P. vivax; more so than P. falciparum. In fact, at temperatures less than optimal for the continued development of P. falciparum sporozoites (<16 °C), P. vivax sporozoites will Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle 11 continue to develop, albeit at a slower pace, in their mosquito hosts, which likely accounts for P. vivax transmission above a 16 °C isotherm but not P. falciparum. The biological attributes of sporozoite development over a large temperature range in an array of susceptible mosquito vector species that over winter combined with delayed relapse genotypes/phenotypes allow the successful transmission of P. vivax as opposed to the other three human malaria species, whether it be P. falciparum, P. malariae, or that other human relapsing parasite, P. ovale, in the more northern latitudes.

3. IN VITRO AND EX VIVO MODELS FOR EXAMINING P. VIVAX BIOLOGY Scientists since Bass and Johns (1912) first reported their attempts at the in vitro propagation of both P. falciparum and P. vivax blood stages have been attempting to cultivate these two parasite species in a continuous in vitro cycle in test tubes, Leighton tubes, tissue culture flasks and a variety of other vessels. It was not until 1976 that Trager and Jensen (1976) and Haynes et al. (1976) reported the continuous in vitro development of P. falciparum blood stages. To date, there is no comparable long-term in vitro continuous culture system to propagate P. vivax-infected RBCs. There have been various attempts, reported and unreported, over the past 30 or so years to culture P. vivax-infected RBCs long-term, in vitro, similarly to methods shown to be successful for P. falciparum. However, most attempts have been no more successful than those of Bass and Johns (1912) reported 100 years ago! The most successful was the attempt by Golenda et al. (1997) when they cultivated P. vivax Chesson strain parasites for six or seven generations with approximately a two-fold multiplication rate by the addition of fresh blood highly enriched with reticulocytes every 48 h and the use of McCoy’s tissue culture medium. This has not been successfully repeated since. Nonetheless, attempts continue presently with efforts largely focused on the routine addition of fresh reticulocytes, either from umbilical cord blood enriched with reticulocytes or produced in vitro from erythroid stem cell cultures (Borlon et al., 2012; Golenda et al., 1997; Grimberg et al., 2012; Lanners, 1992; Mons, 1990; Mons et al., 1988a, 1988b; Noulin et al., 2012; Panichakul et al., 2007; Udomsangpetch et al., 2008). Just as a reliable robust culture system still does not exist for the blood- stage forms of P. vivax, this is also the case for the most closely related simian malaria species, P. cynomolgi. There has been one publication from 30 years ago demonstrating the culture of the Berok strain of this species for up to 12 Mary R. Galinski et al.

3 weeks in vitro in rhesus monkey RBC (Nguyen-Dinh et al., 1981). Our unpublished results support these claims, but this is the same time limit we have achieved so far in our attempts to repeat these results. After 3 weeks, the cultured parasites no longer thrive in vitro despite the routine addition of fresh reticulocyte-enriched target cells. All signs are that the parasite requires from its host, as per the classic definition of parasitism, factors that are not sufficiently supplied in vitro with the addition of rhesus monkey or human serum to medium or fresh reticulocytes. Whether further research will determine what needs to be added to the culture to allow continued robust propagation beyond 3 weeks remains to be seen. A systems biology approach may aid this endeavour and those trying to culture P. vivax long term. Clearly, this same limitation of the current in vitro environment for P. cynomolgi also applies to P. vivax culture investigations and there is a need that goes beyond just the addition of reticulocytes, which will require systematic investigation of the growth requirements of these parasites and their metabolites. Metabolomics comparing in vivo and in vitro-derived samples and environments may provide the data to solve this problem. Regardless, in the absence of long-term cultures, reliable short-term ex vivo blood-stage cultures for growth of developmental stages and merozoite invasion assays have been established since 1989 for P. vivax using strains derived from neotropical New World monkey species infected with this parasite (Barnwell et al., 1989). These cultures and assays have served the purpose of obtaining parasite samples for basic research relevant for discovering diagnostics, vaccine development and drug testing as well as investigation of merozoite biology and for genetic transformation of P. vivax (Pfahler et al., 2006). Similar short-term cultures also exist for P. cynomolgi-infected blood obtained from infected rhesus monkeys (Nguyen-Dinh et al., 1981). Other investigators have tried to develop short-term cultures of P. vivax from ex vivo clinical specimens for invasion assays or drug susceptibility studies but these have not been especially robust (Grimberg et al., 2007; Russell et al., 2003). More recently, Bruce Russell and collaborators have developed a more robust and reliable system to do short-term in vitro culture of P. vivax from ex vivo clinical samples from patients using umbilical cord blood RBCs, which can be cryo-preserved and could prove useful in invasion assays for vaccine studies and growth assays for drug discovery (Russell et al., 2011). We and other investigators are routinely using such methods to mature parasites to the stage of interest for biological and genetic experiments and to carry out short-term merozoite invasion experiments. Through these Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle 13 assays and collaborations, an increasing number of investigators are manag- ing to circumvent the barriers posed by the lack of long-term cultures. In effect, and perhaps as a fortuitous bonus, these investigations are working with parasites that more closely mimic the parasites in vivo than do the parasites after long-term adaptation to culture environment. It is worth noting that ex vivo and in vitro comparisons in P. knowlesi blood-stage gene expression were striking, with hundreds of differentially expressed genes (Lapp et al, unpublished data). It will be useful to examine similar data sets in a rigorous manner for P. falciparum, from in vitro and ex vivo derived parasites samples of the same strain, with Aotus or Saimiri monkeys as hosts. Investigating the liver stages of P. vivax is even more challenging than for studies on the blood stages. Sampling of the liver by biopsy for ex vivo studies even in NHP cannot be done and long-term in vitro hepatocyte cultures that mimic the host liver environment are not yet a reality. Short-term liver-stage in vitro assays for P. vivax are possible using primary human hepatocytes or hepatoma cell lines (Hollingdale et al., 1985; Mazier et al., 1984). However, high-throughput for drug testing and discovery investigations require a reliable and, ideally, an aseptic, sus- tainable source of P. vivax sporozoites. This is quite difficult to come by for P. vivax. Some P. vivax liver-stage and vaccine research has relied in the past on the availability of chimpanzee infections for P. vivax-gametocyte-infected blood to get highly infected Anopheles mosquitoes by membrane feeding to produce millions of sporozoites (Collins et al., 2009). Although strict limitations have since placed these higher primates off limits from conducting most forms of infectious disease research including malaria (Altevogt et al., 2012), New World monkey infections remain an option, though less ideal, for obtaining P. vivax sporozoites. However, these animals are in limited supply and with even greater limitations on the availability of honed expertise and experience for conducting research on P. vivax in these animals (Galinski and Barnwell, 2012). The ideal situation would be to have a robust reliable in vitro culture system, with the added benefit of the continuous production of mosquito infective gametocytes. This added requirement of a (nonexistent) P. vivax culture system, however, is another tall order, as gametocytogenesis tends to turn off in the majority of P. falciparum in vitro cultures, which is also the case for P. knowlesi cultures. Still, the development of P. cynomolgi liver-stage parasites including hypnozoites in primary Macaca fascicularis hepatocytes maintained in vitro for medium throughput assays for drug discovery remains one backup as high numbers of sporozoites can be 14 Mary R. Galinski et al. obtained from mosquitoes fed on infected rhesus monkeys (Dembele et al., 2011). However, this system still is limited for the study of hypnozoites because of its short-term nature due to the limited longevity of primary hepatocytes in vitro and the routine unavailability of aseptic conditions for obtaining sporozoites, which result in the microbial flora of mosquitoes contaminating such cultures. Early models for in vitro study of P. vivax hypnozoites using primary human liver cells (Mazier et al., 1984), or hepatoma cell lines such as HepG2-A2 cells (Hollingdale et al., 1985), while capable of producing both primary schizonts and small forms that appear to be hypnozoites, did not provide sufficient support (long-term host cell viability, enough invasive sporozoites in the cells, appropriate metabolism, etc.) for testing of anti- hypnozoite malarial drugs or truly capable of investigating hypnozoite biology. Production of aseptic P. vivax sporozoites is now feasible for infecting in vitro cultures of HepG2 hepatoma cells and it may be possible to study hypnozoite biology in this system as well as drug screening, though, with limitations that come with the difficulties of sourcing P. vivax sporozoites and the lack of hepatocyte physiology in hepatoma cells (Chattopadhyay et al., 2010).

4. NEOTROPICAL NON-HUMAN PRIMATE MODELS (NEW WORLD MONKEYS) FOR INVESTIGATING THE VARIED BIOLOGY OF VIVAX MALARIA Prior to 1966, any investigations of P. vivax biology had to garner data from naturally acquired clinical cases, clinical infections induced by the bites of infected mosquitoes or infected blood for the treatment of tertiary neurosyphilis, or experimental infections induced in prison and armed forces volunteers (Mueller et al., Chapter 3). Numerous attempts to adapt P. vivax to Old World primates of the Cercopithecidae family had failed miserably and limited use had been made of chimpanzees, which P. vivax can infect. However, as described below, these primates contain their own fantastic array of malaria parasites species, some of which can serve as honest, reliable models for P. vivax. How- ever, in 1966, Young and colleagues reported on the adaptation of P. vivax to small New World monkeys in Panama and could show that these productive infections could be transmitted to human volunteers through mosquitoes and back again to these neotropical primates (Young, 1966). Later that same year, Deane et al. (1966) described the infection Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle 15 of squirrel monkeys in Brazil (Deane et al., 1966). From that time forward, continuing for the next 46 years right up to today, a large number of P. vivax isolates of varied biological characteristics ranging from the frequent early relapsing Chesson strain to the late relapsing North Korean strain have been adapted to grow in neotropical primate species, primarily of the genus Aotus (owl or night monkeys) and Saimiri (squirrel monkeys). These NHP adapted strains have been used in studies to test schizonticidal antimalarial drugs, in malaria vaccine studies for both pre-erythrocytic and erythrocytic candidate antigens as well as transmission blocking vaccines and for studies of P. vivax biology and genetics in both the invertebrate mosquito vector and the primate vertebrate host (Arevalo-Herrera et al., 2011; Galinski and Barnwell, 2012). Today, these models remain as highly relevant as they did in 1966, especially since we still cannot culture P. vivax continuously in vitro in any practical manner. One overriding advantage of these neotropical NHP models for P. vivax is that experimental research designs can be devised to address specific questions about the biology of this parasite within certain limitations of these models.

5. THE RELAPSING MALARIA PARASITES OF SOUTHERN ASIAN MACAQUE MONKEYS AS MODELS FOR P. VIVAX BIOLOGY In the forests of South and Southeast Asia, the varied species of the wonderfully adaptive macaque monkeys, such as Macaca fascicularis, M. nemestrina, M. radiate, M. sinica and other species harbour a varied group of at least seven or eight and probably more species of primate malaria (Coatney, 1971). They are transmitted by forest mosquitoes primarily of the Leucosphyrus group of Anopheles spp, but some can be transmitted by other anopheline species that transmit human malaria in Southern Asia. These simian malaria parasite species present with different phenotypic characteristics that can quite remarkably mimic characteristics of the human species of malaria parasites. However, more remarkably, genetic studies have shown that within this group there is a clade of three parasite species that are not only nearly phenotypically identical to P. vivax but also are genetically close, so much so they must be considered as sibling species of P. vivax (Escalante et al., 2005; Mu et al., 2005). All four of these species (P. vivax, P. cynomolgi, P. simiovale and P. fieldi) share the characteristics of 1) hypnozoites that cause relapse 16 Mary R. Galinski et al. parasitaemia, 2) a preference for invading reticulocytes and 3) the production of CVCs in the membrane of infected erythrocytes. Sometime in the past 70,000 years or so, the ancestor of P. vivax must have jumped from the monkeys dwelling in the forests our ancestors were trekking through on the way to Australia and China. It is perhaps of some interest that P. cynomolgi has two Duffy Binding Protein genes, a major ligand that determines invasion of RBC by merozoites, whereas P. vivax has only one dbp gene (Carlton et al., 2008a; Tachibana et al., 2012). Is it possible that P. vivax lost one of its dbp genes and in doing so lost the ability to invade macaque RBCs? Plasmodium cynomolgi, on the other hand, with two dbp genes also is capable of infecting humans. An alternative scenario is that the ancestor of P. vivax first infected our Homo erectus ancestors, also present in Asia for 1.5 million years until about 50,000 BC, who then passed it on to their Homo sapien cousins. Plasmodium cynomolgi is the best studied of these NHP-relapsing malaria species and because of its genetic relationship along with almost identical biological traits in regards to P. vivax offers much as a model for the human species. In fact, the exo-erythrocytic stages of primate malarias, as well as, the hypnozoite stage were first discovered in P. cynomolgi-infected rhesus monkeys (Krotoski et al., 1980; Shortt and Garnham, 1948) prior to being identified in P. vivax-infected humans and chimpanzees (Krotoski et al., 1982a, 1982c; Shortt et al., 1948). P. cynomolgi, because of its frequent relapse patterns exhibited by the hypnozoite stage in the livers of macaques has been used in many studies for screening 8-aminoquinolines related to primaquine and other compounds for the discovery of liver-stage drugs that will provide radical cure of vivax-like malaria (Bray et al., 1985; Deye et al., 2012; Jiang et al., 1988; Schmidt, 1983). Deye et al. (2012) recently utilised the P. cynomolgi model to screen antimalarial drug combinations for preventing relapses. The P. cynomolgi model will be an invaluable aid for dissecting the biology of the hypnozoite. The fact that P. cynomolgi can be genetically transformed, as shown by the genomic integration and expression of the red fluorescent protein gene (Akinyi et al., 2012) also bodes well for the use of this primate malaria model in being able to experimentally manipulate and follow hypnozoites in the livers of experimentally infected macaque monkeys. Additionally, the development of these imaging tools and purification methods will allow the study of the biology and metabolic make-up of the developing primary trophozoites and schizonts in infected hepatocytes, in addition to the hypnozoite forms. The study of P. cynomolgi, with as many as 14 archived strains, Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle 17 provides the opportunity for expedited progress in this area, compared to the comparably difficult experiments based on experimental P. vivax infections in small New World monkeys (Aotus or Saimiri species) (reviewed in Galinski and Barnwell, 2012). Plasmodium simiovale has been much less studied since its discovery in 1965 (Dissanaike, 1965). However, hypnozoites have been identified in this species in rhesus monkeys and studies with mosquito inoculations show that it can have relapses lasting over nearly 2 years that can be early and frequent or occur late but then with several recurrences at short intervals (Cogswell et al., 1991; Collins and Contacos, 1979, 1974; Collins et al., 1972). In summary, P. cynomolgi and P. simiovale have similar biology to each other and P. vivax and can serve as excellent models of P. vivax infection. This is an important advantage that has enabled and will continue to enable the deeper study of P. vivax, especially in the absence of long-term in vitro culture systems.

6. FROM GENOMICS TO SYSTEMS BIOLOGY: THE BIGGER PICTURE PUZZLES With the advent of genomics, some 20 years ago, scientists aimed to glean what they could from analysis of DNA sequences, and then through comparative genomics. The first P. falciparum genome was published in 2002, P. vivax and P. knowlesi in 2008 and P. cynomolgi just recently (Carlton et al., 2008a; Gardner et al., 2002; Pain et al., 2008; Tachibana et al., 2012); these type of strains have been followed by the availability of sequences from many other P. falciparum genomes and a number of P. vivax genomes, especially since NextGen sequencing has amplified what is possible and brought down the timeframe and cost (Chan et al., 2012). Functional genomics ushered in the “omics” era, with emphasis moving from in vitro to ex vivo transcriptomes and proteomes for P. falciparum (Daily et al., 2007; Dharia et al., 2010; Kuss et al., 2012; LaCount et al., 2005; Lasonder et al., 2002, 2008) and recently initial P. vivax transcriptomes (Bozdech et al., 2008; Westenberger et al., 2010) and proteomes (Acharya et al., 2009, 2011; Roobsoong et al., 2010; Roobsoong et al., 2011) based on clinical samples. We have been com- plementing such data with parasites generated in NHP infections initiated from P. vivax adapted and simian malaria parasite cryo-preserved stocks, from which we can obtain multiple biological replicates of stage-specific samples (Lapp et al., unpublished data). As in P. falciparum, the transcriptome alone cannot precisely predict the expression and timing of protein products; thus, 18 Mary R. Galinski et al. such complimentary work is important. Such studies are also helping groups focus on what genes and proteins are unique to P. vivax (and its comparable simian species) and can represent its unique biology. Others have also begun to investigate the serum proteome of P. vivax-infected individuals, in attempts to associate differentially expressed proteins with pathogenic features of vivax malaria and the immune response (Ray et al., 2012). Metabolomics high-throughput technologies have been developing in parallel as critical tools in this progression of post-genomics inquiry (reviewed in Kafsack and Llinas, 2010). Such powerful methods are setting new sights, standards and expectations ( Jones et al., 2012) for driving in depth investigations into the biology, biochemistry, immunology and pathogenesis of malaria parasitism. Importantly, the malaria research field is rapidly advancing from the use of isolated “omics” technologies and the study of either the host or parasite, to highly integrated systems biology approaches designed to understand the host–parasite interactions and dynamics (reviewed in Le Roch et al., 2012). It is clear that increased efforts in vivax malaria research will rapidly lead to enormous amounts of heterogeneous information from a wide range of fields, reaching from molecular biology and omics to physiology, immunology, pathogenesis and toxicology. These data will be too voluminous to permit optimal analysis with intuitive means and therefore suggest the utilisation of tools that are at the core of the emerging field of systems biology (Voit, 2012). A generic strategy might begin with machine learning techniques capable of discerning static or even dynamic patterns in the very large data sets expected from malaria research laboratories. These patterns are first to be interpreted in terms of simple and complex correlation structures, which in turn will suggest hypotheses regarding the functional networks governing the disease and, in particular, the interactions between pathogen and host, including host cell invasion mechanisms and parasite evasion strategies. These functional interaction networks are most certainly regulated very tightly, probably by both the host and the parasite, and must be expected to change significantly over short and long time horizons. These dynamic and regulatory features imply that the initial static networks must be morphed into fully dynamic systems models. These models will typically be formulated first as differential equations, but may eventually have to account for stochastic effects, such as environmental insults, and discrete interventions, such as treatment regimens and reinfection events. Some techniques for these analyses are yet to be created, but given the rapid advances in systems biology, there is justified expectation that the malaria community will have access to effective means for the integration of Plasmodium vivax: Modern Strategies to Study a Persistent Parasite’s Life Cycle 19 information into adequate dynamical models within the near future. Given the enormous capacity of computational methods, thousands of model settings can be investigated quite rapidly, and their interpretation will allow the laboratory researcher and clinician to prescreen experiments and develop testable hypotheses. Once such models have been constructed and validated; they will not only capture details of the infection process but also allow the simulation of new diagnostics and treatments, possibly even in a person- alised manner (Voit and Brigham, 2008). For vivax malaria research, systems biology approaches may prove to be the best way forward for investigating the unique biology, immunology and pathogenesis associated with P. vivax infections, and NHP experimental models provide ready tools for exploration at a depth not possible with human clinical research. Systems biology approaches can be particularly revealing, especially compared to traditional approaches for studying P. vivax, which in contrast have been painstakingly slow. Fortunately, scientific methods keep advancing from the old fashion one-gene at a time approach to a growing number of “omic” technologies, and now the opportunity to combine the use of omics to achieve a more complete understanding of how a system functions. Questions and hypotheses generated from the field can now be posed to computational biologists who can literally create and model thousands of experimental scenarios to inform the experimental design of actual studies. Plasmodium vivax research, whether through human clinical sampling or NHP in vivo and ex vivo studies, can harness these capabilities and start to maximise research output. The time has come where scientists can dissect the intricacies of the host–pathogen relationship, with all its complex interactions with host cells and factors, to better understand host cell invasion mechanisms, parasite evasion strategies, the immune response and ultimately what factors result in successful parasitism versus death of the parasite or the host. Co-infection dynamics can also be explored with modelling perspectives to ultimately improve upon the treatment for malaria and other infectious diseases. With the sensitivity of metabolomics in identifying thousands of metabolites, and the power of computations involving available metadata, it is reasonable to start to imagine what cultural/social/nutritional and geographical/environmental factors may be potentially associated with the severity of disease and possible stress factors that may also help predict the relapse of hypnozoites. Its particular biological features enable the persistence of this parasite to the extent that this organism is also gaining the interest of ecologists. 20 Mary R. Galinski et al.

7. CONCLUSIONS The rapid pace of science and technology is making inroads that are bringing P. vivax research to the forefront, and no longer leaving it in the dust behind P. falciparum. The potential for a comprehensive understanding of this organism, host–pathogen interactions, and the global nature of this disease is at an all-time high.

ACKNOWLEDGEMENTS The authors acknowledge the financial support of the National Institutes of Health, National Institutes of Allergy and Infectious Diseases, Grant # R01AI24710 and Contract # HHSN272201200031C, as well as the National Center for Research Resources P51RR000165 and the Office of Research Infrastructure Programs/OD P51OD011132. The authors also acknowledge the scientists from the Malaria Host–Pathogen Interaction Center (MaHPIC) for their inspiration and contributions (http://mahpic.emory.edu).

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Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax

Peter A. Zimmerman*,1, Marcelo U. Ferreira†, Rosalind E. Howes‡, Odile Mercereau-Puijalon$ *Center for Global Health & Diseases, Case Western Reserve University, Cleveland, Ohio, USA †Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo (SP), Brasil ‡Department of Zoology, University of Oxford, Oxford, UK $Institut Pasteur, Centre National de la Recherche Scientifique Unité de Recherche Associée, Unité d’Immunologie Moléculaire des Parasites, Paris, France 1Corresponding author: E-mail: [email protected] / Phone 1-216-368-0508

Contents 1. Introduction 28 2. The Era of Great Biological Discovery 29 2.1. Cell Biology and the Germ Theory 29 2.2. Malariotherapy and African-Based Resistance to P. vivax 32 2.3. Human Variation and Blood Groups 34 3. Resistance to P. vivax and Insights on Malaria Red Cell Invasion 35 3.1. Serological Recognition of Duffy (Fy) Blood Group Polymorphism 35 3.2. The Genetic Resistance Factor to P. vivax – Duffy Negativity 38 3.3. The Molecular and Cellular Basis of Duffy Blood Group Polymorphism 40 3.4. The Duffy Binding Protein 45 4. Evolving Perspectives on Resistance to P. vivax 47 4.1. Further Influence of Duffy Polymorphism on Resistance to P. vivax Malaria 47 4.2. Duffy-Independent Red Cell Invasion by P. vivax 49 4.3. Global Distribution of Duffy Polymorphism and the Population at Risk of P. vivax Malaria 54 4.4. Association of Non-Duffy Gene Polymorphisms with P. vivax Resistance 57 4.4.1. G6PD Deficiency 57 4.4.2. Haemoglobinopathies 58 4.4.3. Southeast Asian Ovalocytosis 58 5. Conclusions and Future Directions 60 Acknowledgements 65 References 65

Abstract Resistance to Plasmodium vivax blood-stage infection has been widely recognised to result from absence of the Duffy (Fy) blood group from the surface of red blood cells (RBCs) in individuals of African descent. Interestingly, recent studies from different malaria- endemic regions have begun to reveal new perspectives on the association between Duffy gene polymorphism and P. vivax malaria. In Papua New Guinea and the Americas, heterozygous carriers of a Duffy-negative allele are less susceptible to P. vivax infection than Duffy-positive homozygotes. In Brazil, studies show that the Fya antigen, compared to Fyb, is associated with lower binding to the P. vivax Duffy-binding protein and reduced susceptibility to vivax malaria. Additionally, it is interesting that numerous studies have now shown that P. vivax can infect RBCs and cause clinical disease in Duffy-negative people. This suggests that the relationship between P. vivax and the Duffy antigen is more complex than customarily described. Evidence of P. vivax Duffy-independent red cell invasion indicates that the parasite must be evolving alternative red cell invasion pathways. In this chapter, we review the evidence for P. vivax Duffy-dependent and Duffy- independent red cell invasion. We also consider the influence of further host gene polymorphism associated with malaria endemicity on susceptibility to vivax malaria. The interaction between the parasite and the RBC has significant potential to influence the effectiveness of P. vivax-specific vaccines and drug treatments. Ultimately, the relationships between red cell polymorphisms and P. vivax blood-stage infection will influence our estimates on the population at risk and efforts to eliminate vivax malaria.

1. INTRODUCTION The image of Plasmodium knowlesi ‘pulling’ its way into erythrocytes of Macaca mulatta is iconic in malaria research (Fig. 2.1) and sets the stage for reviewing the mechanisms of human resistance to Plasmodium vivax. Aikawa and colleagues described the events underlying erythrocyte invasion to involve an initial attachment to the red cell membrane by the parasite’s apical end, invagination of the red cell membrane around the merozoite and sealing of the erythrocyte on completion of invasion (Aikawa et al., 1978). As brilliantly shown through their electron micrographs, evidence of a ‘tight junction’ formed through molecular interactions between the parasite and host continues to inspire malaria research. Identifying specific molecules involved in the formation and gliding motility of this junction is central to unravelling the mechanism of Plasmodium species’ invasion of the red cell. Understanding how to inhibit, disrupt or block this inti- mate parasite–host interaction potentially leads to strategies for a vaccine against blood-stage infection, malaria morbidity and mortality. In this chapter, we rely on a wide range of clinical, field and laboratory findings to illustrate our evolving understanding of the factors that influence resistance to P. vivax malaria and the selective barrier that has confronted this parasite. Reviewing this work according to a general chronological Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 29

Figure 2.1 Plasmodium knowlesi invasion of Macaca mulatta red blood cells. A P. knowlesi merozoite has commenced the invasion process through formation of gliding junctions involving merozoite and red cell membranes. This enables invagination of the erythrocyte membrane and movement of the parasite into the parasitophorous vacuole. R, rhoptry; M, micronemes; J, gliding junction; PV, parasitophorous vacuole; E, erythrocyte. (Figure from unpublished data, Hisashi Fujioka) time frame will remind readers how our understanding of P. vivax infection and malaria has developed over the past 95 years. This approach also seeks to emphasise how medical and basic research scientists have applied available experimental strategies in collaborations across multiple generations to solve the important puzzle as to how malaria parasites infect red blood cells (RBCs) and cause a disease that has had significant impact on human health and the evolution of our genome.

2. THE ERA OF GREAT BIOLOGICAL DISCOVERY 2.1. Cell Biology and the Germ Theory The late 1800s to the early 1900s was a revolutionary time period that began the integration of medicine and the sciences. Of paramount importance to this chapter is the germ theory that proposed that microorganisms were the cause of many diseases. Pasteur’s experimental evidence showing that microorganisms in nutrient broth did not arise through spontaneous generation 30 Peter A. Zimmerman et al.

(1860s) significantly demystified the relationship between disease and the microbial world, and Koch’s series of objective criteria provided a formal test to link specific microbes to specific diseases (1890). Following this lead, in 1880, Laveran first linked human malaria to infection of RBCs by plasmodia (Laveran, 1880) (P. falciparum (Welch 1897), P. vivax and P. malariae (Grassi and Feletti, 1890) as well as P. ovale (James, 1929)). During this same time, the medical discipline of psychiatry was coming to understand that infection with the spirochaete bacterium Treponema pallidum, caused syphilis (Schaudinn, 1905; Schaudinn and Hoffman, 1905) and that the resulting disease could advance to cause numerous visceral forms and overlapping clinical outcomes (Merrit et al., 1946). Primary infections (marked by the appearance of a chancre at the site of the infection) would heal in 2 to 6 weeks without leaving a scar. Secondary stages would lead to clinical symptoms in approximately half of all cases, producing skin lesions, rash or other generalised inflammatory symptoms. Following a period of latency that could last for years, approximately one-quarter of patients would go on to develop tertiary stages (paretic and tabetic neurosyphilis; general paralysis of the insane (Brandt, 1985)). Despite an improved understanding of the relationship between the microbe and the natural history of the disease, treatment of syphilis remained based on administration of mercury by mouth, injection, dermatologic application or exposure to its vapours to purge the humour through salivation or sweating. Understandably, optimism greeted Paul Erlich’s development of the arsenicals salvarsan (1910) and neosalvarsan as potential ‘magic bullets’ against syphilis; however, these treatments still exposed patients to significant risk and still failed to ward off or cure neurosyphilis (Stokes and Shaffer, 1924; Arnold, 1984; Jolliffe, 1993). Patients with neurosyphilis were difficult to manage, ‘became completely demented and unable to care for themselves, dying most often in insane asylums’ (Brown, 2000). Accurate syphilis prevalence data is difficult to come by as the disease carried significant social stigma; however, population estimates in Europe and the United States suggested that 15% of the general population had the disease (Stokes, 1918). With at least 10–30% of syphilis patients requiring long-term palliative care, mental institutions were being pushed beyond their effective operating capacities (Stokes, 1918; Chernin, 1984; Hook and Marra, 1992). As a result, there was considerable interest in developing more effective treatments for neurosyphilis. Sporadic reports had been published that mentally ill syphilitic patients who experienced bouts of fever showed signs of recovery or remission (Brown, 2000), and in 1876, Rosenblum reported that approximately 50% of psychiatric patients were cured after an attack of ‘recurrent fever’ Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 31

(Chernin, 1984). It was also noted that observation of neurosyphilis was uncommon in malaria-endemic regions of Africa (Merrit et al., 1946). In an 1887 review, the Viennese psychiatrist Julius Wagner-Jauregg noted 163 incidents of psychoses remitting following typhoid, intermittent fevers or erysipelas. While findings of this nature seemed to encourage treatment of paretic patients by artificially inducing fever, the dangers of experimental treatment of human beings through exposure to agents such as tuberculin or injection of malarial parasites made physicians reluctant to perform these procedures for fear of legal repercussions (Brown, 2000). Wagner-Jauregg’s first treatments of neurosyphilis patients with malariotherapy occurred in 1917, when a shell-shocked soldier from the Mace- donian Front was admitted to the hospital in Vienna. Coincidently, this patient was experiencing malaria fevers and chills. With blood from this patient, Wagner-Jauregg was able to induce fevers in a small number of paretic patients (Withrow, 1990). While improvements were observed in this first series of malaria-treated patients, complications associated with malaria tropica (falciparum malaria) soon became apparent1. After establishing a steady supply of benign tertian malaria (vivax malaria), Wagner- Jauregg reported in 1921 that 25% of his first 200 patients were able to return to work (Brown, 2000). While malariotherapy was not without risk, the success reported by these early trials quickly led to widespread practice throughout Europe and treatment of paretic patients with malariotherapy was first attempted in the United States in 1922. In the United States, equally positive results as experienced in Europe were observed following treatment with tertian malaria as Paul O’Leary and colleagues described in a first report on malariotherapy at the Mayo Clinic (Minnesota)

1 Human malaria is caused by five parasite species (Plasmodium falciparum, P. vivax, P. malariae, P. ovale and recently P. knowlesi in Malaysia (Cox-Singh and Singh, 2008)). Tertian malaria (recurring fevers at approximately 48-hour intervals) caused by P. vivax has been classified as ‘benign’, while disease associated with P. falciparum has been classified as ‘malignant’. While P. vivax was preferred over P. falciparum by practitioners of malariotherapy (Becker, 1949, Chernin, 1984, Withrow, 1990), the literature notes that induced malaria was not without risks for patients. Furthermore, in a note on ‘The Nomencla- ture of Malaria’ by Bruce (1903), it is noted ‘I have left out the commonly used terms, simple, benign, ‘malignant’, pernicious, as they are misleading. The so-called simple tertian may often be more severe than the so-called ‘malignant’ tertian’ (Bruce, 1903). General differences between vivax and falciparum malaria have been described frequently (Zimmerman et al., 2004, Price et al., 2007). Plasmodium vivax shows a selective preference for infecting young red blood cells (reticulocytes) (Kitchen, 1938, Garnham, 1966), where P. falciparum infects a wider range of red cells (Kitchen, 1939); as reticulocytes comprise less than 1% of the circulating red blood cells, this preference may constrain P. vivax parasitaemia. P. vivax and P. ovale are noted to produce dormant liver stages, termed hypnozoites (Krotoski et al., 1982, Krotoski, 1989). Plasmodium falciparum and P. malariae do not produce hypnozoites. Hypno- zoites can be reactivated after clearance of primary infection to cause relapses weeks to years later. 32 Peter A. Zimmerman et al.

(O’Leary et al., 1926). Because of the impact of malaria treatment on neurosyphilis, Wagner-Jauregg was awarded the Nobel Prize in Medicine in 1927 (Withrow, 1990).

2.2. Malariotherapy and African-Based Resistance to P. vivax Treatment of neurosyphilis in the United States significantly expanded the practice of malariotherapy. In the application of malariotherapy to African- Americans in particular, publications documented that certain individuals were observed to exhibit notable immunity to infection by P. vivax. Early evidence of this clinical observation was noted in published discussions of papers presented to the Dermatology and Syphilology section of the Amer- ican Medical Association.

May 19, 1927; Washington, D.C.

Dr. Watson W. Eldridge, Jr., St. Elizabeth’s Hospital, Washington, D.C. – I inoculated the first patient in December, 1922, and we have treated approximately 275 cases since...I should like to know whether, in the group in which the malaria failed to take, reinoculations were successful. I have had several inoculation failures, principally among coloured males. (O’Leary, 1927)

Dr. Paul A. O’Leary, Mayo Clinic, Rochester, Minn. - ...We have reinoculated as many as six times those patients who have not developed chills and fever and have not been successful in obtaining a ‘take’. (O’Leary, 1927) Resistance to vivax malaria was seen to be the primary disadvantage that would prevent malariotherapy from being used as a routine treatment of neurosyphilis. When carefully controlled studies were performed in the context of malariotherapy, details showed that African-Americans and Africans consistently displayed significantly higher levels of resistance to P. vivax strains from numerous geographic origins and inoculation doses compared to Caucasians (Young et al., 1955). Additionally, because African- Americans from nonmalarious regions of the United States were as refractory to P. vivax infection as those from malarious regions, this resistance was suggested to be natural rather than acquired (Boyd and Stratman-Thomas, 1933; Becker et al., 1946; Young et al., 1946; Young et al., 1955; Bray, 1958). Of further interest, to determine if a P. vivax infection once established in an African-American patient would acquire characteristics enabling more successful infection of resistant individuals, Young et al. used blood from a P. vivax-infected African-American to inoculate two resistant individuals of the same race (Young et al., 1955). Despite receiving inocula three to seven Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 33 times higher than that routinely causing blood-stage infection of Caucasian patients, neither of the resistant individuals developed blood-stage parasitaemia. From these results it was concluded that the P. vivax strain would not be transformed to acquire characteristics that would enable subsequent infection of resistant individuals (Young et al., 1955). Interestingly, it was also reported that African-Americans displayed resistance to P. knowlesi and P. cynomolgi in addition to P. vivax. From their studies with patients at the Manhattan State Hospital, Milam and Coggeshall reported that African- American patients experienced significantly milder P. knowlesi infections compared to Caucasian patients as measured by a delayed time to first blood- stage parasitaemia and shorter duration of blood-stage infection (Milam and Coggeshall, 1938). Following their report of accidental human infections with P. cynomolgi (Eyles et al., 1960; Eyles, 1963), Beye et al. wanted to further test whether non-human primates could act as reservoirs of malaria. Their follow-up study included 7 African-American and 13 Caucasian volunteers from the US Penitentiary in Atlanta. An overall summary of their results showed that blood-stage parasites were not observed in any of the African-American study participants, but 12 of the 13 Caucasian patients did exhibit blood-stage parasitaemia (Beye et al., 1961). As P. knowlesi and P. cynomolgi were observed to have difficulties similar to P. vivax for causing malaria in African-American patients and study participants, results have suggested that these parasite species may infect the human RBC by similar invasion pathways. In 1947, Butler and Sapero published an interesting exceptional report on resistance to P. vivax among African-Americans following natural exposure to P. vivax in the South Pacific. The authors wrote that, ‘With the onset of the present war in the Pacific and the arrival of negro [sic] troops on highly malarious bases in the South Pacific (Melanesia), it was hoped that the negro [sic] might be spared the ravages of Pacific vivax malaria because of his racial tolerance to the United States strains’ (Butler and Sapero, 1947). The authors’ study design indicated that the surveyed population included several thousand troops and that 28% were African-American (20–35 years of age; primarily from the Carolinas and Georgia), and virtually no malaria was noted among the study group during training procedures in the United States. Significant exposure to Anopheles farauti was noted once the troops were deployed in the South Pacific and while suppressive atabrine was provided, a sizable incidence of initial and recurrent malaria attacks indicated that this prophylactic treatment was not taken regularly. Plasmodium species infections were calculated monthly for primary and recurrent attacks. While these infections were not differentiated according to racial groups, the study 34 Peter A. Zimmerman et al. observations suggested that this was not a serious omission. The results from the study showed that 90.5% of all re-admissions were due to P. vivax and that 41.5% of the re-admissions occurred in the African-American group. The authors conceded that if the entire 9.5% of the non-vivax re-admissions occurred in the African-American group, 32% of African-American re-admissions would have correlated with P. vivax infection (Butler and Sapero, 1947). In contrast to reports from malariotherapy trials, the conservative evaluation of this study population suggested that African-American troops were highly susceptible to blood-stage infection by Pacific strains of P. vivax when naturally exposed to the parasite.

2.3. Human Variation and Blood Groups A theoretical synthesis similar to the one occurring in the cell biology and infectious disease world also gained momentum in genetics and evolution at the dawn of the twentieth century. At this time, Darwin’s On the Origin of Species had stimulated wide-ranging controversy throughout the scientific community. Although Mendel’s re-discovered work with garden peas provided a foundation for understanding the dynamics of heredity, opposing factions debating the role of natural selection in evolution took his observations to argue that evolution resulted from successive leaps or gradual change; the role of mutation in natural selection of new species was at first questioned (Mayr and Provine, 1981). The population biologists Fisher, Wright and Haldane promoted ideas that genes worked together to bring about genetic variation in populations. From this population-based perspective, genetic variations that optimised fitness would be transmitted from one generation to the next (Mayr and Provine, 1981). The Malaria Hypothesis, first proposed by Haldane, is a well-known but counter- intuitive twist of population biology and selection theory. This hypothesis proposes that otherwise harmful mutations (e.g. the thalassaemias) are transmitted at higher frequencies in some populations because they confer selective advantages against plasmodia and balance susceptibility to malaria (Haldane, 1949). Practical implications regarding the influence of mutation on phenotype, heritability and human population biology came to light in studies on blood transfusion, where many of the first human genetic polymorphisms were identified through serological cross-reactivity, recognising variations in blood group antigens (Race and Sanger, 1950; Mourant et al., 1976). Karl Landsteiner made the first observations that serum from healthy humans had an agglutinating effect on the blood corpuscles Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 35 of other humans, leading to the identification of the ABO blood group system in 1901 (Landsteiner, 1901). Blood group systems enabled early human geneticists to test heritability of these polymorphic traits, provided consistent opportunity to test population genetic hypotheses and allowed them to make some of their first observations regarding genetic similarities and differences between races and ethnicities. At present, there are 32 blood group systems inclusive of over 600 specific antigens, encoded by 42 genes; overall polymorphism is captured among 1312 alleles (Table 2.1). Cross-reacting antibodies recognise extracellular epitopes of proteins imbedded in the RBC membrane. Malarial parasites would naturally encounter these epitopes when contacting the red cell prior to blood- stage infection. Therefore, it is not surprising that a number of these blood group proteins have been implicated in influencing susceptibility to malaria (Table 2.1).

3. RESISTANCE TO P. VIVAX AND INSIGHTS ON MALARIA RED CELL INVASION

Investigator Profile: An Interview with Louis Miller, M.D., with Vicki Glaser. (Glaser, 2004)

[In the early 1970s] we knew that invasion was very specific – each type of Plasmodium goes to a specific host – but we did not know why. At that time I was working on a monkey parasite, P. knowlesi… The parasite would only invade certain types of RBCs [in tissue culture], such as human or monkey cells, and it would not invade others such as mouse… As host red cell specificity was likely based on surface molecules that act as receptors, I began to study red cells…null for various blood groups in the hope that one would be the receptor for P. knowlesi invasion of human red cells. I found that P. knowlesi was not able to invade Duffy blood group negative red cells. I went to the library that night, and I knew right away that I had discovered the missing factor for the resistance of West Africans to P. vivax. (Louis Miller)

3.1. Serological Recognition of Duffy (Fy) Blood Group Polymorphism The Duffy blood group antigen (Fy a) was first observed in 1950 on erythrocytes using allo-antisera found in a multiply transfused haemophiliac (named by permission of the patient) at the time a haemolytic transfusion reaction was observed (Cutbush et al., 1950). The expected Fyb antisera was discovered in Berlin shortly thereafter (Ikin et al., 1951); surveys of European 36 Peter A. Zimmerman et al. Discovered 1900 1927 1927 1940 1945 1946 1946 1950 1951 1953 1956 1962 1962 1965 1967 1942 1962 Yes Yes – – – – – – Yes – – – – – – – – Malaria – CD235 CD240 CD239 CD238 CD233 – – CD234 – – CD99 – CD297 – CD242 – CD 9q34.2 4q31.21 1p36.11 19q13.32 7q34 17q21.31 22q11.2-qter 19p13.3 1q23.2 18q12.3 7q22.1 Xp22.33 1p34.2 12p12.3 7pl4.3 19p13.2 6p21.3 Chromosomal Chromosomal Location GYPE ABO GYPB, GYPA, RHCE RHD, LU KEL SLC4A1 FUT3 DARC SLC14A1 ACHE MIC2 XG, ERMAP ART4 AQP1 ICAM4 C4A, C4B C4A, Gene Name(s) ABO MNS RH LU KEL DI P1 LE FY JK YT XG SC DO CO LW CH/RG System System Symbol 4 1 6 6 3 2 2 7 6 3 3 9 46 50 19 31 21 No. of No. Antigens Blood Group Systems Blood Group

ABO MNS P Rh Lutheran Kell Lewis Duffy Kidd Diego Yt (Cartwright) Xg Scianna Dombrock Colton System Name System Landsteiner-Wiener Chido/Rodgers 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 No. 16 17 Table 2.1 Table Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 37 1952 1977 1960 1965 1970 1973 1979 – 1978 – – – – http://www.ncbi.nlm. – – – Yes Yes – – – – – – – – CD173 CD55 – CD236 CD35 CD44 CD147 CD151 CD108 – – – CD241 19q13.33 1q32.2 Xp21.1 2q14.3 1q32.2 11p13 19p13.3 11p15.5 15q24.1 6p24.2 3q26.1 9p13.3 6p21-qter FUT1 CD55 XK GYPC CR1 CD44 BSG CD151 SEMA7A GCNT2 B3GALT3 AQP3 RHAG H CROM XK GE KN IN OK RAPH JMH I GLOB GIL RHAG . 1 1 8 9 4 1 1 5 1 1 1 3 15 H Kx (McLeod syndrome) Gerbich Cromer Knops Indian Ok Raph Milton Hagen John I Globoside Gill Rh-associated glycoprotein 18 website. please consult the following the latest figures, For 42 genes and 1312 alleles. group systems, 32 blood are there 2012, 5, As of March nih.gov/projects/gv/rbc/xslcgi.fcgi?cmd=bgmut/summary 19 20 21 22 23 24 25 26 27 28 29 30 38 Peter A. Zimmerman et al. populations suggested frequencies for the co-dominantly expressed Fya and Fyb antigens of 35% and 65%, respectively (Cutbush et al., 1950; Ikin et al., 1951). Upon screening, a series of blood samples from African-American donors to the Knickerbocker Blood Bank of New York City, Sanger et al. observed that 68% of the samples did not react with either the Fy a or Fy b antisera (Sanger et al., 1955). Additional analysis of Nigerian families showed that the null phenotype was inherited in Mendelian manner and provided an opportunity to investigate Fy a copy number. In an earlier study, Race et al. found that the antiserum Pri reliably distinguished between single and double donors for Fy a (Race et al., 1953). Results obtained from tests on three African-Americans who were phenotypically Fy(a+b+) and two who were Fy(a+b−) all suggested that Fy a was observed to be present in single-dose quantity, as compared to double-dose quantities observed in Fy(a+b−) Europeans (Sanger et al., 1955) (Duffy blood group nomenclature is summarised at the conclusion of Section 3.3; Table 2.2). These observations suggested that those of African ancestry possessed either a different antigen, Fyc, or they did not express the Duffy antigen and carried a Duffy-null allele, Fy0. Blood group researchers hypothesised that identification of an Fyc antigen would be forthcoming as a result of large numbers of blood transfusions involving African donor and Caucasian recipient pairs, or through their attempts to stimulate anti-Fyc reactivity through injection of Fy(a−b−) red cells into European volunteers (Sanger et al., 1955). The failure to discover the Fyc antigen and therefore the possibility of a Duffy- null phenotype was not unanticipated. A comparable observation had been made previously in the MNSs system (Table 2.1), where an occasional Afri- can blood sample reacted with neither the anti-S nor the anti-s antisera (Sanger et al., 1955).

3.2. The Genetic Resistance Factor to P. vivax – Duffy Negativity The impressive distribution of the Fy(a−b−) phenotype in diverse Afri- can populations has been a fascination to population geneticists, evolutionary biologists and infectious disease physicians and biologists. Based on the overlapping distribution of the Fy(a−b−) phenotype, the very low prevalence of P. vivax in African populations (Bray, 1957) and the desire to identify ‘receptors’ used by malarial parasites to invade human erythrocytes (Butcher et al., 1973; Miller and Dvorak, 1973), Louis Miller and colleagues at the US National Institutes of Health performed a series of studies in the mid-1970s to determine if red cells deficient for any of the human Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 39 blood group systems would resist infection using newly developed abilities to culture malarial parasites in the laboratory (Butcher and Cohen, 1971). In their studies, Miller and colleagues first observed that the non-human primate malarial parasite P. knowlesi was not able to infect erythrocytes from Fy(a−b−) African-Americans in vitro (Miller et al., 1975), while the parasite easily infected erythrocytes from Fy(a+b−), Fy(a+b+) and Fy(a−b+) donors. From these results, Miller et al. hypothesised that Fy(a−b−) would explain the absence of P. vivax from West Africa where blood group system surveys were reporting that Fy(a−b−) frequency was nearly 100% (Mourant et al., 1976). Miller’s in vitro findings led directly to a study testing the in vivo susceptibility of Duffy-negative and Duffy-positive individuals to P. vivax blood-stage infection (Miller et al., 1976). In this study, 17 consenting prisoner volunteers were first characterized for their Duffy blood group phenotype serologically. P. vivax-infected mosquitoes were then allowed to take blood meals, first from Fy(a−b−) African-Americans, and then following interruption, were allowed to continue feeding on Fy(a+b−), Fy(a+b+) and Fy(a−b+) Caucasian and African-Americans. Results showed that none of the five Fy(a−b−) study subjects developed blood-stage parasitaemia despite evaluation of daily blood smears for 90–180 days, while all 12 of the Duffy- positive individuals developed blood-stage infection within 15 days (Miller et al., 1976). While this study showed strong evidence that P. vivax required the Duffy blood group antigen to be present on the erythrocyte surface to invade the cell successfully and continue its life cycle, no information beyond basic susceptibility to blood-stage infection was produced. Studies at this point of the investigation neither tested for differences in susceptibility based on Fya vs. Fyb, nor were they on individuals who were heterozygous for the Duffy- negative allele, expressing a single gene dose of the Duffy blood group protein. Further comparisons regarding P. vivax susceptibility among these Duffy phenotypes could have provided important insight towards understanding the relative selective differences among the Fy a, Fyb and Duffy- negative alleles. In an attempt to explain how the frequency of Duffy blood group negativity had risen to 100% corresponding with the absence of P. vivax from vast regions of malaria-endemic West Africa, Miller and colleagues offered the following hypotheses. ‘Although P. vivax infection rarely causes death, it may decrease survival in African children with malnutrition and other endemic diseases… as the frequency of the Duffy-negative gene increased in the population, the number of susceptible persons decreased below a 40 Peter A. Zimmerman et al. critical level, and P. vivax disappeared from the region’ (Miller et al., 1976). This hypothesis suggests that the Duffy-negative phenotype increased the fitness of human populations against vivax malaria and would have led to the evolution of a human host population in which P. vivax was not able to reproduce with enough success to maintain its life cycle. This, and queries about other pathogens that may interact with the Duffy antigen have fuelled the debate surrounding the relationship between P. vivax and evolution of the Duffy-negative phenotype for decades. (Livingstone, 1984; Carter, 2003; Kwiatkowski, 2005; Rosenberg, 2007).

3.3. The Molecular and Cellular Basis of Duffy Blood Group Polymorphism As identification of Fyc was not forthcoming, understanding the molecular differences responsible for this and the other Duffy blood group polymorphisms would rely on the advance of molecular biology. Methodical progress towards cloning the Duffy gene can be marked through attempts to purify the Duffy protein (Moore et al., 1982; Hadley et al., 1984; Chaudhuri et al., 1989) and identification of a series of Duffy epitopes: Fy3 (Albrey et al., 1971), Fy4 (Behzad et al., 1973), Fy5 (Colledge et al., 1973) and Fy6 (Nichols et al., 1987) and their respective antisera. These antisera have been used to illustrate that the Duffy protein was characterized by a number of different epitopes. It is therefore important to note that all these epitopes were absent from Fy(a−b−) African individuals. Beginning in 1988, Chaudhuri and colleagues at the New York Blood Centre described a series of experiments using the murine anti-Fy6 monoclonal antibody to affinity purify Duffy antigens from solubilised erythrocytes (Chaudhuri et al., 1989). Chaudhuri et al. further studied these Duffy peptides by amino acid sequencing and synthesis of a DNA probe to identify a gene-specific cDNA molecule (Chaudhuri et al., 1993). Through this process, they identified a 338 codon open reading frame (ORF) sequence exhibiting significant homology to the human interleukin 8 receptor, predicting seven transmembrane segments, an extracellular amino terminus, three extracellular loop domains, three intracellular loop domains and a carboxy-terminal cytoplasmic tail (Fig. 2.2) (Chaudhuri et al., 1993). Further studies on the genomic organisation of the Duffy gene sequence confirmed early predictions that the gene locus was present in a peri-centromeric region of human chromosome 1 (1q22-23) (Donahue et al., 1968; Dracopoli et al., 1991; Mathew et al., 1994). While the role of the Duffy blood group antigen is potentially of great interest in allergy (Vergara et al., 2008), cardiovascular disease Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 41

(Reich et al., 2009), cancer biology (Shen et al., 2006) and HIV-AIDS (He et al., 2008), we will not cover these topics here. Additional details related to Duffy antigen chemokine receptor biology (Pruenster et al., 2009) are provided in the legend to Fig. 2.2. Specific analysis of Duffy cDNA molecules (5′-RACE) produced evidence that the gene was composed of two exons (Iwamoto et al., 1996). Exon 1 was observed to encode seven amino acids, MGNCLHR; exon 2 was found to encode 338 amino acids. It was subsequently shown that the primary transcript of the Duffy gene was composed of codons 1–7 from exon 1 joined to codons 10–338 from exon 2 encoding a protein of 336 amino acids; this splice variant is expressed in erythroid lineage cells. Additional studies characterizing the Duffy gene were also successful in identifying a single nucleotide polymorphism (SNP) in codon 42 associated with the Fy a (GGT; encodes glycine) and Fyb (GAT; encodes aspartic acid) antigens (Chaudhuri et al., 1995; Iwamoto et al., 1995; Tournamille et al., 1995b). Then, after acknowledging no additional polymorphism compared to the FY*B allele, suggesting that Africans carried no important disruption in the Duffy ORF (Chaudhuri et al., 1995; Tournamille et al., 1995a), Tournamille et al. discovered a T to C SNP 33 nucleotides upstream from the primary transcription starting position (−33) in the Duffy gene promoter (originally positioned at nucleotide −46) (Tournamille et al., 1995a), resulting in an FY*BES allele (ES = erythroid silent). Duffy-negative Afri- cans were homozygous for this polymorphism that was shown to occur in a tissue-specific GATA1 transcription-factor-binding motif. In vitro assays showed that this polymorphism blocked gene expression in erythroid lineage cells but did not block expression in non-erythroid cells. Results of this study provided the molecular genetic explanation for erythrocyte Duffy negativity. Since the identification of the Fy a, Fyb and Duffy-negative SNPs, addi tional less-common variants have been identified to provide a more complete description of Duffy genotype and serological phenotype polymorphisms. Following identification of the FY*BES allele, Zimmerman et al. sought to determine if the Duffy gene in Papua New Guineans living in P. vivax- endemic regions would be characterized by accumulation of any functional polymorphism. A survey of the Duffy gene promoter and ORF polymorphisms identified above revealed that the same promoter SNP found on the African FY*BES allele was observed on the resident Papua New Guinea (PNG) FY*A allele (suggests FY*AES) (Zimmerman et al., 1999). Flow cytometry comparing anti-Fy6 (Nichols et al., 1987) antibody binding to 42 Peter A. Zimmerman et al.

Figure 2.2 The Duffy antigen. The diagram illustrates the primary structure of the 236-amino-acid 36–46-kDa Duffy antigen with seven predicted transmembrane domains and extracellular and intracellular domains. Amino acids comprising the Fy6 and Fy3 antibody-binding domains are marked by brackets. Amino acid sequence polymorphisms are identified at residues 42 (G vs. D; Fya vs. Fyb), 89 (R vs. C; Fyb vs. Fybweak) and 100 (A vs. T) and the two premature termination codons (W vs. X) at residue positions 96 and 134. Glycosylation sites are identified at amino acid residues N16 and N27. Disulfide bonds occurring between C129 (extracellular loop 2) and C195 (extracellular loop 3) and between C51 (amino terminal head) and C276 (extracellular loop 3) are predicted to contribute to further tertiary structure within the cell membrane as depicted in the inset. Amino acids predicted to comprise the P. vivax binding region are identified in red (Chitnis et al., 1996). Duffy Antigen Function – The Duffy antigen receptor for chemokines (DARC) is a ‘silent’ 7-transmembrane receptor. This results from the absence of a DRYLAIV amino acid motif in the second intracellular loop needed to couple with G-proteins that initiate intracellular signalling cascades (Murphy, 1996). Duffy is one of a few chemokine receptors that bind to inflammatory chemokines, categorised by structural features into two different groups, α (amino acid motif -CC-) and β (amino acid motif –CXC-). On erythrocytes, the Duffy antigen is proposed to act as a sink that binds to excess chemokines and limits inflammation (Darbonne et al., 1991). Reciprocally, Duffy binding of chemokines prevents their diffusion into organs and peripheral tissue Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 43 erythrocytes from six PNG homozygous wild-type individuals and six PNG heterozygous individuals showed that individuals with two erythroid-functional alleles expressed approximately twice the amount of the Fy a antigen compared to individuals with one erythroid-functional allele. Since the time that FY*AES was reported in PNG, this allele has also been found in Tunisia (Sellami et al., 2008). Before describing additional polymorphism, the Duffy- negative phenotype has been observed in association with a 14-nucleotide deletion in the Duffy coding sequence (Mallinson et al., 1995). Further variation in Duffy serology, originally described by Chown et al., was observed as a result of low Fyb expression levels (termed Fyx) (Chown et al., 1965), observed primarily in Caucasian families. Again, application of molecular genetic strategies enabled identification of nucleotide sequence changes associated with this serological phenotype. First, descriptions of the mutation underlying the Fyx, or Fybweak, variant identified polymorphism in codon 89 of the FY*B allele, changing the amino acid sequence from arginine (codon CGC) to cysteine (codon TGC) in Fyb and Fybweak antigens (Olsson et al., 1998; Parasol et al., 1998; Tournamille et al., 1998). This polymorphism has been observed in association with an alanine to threo- nine amino acid substitution at codon 100 (GCA to TCA), and an additional alanine to serine substitution at codon 49 (GCA to TCA) (Castilho 2004). The substitution these FY*X alleles all share is arginine to cysteine at codon 89; to date, this polymorphism has not been observed on the FY*A allele. The Fyx polymorphism occurs within the first intracellular loop of the Duffy protein and is associated with reduced cell surface expression of Duffy (Olsson et al., 1998; Tournamille et al., 1998). The frequency of the space and in this way acts as a reservoir of chemokines in the circulating blood (Fukuma et al., 2003). Duffy is also expressed on a variety of non-erythroid cells including venular endothelial cells; in this context recent studies suggest two potential roles for Duffy. On venular endothelial cells, Duffy has been proposed to act as a chemokine intercep- tor (internalisation receptor) by internalising and scavenging chemokines (Nibbs et al., 2003). Alternatively, Pruenster et al. have shown that Duffy acts to mediate chemokine transcytosis (Pruenster et al., 2009). In their in vitro system, Duffy-mediated chemokine transcytosis led to apical retention of intact chemokines and leukocyte migration across Duffy-expressing endothelial cell monolayers. How these complex roles of the Duffy antigen are regulated and influence human health remains to be determined.(Originally published in Zimmerman 2004. The enigma of vivax malaria and erythrocyte Duffy-negativity, in: Dronamraju, K.R., (Ed.), Infectious Disease and Host-Pathogen Evolution.Cambridge Uni- versity Press, New York, pp 141–172.) (Reproduced with permission from Cambridge Univer- sity Press and Krishna R. Dronamraju). (For interpretation of the references to colour in this figure legend, the reader is referred to the online version of this book.) 44 Peter A. Zimmerman et al.

FY*Bweak allele is approximately 2% in Caucasians (Chown et al., 1965; Olsson et al., 1998). Finally, weak expression of Fyb has been reported in association with deletion of a ‘C’ nucleotide residue, between −76 and −74, of the Duffy gene promoter in an Sp1 regulatory site (Moulds et al., 1998). Overall, flow cytometry studies testing the association between Duffy promoter and Fybweak polymorphisms have demonstrated consistent relationships. Relative levels of erythroid expression have shown that heterozygous carriers of a Duffy-negative allele express approximately 50% the level of the Duffy antigen on their red cells compared to the red cells from individuals homozygous for Duffy-positive alleles. The FY*X allele is associated with approximately 10% of the expression compared to the FY*A and FY*B alleles (Tournamille et al., 1998). The overall Duffy phenotype is dependent on both promoter and coding region SNPs. Expression phenotypes relative to the 15 different genotypes possible from the five known Duffy alleles (FY*A, FY*B, FY*X, FY*AES, FY*BES ) are summarised in Table 2.2. Additional observations that Duffy expression is highest on reticulocytes (Woolley et al., 2000; Woolley et al., 2005) may contribute to the observation of preferential invasion of these immature red cells by P. vivax (Kitchen, 1938). Comparative sequence analyses of Duffy gene orthologues in non-human primates have shown that the FY*B allele is the ancestral state (Palatnik and Rowe, 1984; Chaudhuri et al., 1995; Li et al., 1997; Tournamille et al., 2004; Demogines et al., 2012; Oliveira et al., 2012). The Duffy-negative allele FY*BES bears clear signatures of strong and recent positive selection in African populations (Hamblin and Di Rienzo, 2000; Hamblin et al., 2002). Interestingly, the FY*A allele predicted to have arisen after FY*BES (Li et al., 1997) has also been characterized by levels of polymorphism lower than would be expected by a neutral model of evolution in a sample of Chi- nese individuals (Hamblin et al., 2002). Phylogenetic comparisons of this nature for the FY*X and FY*AES alleles have not yet been performed. That P. vivax would be considered to be the agent behind the selection observed in human-specific alleles is curious given the long-held considerations that P. vivax infection is rarely lethal in humans. As a growing number of clinical research studies are establishing connections between P. vivax and severe malaria and malaria mortality (e.g. (Genton et al., 2008; Tjitra et al., 2008; Anstey et al., 2009; Baird, 2009; Kochar et al., 2009)), further consideration is necessary to determine how vivax malaria exerts its pressure as an agent of natural selection (Anstey et al. discuss details of clinical vivax malaria in Chapter 3 in Volume 80 of this special issue). Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 45

Table 2.2 Working Guidelines for Duffy* blood group nomenclature† Phenotype Allele Antigen Genotype‡ Serologic Expression$ FY*A Fya FY*A/FY*A Fya+/b− 2×Fya, 0×Fyb FY*B Fyb FY*A/FY*AES – 1×Fya, 0×Fyb FY*X Fybweak FY*A/FY*BES – 1×Fya, 0×Fyb FY*AES – FY*B/FY*B Fya−/b+ 0×Fya, 2×Fyb FY*BES – FY*B/FY*X – 0×Fya, 1.1×Fyb – – FY*B/FY*AES – 0×Fya, 1×Fyb – – FY*B/FY*BES – 0×Fya, 1×Fyb – – FY*X/FY*X Fya−/b+weak 0×Fya, 0.2×Fyb – – FY*X/FY*AES – 0×Fya, 0.1×Fyb – – FY*X/FY*BES – 0×Fya, 0.1×Fyb – – FY*A/FY*B Fya+/Fyb+ 1×Fya, 1×Fyb – – FY*A/FY*X – 1×Fya, 0.1×Fyb – – FY*AES/FY*AES Fya−/Fyb− 0×Fya, 0×Fyb – – FY*AES/FY*BES – 0×Fya, 0×Fyb – – FY*BES/FY*BES – 0×Fya, 0×Fyb

*Alternate gene name = Duffy antigen/receptor for chemokines (DARC) †Consistent with International Society of Blood Transfusion blood group terminology (web address provided below) www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blooct- group-terminology/blood-group-terminology/#c581 ‡ES, erythrocyte silent – attributed to the T to C transition at nucleotide −33 in the Duffy gene promoter $Expression phenotypes based on composite of flow cytometry and chemokine binding. (Ménard et al., 2010)

3.4. The Duffy Binding Protein Several studies have now described the parasite ligand interacting with the Duffy blood group antigen. P. knowlesi Duffy binding proteins (Haynes et al., 1988; Adams et al., 1990) and P. vivax DBP (PvDBP) (Wertheimer and Barnwell, 1989; Fang et al., 1991) have molecular weights of approximately 140 kD; P. knowlesi α (Pkα) binds to the Duffy antigen (Chitnis and Miller, 1994). A 330-amino acid cysteine-rich region of the DBP is predicted to be the protein domain responsible for binding to Duffy-positive human RBCs (Ranjan and Chitnis, 1999). The DBP is expressed in the micronemes and on the surface of P. knowlesi, and by homology, P. vivax merozoites. A number of structural features of the DBPs are shared with erythrocyte binding proteins of other malarial parasites. As such, these proteins have come to be known as Duffy-binding-like erythrocyte binding proteins (DBL- EBP) (Adams et al., 1992) Shared features among EBPs include two cysteine-rich regions (Region II – containing 12 conserved cysteine residues; Region VI containing 8 conserved cysteine residues), a highly polymorphic 46 Peter A. Zimmerman et al. region (Regions III to V) and numerous aromatic amino acids (tryptophan, phenylalanine and tyrosine) (Adams et al., 1992). Because of the overall significance of EBPs in blood-stage infection, it is important to consider further the interaction between the PvDBP and the Duffy antigen. Despite the difficulties in growing P. vivax in culture, a number of in vitro studies have built a body of information on the importance of DBP–Duffy antigen interaction to invasion of the RBC. As noted above, while P. knowlesi merozoites would successfully contact and reorient their apical surfaces in apposition to the membrane of both Duffy-positive and Duffy-negative erythrocytes, the tight junction between merozoite and erythrocyte membranes did not form with Duffy-negative cells (Miller et al., 1979). These observations have suggested that some aspect of the parasite’s invasion mechanism failed to engage in the absence of the Duffy antigen. Experiments more specifically focused on the P. vivax DBP have demonstrated competi- tive interference between recombinant PvDBP expressed on COS cells and the Duffy receptor on donor erythrocytes using a 35-amino-acid peptide (amino acid 8–42) from the receptor’s NH2-terminal domain, the monoclonal antibody anti-Fy6 and the chemokine MGSA (CXCL1) (Chitnis and Miller, 1994; Chitnis et al., 1996). These same studies have also demonstrated that sulphation of tyrosine 41 is critical for optimal DBP binding to the Duffy receptor in vitro (Choe et al., 2005). Further studies employ- ing this COS-cell-binding affinity assay showed that Duffy-negative heterozygous, compared to homozygous positive, erythrocytes exhibit consistently lower affinity for PvDBP transfected cells (Michon et al., 2001). Additionally, elevated levels of amino acid sequence polymorphism in the DBP binding region, as well as antibody responses from people living in P. vivax-endemic regions recognising DBP, suggest that this molecule may be under selective pressure (Tsuboi et al., 1994; Ampudia et al., 1996; Fraser et al., 1997; Michon et al., 1998; Cole-Tobian et al., 2002) by the human immune system. A number of years later, Singh et al. have now shown that P. knowlesi merozoites of Pkα knockout strain are not able to form a junction with or invade Duffy-positive human RBCs (Singh et al., 2005). That the Pkα knockout continues to successfully invade rhesus erythrocytes suggests that the P. knowlesi β and γ proteins bind other receptors and enable Duffy-independent red cell invasion (Chitnis and Miller, 1994; Ranjan and Chitnis, 1999). Interrogation of the parasite ligand–host receptor relationship has since been examined through studies to characterize more specifically the interaction of PvDBP with the Duffy antigen. Recombinant chimeric proteins have been used to localise PvDBP-Duffy binding to a 170-amino-acid Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 47 segment of the parasite ligand between cysteines 4 and 7 (Ranjan and Chit- nis, 1999). Further studies have gone on to suggest that there are discon- tinuous epitopes within this segment that are predicted to be important for Duffy antigen interaction (VanBuskirk et al., 2004; Hans et al., 2005) and that receptor binding residues and polymorphic residues under immune pressure map to opposing surfaces of PvDBP (Singh et al., 2006). More recent studies have shown that both patient-derived and polyclonal rabbit antibodies specific for PvDBP are able to inhibit P. vivax invasion of human RBCs in vitro (Grimberg et al., 2007; Russell et al., 2011).

4. EVOLVING PERSPECTIVES ON RESISTANCE TO P. VIVAX Molecular and cell biology technologies have provided powerful strategies for cloning and expressing proteins from malarial parasites to study their interactions with human red cells in vitro (Adams et al., 1992). Molecular diagnostic methods for interrogating genetic polymorphisms and diagnosing infection have transformed strategies for studying the epidemiology of malaria (Greenwood, 2002). Over the past 20 years, application of these methods has significantly influenced our perspectives on the frequency and distribution of P. vivax as well as the role played by genetic polymorphism and the interaction between P. vivax and the human RBC.

4.1. Further Influence of Duffy Polymorphism on Resistance to P. vivax Malaria Discovery of the FY*AES allele in PNG (Zimmerman et al., 1999) provided a new opportunity to evaluate the association between a Duffy-negative allele and susceptibility to P. vivax infection and disease. Although no individual has been identified to be homozygous for the FY*AES allele in PNG, the opportunity was provided to determine if there was any selective advantage associated with being a heterozygous carrier of a Duffy-negative allele. In these studies, Kasehagen and colleagues performed cross-sectional malaria prevalence surveys in the same PNG communities where the FY*AES allele had been identified. These Wosera villages, north of the PNG Central Ranges, are highly endemic for all four human malarial parasite species (Genton et al., 1995a; Kasehagen et al., 2006; Lin et al., 2010). Plas- modium species infection status was evaluated by conventional blood smear light microscopy and semi-quantitative polymerase chain reaction (PCR)- based strategies. In both unmatched and matched (adjusted for age, sex 48 Peter A. Zimmerman et al. and village of residence) longitudinal cohort analyses, results showed that Duffy-negative heterozygotes (FY*A/*AES) were partially protected from P. vivax blood-stage infection compared to those homozygous for wild-type alleles (FY*A/*A) (Kasehagen et al., 2007). In these same study cohorts, there were no differences in susceptibility to P. falciparum infection between FY*A/*AES and FY*A/*A study participants (Kasehagen et al., 2007). Additional analyses evaluated parasitaemia by a semi-quantitative PCR assay between FY*A/*AES and FY*A/*A study participants, in age group categories less than and greater than 15 years. Among FY*A/*AES children under 15 years of age, the mean P. vivax fluorescent signal intensity (corresponds with parasitaemia (McNamara et al., 2006)) was significantly ES lower among FY*A/*A (mean = 2.37, log10 transformed) compared to FY*A/*A children (mean = 2.96) (Mann–Whitney U:P = 0.023). Interest- ingly, this was similar to the difference in parasitaemia observed between FY*A/*A individuals older vs. younger than 15 years of age (mean fluorescent signal intensity 2.23 vs. 2.81, respectively; Mann–Whitney U:P < 0.0001). This suggested that the difference in parasitaemia attributed to the difference in genotype (FY*A/*AES and FY*A/*A) was similar to the difference that would otherwise be attributed to acquired immunity of older individuals. Similar to results from the longitudinal cohort studies, no association was observed between susceptibility to P. falciparum parasitaemia and the Duffy genotype. The findings from this study provided the first evidence that Duffy-negative heterozygosity reduced erythrocyte susceptibility to P. vivax infection (Kasehagen et al., 2007). Reduced susceptibility to P. vivax was not associated with an increased susceptibility to P. falciparum malaria as may have been predicted by studies that previously reported reduced severity of falciparum malaria conferred by exposure to P. vivax. Similarly, cross-sectional studies in the Amazon Basin of Brazil (Cavasini et al., 2007; Sousa et al., 2007) and Rio Grande do Sul (Albuquerque et al., 2010) have shown significantly reduced prevalence of P. vivax infection among Duffy-negative heterozygotes (either FY*A/*BES or FY*B/*BES) when compared with subjects from the same endemic areas with the FY*A/*A, FY*B/*B or FY*A/*B genotypes. These results suggest that Duffy-negative heterozygosity confers significant protection from vivax malaria and may provide some insight regarding a selective advantage that led to the FY*BES allele reaching genetic fixation in Africa. The frequency of the FY*X allele at 2% in Caucasian populations may limit opportunities to perform epidemiological studies to determine if the Fybweak antigen is associated with reduced risk of P. vivax malaria. In contrast, Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 49 the FY*A allele is widely distributed in Southeast Asia (Howes et al., 2011), where P. vivax is proposed to have evolved from origins as a parasite of Old World monkeys (Carter, 2003; Escalante et al., 2005; Culleton et al., 2011), and in South America, where P. vivax is the predominant malaria parasite in human infections (Oliveira-Ferreira et al., 2010; Arevalo-Herrera et al., 2012). In vitro studies have shown that the P. knowlesi DBP interacts with stronger affinity to the Fyb compared to the Fya antigen. After observing similar results with the PvDBP (40–50% decreased binding to Fya+b− vs. Fya−b+ erythrocytes (King et al., 2011); P < 0.0001), King et al. performed a cohort study in the Brazilian Amazon to determine if the in vitro results translated to in vivo protection from clinical vivax malaria in association with the FY*A compared to the FY*B allele (King et al., 2011). Study participants (n = 400; 5–74 years of age) lived along the Iquiri River where the annual incidence rates of P. vivax and P. falciparum malaria during the 14-month study period were 0.31 and 0.17, respectively (124 cases of P. vivax, 66 cases of P. falciparum, 31 cases of P. vivax + P. falciparum malaria). Overall, when compared to the FY*A/*B genotype (n = 140), individuals with the FY*A/*BES and FY*A/*A genotypes experienced 80% (n = 35; risk ratio, 0.204 (95% confidence interval (CI), 0.09–0.87)) and 29% (n = 52; risk ratio, 0.715 (95% CI, 0.31–1.21)) reduced risk of clinical vivax malaria, respectively. Consistent with stronger affinity between PvDBP and the Fyb antigen, individuals with the FY*B/*BES and FY*B/*B genotypes experienced 220–270% increased risk of clinical vivax malaria, respectively, when compared to FY*A/*B (FY*B/*BES: n = 76; risk ratio, 2.17 (95% CI, 0.91–4.77); FY*B/*B: n = 87; risk ratio, 2.70 (95% CI, 1.36–5.49). As in the studies performed by Kas- ehagen et al., there was no association between the FY genotype and risk for P. falciparum in the multivariate analysis (overall risk ratio 1.08, 95% CI 0.87–2.38, P = 0.42). While it will be helpful to see if additional epidemiological studies corroborate these findings, results from Cavasini et al. are of interest (Cavasini et al., 2007). In their studies, FY*A/*BES was observed less frequently among 312 P. vivax patients (10.9%) than in 330 healthy blood donors (Brazilian blood bank; 18.8%). Results from King et al. would suggest that like the FY*BES allele, the frequency of FY*A has increased in frequency (reaching genetic fixation in many populations) to improve human fitness against P. vivax malaria (King et al., 2011).

4.2. Duffy-Independent Red Cell Invasion by P. vivax With consistent observation of resistance to P. vivax malaria associated with Duffy negativity, reduced susceptibility to P. vivax associated with lower 50 Peter A. Zimmerman et al.

Duffy expression and/or reduced affinity between the Duffy antigen and the parasite invasion ligand, it is understandable that the Duffy antigen has come to be regarded as an essential receptor for P. vivax red cell invasion. It has therefore been of keen interest that an increasing number of studies have reported P. vivax PCR positivity in Duffy-negative people. These studies have included P. vivax in Duffy-negative people in the Nyanza Province of Western Kenya (Ryan et al., 2006) and the Amazon Basin (Brazil) (Cavasini et al., 2007). However, another large-scale PCR-based survey covering nine different African countries detected only one P. vivax-positive person in over 2500 samples, and this individual was Duffy positive (Culleton et al., 2008). With a history of clinical reports of P. vivax malaria occurring in Europeans returning from holiday or business travel to Africa (Phillips- Howard et al., 1990; Gautret et al., 2001; Mendis et al., 2001; Muhlberger et al., 2004; Guerra et al., 2010), new questions have arisen with regard to the Duffy-negative P. vivax resistance factor (Rosenberg, 2007). In an effort to understand the epidemiology of malaria throughout Madagascar, Ménard and colleagues initiated a series of blood sample collections in 2006 from the country’s four major malaria-transmission regions (Ménard et al., 2010). These surveys provided new insight into the basic prevalence of Plasmodium species infections and drug resistance. They also opened the opportunity to investigate the intersection of malaria infection in a human population characterized by unique origins and admixture. The peopling of Madagascar is recent in human history and is suggested to have been initiated by sea-faring people of Indonesia or Malaysia (Nias Island of western Sumatra or Borneo, respectively) with evidence that founding individuals arrived 2300 years before present (Burney et al., 2004). Upon Bantu migration from Africa (Tanzania and Mozambique) during the second and third centuries and new waves of Malayo-Indonesian immigration from the eighth century onwards, significant cultural assimi- lation and genetic admixture has occurred. Malaria is likely to have been transported to Madagascar through the earliest human settlers more than 2000 years ago. It is more difficult to predict when during the first mil- lennium of human settlement the human population numbers and density became favourable to support endemic transmission of the four common species of human malaria parasites that are observed in Madagascar today. In this setting, surveys of school-aged children revealed P. vivax PCR positivity in 8.8% of asymptomatic Duffy-negative children (n = 476) (Ménard et al., 2010). During surveys to assess in vivo efficacy of drugs recommended Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 51 by the Madagascar Ministry of Health to treat malarial illness, nine Duffy- negative people were identified who had PCR-confirmed, mono-infection P. vivax malaria (4.9% of 183 participants). Given the unusual prevalence of vivax malaria in people considered to be resistant to this disease, Ménard et al. took additional steps to validate this finding by microscopy to provide the first evidence to confirm Duffy-independent blood-stage infection and development by P. vivax (Fig. 2.3) (Ménard et al., 2010). Microscopy results included the observation of sexual-stage gametocytes necessary to continue the parasite life cycle through mosquito transmission. Consistent with observations reported by many blood group laboratories, flow cytometry analysis of erythrocytes from Malagasy study participants who had experienced clinical P. vivax malaria showed that Duffy-negative genotype and phenotype

Figure 2.3 Standard Giemsa-stained thin smear preparations of P. vivax infection and development in human Duffy-negative erythrocytes. Panels A and B originated from a 4-year-old female, genotyped as Duffy negative FY*B( ES/*BES), who presented at the Tsiroanomandidy health center with fever (37.8 °C), headache and sweating without previous anti-malarial treatment. Standard blood smear diagnosis revealed a mixed infection with P. vivax (parasitaemia = 3040 parasitised red blood cells [pRBC]/µl) and P. falciparum (parasitaemia=980 pRBC/µl). PCR-based Plasmodium species diagnosis confirmed the blood smear result; P. malariae and P. ovale were not detected. (A) a P. vivax early-stage trophozoite with condensed chromatin, enlarged erythrocyte volume, Schüffner stippling and irregular ring-shaped cytoplasm. (B) aP. vivax gametocyte – lavender parasite, larger pink chromatin mass and brown pigment scattered throughout the cytoplasm are characteristics of microgametocytes (male). Panel C originated from a 12- year-old Duffy-negative FY*B( ES/*BES) male, who presented at the Miandrivazo health centre with fever (37.5 °C) and shivering without previous anti-malarial treatment. Stan- dard blood smear diagnosis and light microscopy revealed infection with only P. vivax (parasitaemia = 3000 pRBC/µl). PCR-based Plasmodium species diagnosis confirmed this blood smear result; P. falciparum, P. malariae and P. ovale were not detected. The parasite featured shows evidence of a P. vivax gametocyte – large blue parasite, smaller pink chromatin mass and brown pigment scattered throughout the cytoplasm are characteristics of macrogametocytes (female). (Adapted from Ménard et al, 2010. Plasmodium vivax clinical malaria is commonly observed in Duffy-negative Malagasy people. Proc. Natl. Acad. Sci. U. S. A. 107, 5967–5971). (For interpretation of the references to colour in this figure legend, the reader is referred to the online version of this book.) 52 Peter A. Zimmerman et al. were 100% concordant. Additionally, DNA sequence analysis of the Duffy gene confirmed that the Duffy-negative allele identified in Madagascar was identical to the FY*BES allele observed in West Africa (included > 2550 bp of the gene’s proximal promoter and full coding sequence). Population-level observations from Ménard et al. provide further insight regarding the unique parasite–host relationships discovered in Madagascar (Ménard et al., 2010). In the communities with the highest frequencies of Duffy negativity, there was little to no prevalence of P. vivax infection detected in either Duffy-positive or Duffy-negative people. In contrast, with an increase in the frequency of Duffy positivity, P. vivax prevalence increased and was not significantly different between Duffy-positive and Duffy-negative people (Fig. 2.4; χ2 results: Miandrivazo P = 0.733; Mae- vatanana P = 0.278; Tsiroanomandidy P = 0.09). These results suggest that high population levels of Duffy negativity may act similarly to herd immunity to reduce transmission and consequently protect Duffy-positives from vivax malaria. In populations with higher frequencies of Duffy positivity, opportunities for the parasite to attempt invasion of the Duffy-negative RBCs are more frequently available as antibody reactivity against P. vivax merozoites, PvDBP and PvMSP-1 indicate that liver-stage infection (and therefore hypnozoite formation) commonly occurs in Duffy-negative people (Spencer et al., 1978; Michon et al., 1998; Herrera et al., 2005; Culleton et al., 2009). It is important to note that a survey of clinical malaria from this Madagascar study did suggest that Duffy negativity was associated with protection from malarial illness. Finally, it is of interest that genotyping results from six unlinked P. vivax-specific microsatellites suggested that multiple P. vivax strains were present in the blood samples from Duffy-negative infections. With additional reports of P. vivax PCR-positive Duffy-negative people from Equatorial Guinea (Rubio et al., 1999; Mendes et al., 2011), Gabon (Mendes et al., 2011) and Mauritania (Wurtz et al., 2011), evidence of Duffy-independent P. vivax blood-stage infection has been observed across geographically distant communities of the Duffy-negative population of sub-Saharan Africa. It will be important to continue surveillance of P. vivax strains capable of Duffy-independent red cell invasion because of implications regarding the potential that P. vivax may compound the burden of clinical malaria in Africa and complicate P. vivax-specific vaccine development and malaria elimination. Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 53

Figure 2.4 Frequency distribution of P. vivax infections and clinical cases identified in Duffy-positive and Duffy-negative Malagasy people. Pie graphs show the prevalence of Duffy-positive (dark/light green) and Duffy-negative (red/pink quadrants) phenotypes in the eight Madagascar study sites. Prevalence of P. vivax infection observed in the survey of school-aged children is shown in red and dark green; population subsets not infected with P. vivax are pink and light green. Study sites identified by a red star indicate that clinical vivax malaria was observed in Duffy-negative individuals. A green star indicates that vivax malaria was observed in Duffy-positive individuals only (Ejeda). In Ihosy, clinical malaria was observed in one individual with a mixed P. vivax/P. falciparum infection. P. vivax malaria was not observed in Andapa and Farafangana (black star). Malaria transmission strata are identified as tropical (lightest grey), sub- desert (light grey), equatorial (middle grey) and highlands (dark grey). (Adapted from Ménard et al, 2010. Plasmodium vivax clinical malaria is commonly observed in Duffy- negative Malagasy people. Proc. Natl. Acad. Sci. U. S. A. 107, 5967–5971). (For interpretation of the references to colour in this figure legend, the reader is referred to the online version of this book.) 54 Peter A. Zimmerman et al.

4.3. Global Distribution of Duffy Polymorphism and the Population at Risk of P. vivax Malaria Much like the maps used to illustrate an overlap between malaria endemicity and the distribution of the sickle cell allele (Piel et al., 2010), α- and β-thalassaemias (Weatherall and Clegg, 2001) and glucose-6-phosphate dehydrogenase (G6PD) deficiency (Howes et al., 2012), population surveys of Duffy blood group variants have been used to map the polymorphisms’ spatial distribution. These maps provide an important overview of the ongoing relationship between P. vivax and human malaria. Recently, Howes et al. have collated a comprehensive geographically referenced database of available Duffy phenotype and genotype survey data to refine the global cartography of the common Duffy variants (FY*A, FY*B and FY*BES) (Howes et al., 2011). Results of this effort are summarised in Fig. 2.5. (For information on source data for this study, see Howes et al. Supplemental Information for 320 references). Recalling that non-human primate studies provide evidence that FY*B (green) is the ancestral allele in the hominid lineage, this map suggests that the FY*BES (red to orange) originated in Africa and has spread across a vast geographical and ethnic landscape. The map also indicates that FY*A (blue) has reached genetic fixation in regions of the world where the non-human malaria parasite ancestors originated and dispersed (noted above). From these potential Asian origins, FY*A has admixed with FY*B and also spread into the Americas. Areas of the map appearing in different shades of grey identify regions where populations are characterized by heterogeneous frequencies of FY*A, FY*B and FY*BES ranging from 20–50%. In these latter regions, P. vivax is likely to have been exposed to a range of RBC phenotypes with varying contact affinities between PvDBP and the Duffy receptor, including heterozygous and homozygous Duffy negativity. In the struggle to survive, it seems reasonable to hypothesise that P. vivax strains have optimised effective contact of the apical invasion mechanism across a gradient of Duffy polymorphism, which now includes absence of this receptor, to engage the moving junction needed for successful red cell invasion. With increasing evidence that P. vivax is not restricted to a Duffy- dependent invasion pathway, it is important to consider how this might affect the estimation of the population at risk of P. vivax malaria (PvPAR). To date, Malaria Atlas Project estimates of the PvPAR have applied a biological exclusion criterion based on 100% protection from P. vivax infection by Duffy negativity (Guerra et al., 2010). With potential that a model Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 55 - FY*B (when co- m resolution on resolution m 10 k 10

FY*A or allele and either 50%) are represented by a colour gradient gradient colour a by represented are 50%) ES

≥

FY*B Areas predominated by a single allele (frequency allele single a by predominated Areas ). Areas of allelic heterogeneity where no single allele predominates, but two of or alleles ). no allelic more each Areas where single heterogeneity allele predominates, ES alleles. FY FY*B ). Median values of the predictions were generated for each allele frequency at a 10 a at frequency allele each for generated were predictions the of values Median ). 20%, are 20%, shown palest are in heterogeneity between for grey-scale: the silent ; red/yellow, FY*B ; red/yellow,

≥

Global frequencies of the of frequencies Global

; green, FY*A ; green, Howes et al., 2011 et al., ( Howes described ously inherited, inherited, these do not generate new phenotypes), and darkest being co-occurrence of all three alleles (and correspondingly the greatest genotypic and phenotypic diversity). Overall surfacepercentage area of each class The is probability listed distribution in the based legend. previ as dataset, input the to best corresponds this as value, median the case, this in statistic: single a as summarised is model Bayesian a on Figure 2.5 Figure (blue, frequencies have a global grid with GIS software (ArcMap 9.3; ESRI). (For interpretation of the to references colour in this figure legend, thereader is referred of this book.) the online version to 56 Peter A. Zimmerman et al. based on this conservative perspective may significantly underestimate the global PvPAR, we were interested in considering how reduced Duffy- negative exclusion would alter this metric in global and regional estimates. Figure 2.6 shows that even if Duffy-negative protection were reduced to 50%, the overall global burden of P. vivax infection would remain heavily

Figure 2.6 Estimated change in the P. vivax population at risk before (A) and after (B) relaxing the level of resistance conferred by Duffy negativity from 100% to 50%, respectively. Overall increases in the percentage of PvPAR (C). The population-at-risk exclusions are based on the methods developed by Guerra et al. in 2010 (Guerra et al., 2010) and subsequently refined by Gething et al. To define the P. vivax population at risk (PvPAR), Gething et al. imposed several layers of exclusion from the countries with endemic P. vivax transmission (95 countries). Annual parasite incidence (API) data were used to refine the sub-national levels of transmission along administrative boundaries, classify- ing areas into unstable (<0.1 cases per 1000 population per year), stable transmission (≥0.1 cases per 1000 population per year) and malaria free (Guerra et al., 2010). Tem- perature exclusion based on minimum requirements for parasite sporogony modelled in relation to vector lifespan (Gething et al., 2011). Aridity mask to exclude areas too dry to sustain transmission by restricting vector survival and availability of ovipositioning sites. The aridity mask was derived from the bare ground areas in the GlobCover land cover imagery (Guerra et al., 2008). Medical intelligence was used to further exclude malaria- free urban areas (modulated with knowledge of the local Anopheles vectors) (Guerra et al., 2010). All these methods have been described in greater detail by Gething et al. (2012) and in Chapter 1 in Volume 80 of this special issue. Extensive data collection was necessary for the API exclusions, and individuals who contributed data to this process are acknowledged on the Malaria Atlas Project website (MAP: www.map.ox.ac.uk/). (For a colour version of this figure, the reader is referred to the online version of this book.) Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 57 focused in Asia. Not surprisingly, the most significant changes in PvPAR would be observed in the Africa, Saudi Arabia and Yemen region (Africa+) with a projected fivefold increase. Interestingly, this increased PvPAR in Africa would surpass the estimated risk in the Americas by threefold.

4.4. Association of Non-Duffy Gene Polymorphisms with P. vivax Resistance Recently, a number of groups have begun to consider the possibility that non-Duffy human gene polymorphisms associated with protection against falciparum malaria may also affect susceptibility to infection and disease attributable to P. vivax.

4.4.1. G6PD Deficiency Among the RBC variants considered to date, G6PD deficiency is important to examine for a combination of reasons. The enzyme catalyses the first reaction in the pentose phosphate pathway leading to the formation of NADPH needed by cells to counter oxidative stress (Cappellini and Fiorelli, 2008). The enzyme deficiency was discovered because of its association with haemolytic anaemia following administration of primaquine (Beutler, 1994), the only clinically validated medication against P. vivax hypnozoites (Wells et al., 2010). Because of the widespread distribution of G6PD deficiency in malaria- endemic regions, administration of primaquine and therefore, elimination of P. vivax is problematic. The gene (13 exons distributed over 18.5 kb; ≥140 mutations (Cappellini and Fiorelli, 2008; Minucci et al., 2012)) is located in the telomeric region of the human X chromosome and is therefore characterized by classical X-linked inheritance patterns, hemizygosity in males and X-inactivation in females (Beutler, 1996). The most common West African G6PD variant, G6PD A- (Val → Met, codon 68; moderate (10–60%) activity variant), has been associated with significant reduction in the risk of severe falciparum malaria in male hemizygotes (Ruwende et al., 1995; Guindo et al., 2007) and in heterozygous females (Ruwende et al., 1995). More recently, Louicharoen et al. found that the G6PD-Mahidol487A mutation (Gly → Ser, codon 163; moderate (10–60%) activity variant) was associated with reduced P. vivax, but not P. falciparum, parasite density (Louicharoen et al., 2009). Whether the G6PD-Mahidol487A mutation reduces the severity of clinical vivax malaria was not reported. Leslie et al. have more recently reported that the G6PD-Mediterranean type (Med; Ser → Phe, codon 188; severely deficient (1–10% activity)) is associated with protection from clinical vivax in a case-control study of Afghan refugees living in Pakistan (Leslie et al., 2010). 58 Peter A. Zimmerman et al.

Howes et al. further discuss details regarding the complexities of G6PD deficiency and vivax malaria in Chapter 4 of this Volume.

4.4.2. Haemoglobinopathies To date, very few studies have been performed to investigate associations between the major haemoglobinopathies and P. vivax (Taylor et al., 2012). Increased susceptibility to P. vivax infection has been observed in association with α-thalassaemia (−α/−α) in Vanuatu (Williams et al., 1996) and PNG (Allen et al., 1997) and with HbE β-thalassaemia in Sri Lanka (O’Donnell et al., 2009). It is suggested that α-thalassaemia may increase susceptibility to P. vivax infection because of higher overall red cell turnover increasing reticulocytaemia (Weatherall and Clegg, 2001). As P. vivax shows a strong preference for infecting reticulocytes (Kitchen, 1938), increased reticulocyte counts associated with the thalassaemias would produce more target cells for the merozoites to infect. In one additional study in India, reduced susceptibility to P. vivax infection was reported in association with HbE (Kar et al., 1992). Although red cell remodelling and pathogenesis is markedly different among human malaria species parasites, the association of reduced susceptibility to P. vivax with HbE may be more consistent with observations from falciparum malaria studies where α-thalassaemia confers protection against malaria. This protection is proposed to occur because erythrocytes bind higher levels of antibody from sera of malaria-exposed individuals (Luzzi et al., 1991) and were observed to be more readily phagocytised by blood monocytes (Yuthavong et al., 1988) compared with normal red cells. Interestingly, the concept that α-thalassaemia can increase susceptibility to P. vivax infection in young children while being associated with decreased susceptibility to P. falciparum has led to a hypothesis that the predilection to P. vivax may lead to cross-immunity between parasite species, which protects against falciparum malaria later in life. This hypothesis has stirred a lively debate regarding benefits and risks of cross-species infections (Bruce and Day, 2003; Snounou, 2004; Zimmerman et al., 2004). While this debate will continue, it must be acknowledged that P. vivax is responsible for causing severe illness and death (Baird, 2009) and that mixed infections with P. vivax and P. falciparum can be more severe than mono-infections by these same two species (Tjitra et al., 2008).

4.4.3. Southeast Asian Ovalocytosis More consistent with P. vivax and the Duffy receptor interactions, there is significant interest in the influence of mutations contributing to Southeast Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 59

Asian ovalocytosis (SAO) and protection against vivax malaria. Two proteins associated with red cell ovalocytosis in Melanesians include the solute carrier family 4, anion exchanger, member 1 (SLC4A1, also known as erythrocyte membrane protein band 3) and glycophorin C (GYPC). SLC4A1 is expressed in the red cell membrane and functions as a chloride/bicarbonate exchanger involved in CO2 transport from tissues to lungs. Three domains of the protein are structurally and functionally distinct. First, the 40 kDa N-terminal cytoplasmic domain (400 amino acids) contains an 11-amino- acid segment (residues 175–185) that acts as an attachment site for the red cell skeleton by binding ankyrin (Chang and Low, 2003; Stefanovic et al., 2007). Second, a hydrophobic, polytopic transmembrane domain (481 amino acids; 14 membrane-spanning segments) carries out anion exchange (Abdalla et al., 1980). Third, the cytoplasmic tail at the extreme C-terminus of the membrane domain (41 amino acids) binds carbonic anhydrase II. Association with glycophorin A (GYPA) promotes the correct folding and translocation of the SLC4A1 protein. This protein is predominantly dimeric but forms tetramers in the presence of ankyrin (Alper, 2009). A number of single amino acid substitutions in SLC4A1 give rise to the Diego blood group system (Poole, 2000) and a 27 base pair deletion removing 9 amino acids (SLC4A1Δ27; codons 400–408) near the first transmembrane region of the protein leads to SAO ( Jarolim et al., 1991; Mgone et al., 1996; Mgone et al., 1998). GYPC is a physiologically important monomer (128 amino acids, apparent 35 kDa, integral membrane sialoglycoprotein) (Car- tron et al., 1993) that interacts with the peripheral membrane protein 4.1 to mediate attachment of the submembranous cytoskeleton to the erythrocyte membrane. Deletion of GYPC exon 3 (GYPCDex3) results in the Gerbich-negative blood group phenotype (Colin et al., 1989; High et al., 1989) and has also been associated with ovalocytosis in PNG in the absence of SLC4A1Δ27 (Patel et al., 2001). In vitro studies have shown that SAO compared to normal RBCs show resistance to both P. falciparum and P. knowlesi (Kidson et al., 1981; Hadley et al., 1983). Although trypsin treatment had previously been shown to render resistant Duffy-negative red cells susceptible to P. knowlesi infection (Mason et al., 1977; Miller et al., 1979), this same experimental strategy was not successful with SAO cells (Hadley et al., 1983). The mechanism of resistance was suggested to be increased rigidity of the RBC membrane (Mohandas et al., 1984). Both SAO and Gerbich negativity have been shown to reduce the severity of falciparum malaria (Cattani et al., 1987; Serjeantson, 1989; Genton et al., 1995b; Allen et al., 1999). These overall 60 Peter A. Zimmerman et al. in vitro and in vivo observations prompted the investigation of the impact of SAO on susceptibility to P. vivax malaria in PNG. It is important to note that P. vivax malaria in PNG is observed to peak by approximately 3 years of age (Kasehagen et al., 2006; Mueller et al., 2009) and evidence of significant protection from clinical symptoms associated with P. vivax is observed in young school-aged children (Michon et al., 2007). The impact of SAO was studied through multiple childhood cohorts. Results from this study showed that in a cohort of infants 3–21 months of age SAO was associated with a 55% reduction in the risk of clinical P. vivax episodes with parasitaemia greater than 500 infected cells/µl (adjusted IRR (incidence rate ratio) = 0.54; CI95 (0.34, 0.59), P < 0.0001). Additionally, in a treatment-time to re-infection cohort of 5–14 year olds, SAO children experienced a 52% reduction in P. vivax re-infection diagnosed by light microscopy (CI95 (23, 87), P = 0.014) (Rosanas-Urgell et al., 2012). Further studies showed that while Duffy antigen expression was not significantly different on SAO compared to normal erythrocytes, high-level PvDBP-specific binding inhibitory antibodies (>90% binding inhibition) were observed significantly more often in sera from SAO than non-SAO children (SAO, 22.2%; non-SAO, 6.7%; P = 0.008)(Rosanas-Urgell et al., 2012). Consistent with in vitro observations, interactions leading to reorientation and apical contact of the P. vivax merozoite are likely to occur in vivo. Results indicating that PvDBP-specific binding inhibitory antibodies were more common in SAO children suggest that PvDBP exposure to the immune system is somehow different in SAO compared to non-SAO children and stimulates production of higher quality antibody recognition of this important parasite invasion ligand.

5. CONCLUSIONS AND FUTURE DIRECTIONS Very early experience with malariotherapy in the United States revealed that high-level resistance to blood-stage infection with P. vivax was observed in many, but not all, African Americans (Fig. 2.7). Although not considered at that time, observations of syphilologists of the 1920s initiated efforts to explain this curious occurrence and launched investigations that have revealed the mechanisms that malarial parasites use to infect the RBC. The first critical breakthroughs identifying components of these red cell invasion mechanisms were made by Louis Miller and colleagues in the mid-1970s when the Duffy blood group antigen was identified as the receptor for P. knowlesi and P. vivax. Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 61

Figure 2.7 Summary of major events providing insight on resistance to P. vivax. (For a colour version of this figure, the reader is referred to the online version of this book.)

As in other fields of biomedical research, invention of the PCR by Kerry Mullis in 1983 transformed many aspects of malaria research. The first published application of PCR demonstrated how sickle cell anaemia could be diagnosed by amplification of a small segment of the β-globin gene (Saiki et al., 1985). Amplification of malarial parasites from human blood samples was performed in the early 1990s to diagnose species with greater sensitivity than conventional blood smear methods (Barker et al., 1992; Barker et al., 1994) and to identify P. falciparum strains that were carrying mutations associated with drug resistance (Zolg et al., 1989). As powerful high-throughput multiplex assays for diagnosing malarial infections have become routinely available, perspectives on species complexity of malarial infections have changed significantly. PCR applications have improved our understanding of the epidemiology of vivax malaria, in particular, where blood smear diagnosis is hampered by lower sensitivity, and in differentiating P. vivax from P. ovale (which share numerous morphological similarities). Reliable diagnoses of P. vivax have brought into question preconceptions that Africans are always fully resistant to P. vivax erythrocyte infection and that the parasite is absent from sub-Saharan Africa. PCR diagnostic and genotyping strategies have therefore played significant roles in identifying P. vivax infections in Duffy-negative people (Ryan et al., 2006; Cavasini et al., 2007; Ménard et al., 2010; Mendes et al., 2011; Wurtz et al., 2011) and 62 Peter A. Zimmerman et al. in the performance of the field-based studies that have identified new vivax malaria resistance factors (Cavasini et al., 2007; Kasehagen et al., 2007; Sousa et al., 2007; Louicharoen et al., 2009; Albuquerque et al., 2010; King et al., 2011; Rosanas-Urgell et al., 2012). Do these recent observations suggest that P. vivax is now evolving new capacity to infect human RBCs or has this parasite always had this capacity? Species naturally evolve, particularly when confronted with a selective barrier that threatens their ability to reproduce – Duffy negativity clearly represents this kind of barrier for P. vivax. Evidence suggests that the parasite’s human red cell invasion mechanism has not been restricted to the Duffy antigen. Clinical observations for decades have reported that Duffy- positive Caucasians return from travels to Duffy-negative Africa with P. vivax infections (Phillips-Howard et al., 1990; Gautret et al., 2001; Mendis et al., 2001; Muhlberger et al., 2004; Guerra et al., 2010), and records of African or African-American individuals infected with P. vivax (albeit lacking Duffy phenotype data) have appeared periodically (Butler and Sapero, 1947; Hankey et al., 1953; Bray, 1958). The alternative explanation for the sudden increase in P. vivax-positive Duffy-negative people is that molecular diagnostic methods now provide clinicians and researchers with tools that are more sensitive than the parasitological methods previously employed. To understand the true epidemiology of vivax malaria in Africa, future population studies should include surveillance for P. vivax in molecular diagnostic assays routinely. With confirmation that P. vivax can infect Duffy-negative red cells, it is important to know how the parasite is progressing through the critical steps leading to reticulocyte invasion (Fig. 2.8). What then are the components of the P. vivax Duffy-independent invasion mechanism? The electron microscopy that has so vividly captured P. knowlesi interactions with Duffy-positive and Duffy-negative red cells has shown that the parasite is able to reorient its apical end in apposition to the red cell membrane of both Duffy-positive and Duffy-negative cells. However, absence of the Duffy antigen limits further junction formation that sets in motion the events required to complete invasion. In the absence of the Duffy antigen, what red cell protein(s) enable the junction formation needed for further downstream events – what is the new invasion receptor? The Duffy antigen has been shown to reside in a cluster of other red cell membrane proteins as part of a protein 4.1R mul- tiprotein complex (Mohandas and Gallagher, 2008). This complex includes Band 3 (link to SAO), glycophorin C and other blood group proteins (Kell, reticulocyte binding homologues (Rh), XK). One wonders, if through its Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 63

Figure 2.8 Overview of P. vivax merozoite interaction with the human red blood cell. Initial attachment occurs between any part of the merozoite (blue) and erythrocyte (red). The merozoite reorients, positioning its apical end for attachment to the red cell membrane. A junction forms between the apical end of the merozoite and the erythrocyte membrane of Duffy-positive cells (first call-out box). In contrast, P. knowlesi electron microscopy has shown thin filaments between the merozoite apical end and the Duffy-negative red cell membrane; however, the merozoite is not drawn into contact with the red cell and the junction fails to form. This has implied that junction formation fails to occur between P. vivax and the Duffy-negative red cell membrane as well (second call-out box). Once a durable junction has formed between the merozoite and the red cell, micronemes (green) and rhopteries (dark blue) release their contents, the red cell membrane invagi- nates and the merozoite moves into the parasitophorous vacuole (third call-out box). Movement of the gliding junction is complete once the merozoite is engulfed within the parasitophorous vacuole and the orifice at the red cell membrane is sealed (fourth call-out box). (For interpretation of the references to colour in this figure legend, the reader is referred to the online version of this book.) 64 Peter A. Zimmerman et al. dependence on interaction with the Duffy antigen, whether the parasite’s DBP has gained or optimised a molecular connection with other members of the 4.1R complex. From the parasite’s vantage point, an equally important piece to the Duffy-independent puzzle must be identified. Unlike P. knowlesi, the DBP in P. vivax is found only as a single-copy gene (Carlton et al., 2008). It is possible that new PvDBP polymorphism could enable this protein to interact with alternative receptors; however, studies performed to date have not identified PvDBP variants that are able to bind Duffy-negative cells. Recent cell biology studies indicate that parasite proteins released in succession from the micronemes, rhopteries and dense granules are integral to parasite-host cell recognition, attachment and junction motility (Carruthers and Sibley, 1997). While AMA1 and RON protein interactions are critical components of the moving junction (Richard et al., 2010; Srinivasan et al., 2011), it is clear that some parasite-host interaction is required to secure initial contact by the parasite’s apical end before the junction can be formed. As a number of reticulocyte binding proteins have been localised to the micronemes (Meyer et al., 2009), these proteins would appear to be the likely alternative ligands if PvDBP has been rendered irrelevant in a Duffy-independent invasion mechanism. In P. falciparum evidence exists for functional redundancy of erythrocyte binding antigen and Rh, so that inhibition of one pathway is compensated for by the functioning of others (Stubbs et al., 2005; Triglia et al., 2009). Once an invasion pathway can no longer be used because the receptor is absent or the gene for the dominant ligand has been deleted, P. falciparum is able to use alternative pathways by re-deploying the expressed suite of ligands (Baum et al., 2005) or by differential gene expression of PfRh genes (Stubbs et al., 2005). It is possible that vivax parasites are able to use alternative invasion pathways that remain cryptic in the presence of the Duffy receptor and become operational in its absence. Answers to the questions prompted by Duffy-independent infection by P. vivax are of critical importance to development of a vivax-specific vaccine. The challenge confronting this mission is a familiar one: P. vivax is reluctant to grow in laboratory cultures and this precludes many of the experimental approaches that have been so effective in studying red cell invasion by P. falciparum (Cowman and Crabb, 2006). As cellular invasion mechanisms are highly conserved across apicomplexa, new strategies available through parasite genomics and proteomics will be important to mine. It may also be Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax 65 important to keep in mind the important insights gained through studies on P. knowlesi (Aikawa et al., 1978).

ACKNOWLEDGEMENTS We wish to acknowledge the contributions of thousands of study volunteers who have contributed to the studies reviewed here. We are grateful for the contributions of Peter Gething for sharing methodologies and unpublished data related to adjusted PvPAR predictions, Hisashi Fujioka, Didier Ménard, Arsène Ratsimbasoa, Yves Colin and Christiane Bouchier for developing critical data for this review, and Samantha Zimmerman for graphic design. We thank Louis Miller and David Serre for helpful comments that improved the clarity of this manuscript. PAZ has been supported through the US National Institutes of Health (NIH) (AI46919, AI089686, AI093922) and the Fogarty International Center (TW007377, TW007872). MUF is supported through the US National Institutes of Health (AI 075416) and the Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (03/09719-6, 05/51988-0, and 07/51199-0). REH was funded by a Wellcome Trust Biomedical Resources Grant (#085406). OMP has been supported through the 7th European Framework Program (FP7/2007-2013, contract 242095, Evimalar) and the Agence Nationale de la Recherche (contract ANR-07-MIME-021-0).

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Natural Acquisition of Immunity to Plasmodium vivax: Epidemiological Observations and Potential Targets

Ivo Mueller*, Mary R. Galinski**, Takafumi Tsuboi†, Myriam Arevalo-Herrera‡, William E. Collins$, Christopher L. King¶ *Walter + Eliza Hall Institute, Infection & Immunity Division, Parkville, Victoria, Australia; Barcelona Centre for International Health Research (CRESIB, Hospital Clínic-Universitat de Barcelona), Barcelona, Spain **Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine; Emory Vaccine Center and Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, USA †Cell-Free Science and Technology Research Center and Venture Business Laboratory, Ehime University, Matsuyama, Ehime, Japan ‡Caucaseco Research Center, Cali, Colombia $Institutional Association: Malaria Branch, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia ¶Center of Global Health & Diseases (CGHD), Case Western Reserve University, Veterans Affairs Medical Center, Cleveland, OH, USA

Contents 1. Overview of Naturally Acquired Immunity to Malaria 78 2. Differential Acquisition of Immunity to P. vivax and P. falciparum Under Natural Exposure 81 3. Acquisition of Immunity in Experimental Infections – Lessons from Malaria Therapy Patients and Irradiated Sporozoites 84 4. Unique Biological Characteristics of P. vivax that Contribute to NAI 88 4.1. Relapses 88 4.2. Force of Blood-Stage Infection 89 4.3. The Infected Red Blood Cell Membrane and Variant Surface Antigens 89 4.4. Critical Red Cell Invasion Ligands 91 4.5. Immune Regulation 93 5. Effector Mechanisms for Blood-Stage Immunity 93 6. Targets of Blood-Stage Immunity 94 6.1. PvMSP1 97 6.2. PvMSP3 98 6.3. PvMSP9 99 6.4. PvAMA1 100 6.5. PvRBPs 101 6.6. PvDBP 102 6.7. Role of T-Cell Immune Responses in Acquired Immunity 103 6.8. Immunological Memory and Duration of Clinical Immunity 105

7. Immune Responses to Malaria Pre-Erythrocytic Stages 106 8. Sexual Stage Parasites and Transmission-Blocking Immunity 111 8.1. Evidences of Naturally Acquired TBI of P. falciparum 112 8.2. Evidences of Naturally Acquired TBI of P. vivax 112 8.3. Towards the Discovery of the Target Molecules against TBI of P. vivax 113 9. Conclusions 114 10. Future Directions 114 References 115

Abstract Population studies show that individuals acquire immunity to Plasmodium vivax more quickly than Plasmodium falciparum irrespective of overall transmission intensity, resulting in the peak burden of P. vivax malaria in younger age groups. Similarly, actively induced P. vivax infections in malaria therapy patients resulted in faster and generally more strain-transcending acquisition of immunity than P. falciparum infections. The mechanisms behind the more rapid acquisition of immunity to P. vivax are poorly understood. Natural acquired immune responses to P. vivax target both pre-erythrocytic and blood-stage antigens and include humoral and cellular components. To date, only a few studies have investigated the association of these immune responses with protection, with most studies focussing on a few merozoite antigens (such as the Pv Duffy binding protein (PvDBP), the Pv reticulocyte binding proteins (PvRBPs), or the Pv merozoite surface proteins (PvMSP1, 3 & 9)) or the circumsporozoite protein (PvCSP). Naturally acquired transmission-blocking (TB) immunity (TBI) was also found in several populations. Although limited, these data support the premise that developing a multi-stage P. vivax vaccine may be feasible and is worth pursuing.

1. OVERVIEW OF NATURALLY ACQUIRED IMMUNITY TO MALARIA A major controlling force that determines the incidence and prevalence of malaria infection and disease in endemic areas is the parasitological and clinical immunity collectively referred to as naturally acquired immunity (NAI). Generally, NAI determines not only the age-specific incidence and prevalence of P. falciparum (Pf ) and P. vivax (Pv) infections but also the expression of pathological processes that underlie the clinical manifestations of infection. Improved understanding of the development and maintenance of NAI and the ability to cope with the severe manifestations of malaria are now particularly important, since effective public health interventions that reduce transmission are being deployed. The microbiologist Robert Koch in 1899/1900 first outlined the basic elements of NAI to malaria during an expedition to the Dutch East Indies (Indo- nesia) and German New Guinea (now Papua New Guinea (PNG)) (Koch, 1900a, 1900b, 1900c, 1900d). By studying malaria infections in both indigenous Natural Acquisition of Immunity to Plasmodium vivax 79 and immigrant clinical cases and asymptomatic individuals, he determined that (1) in endemic areas, malaria is most apparent in young children and the parasites may be completely absent from the blood of adults; (2) individuals who are constantly exposed to infection develop immunity to malaria with immigrants from non-endemic areas, achieving an “unmistakable degree of immunity just as is the case in the children of natives” after 3–4 years of exposure; (3) in people who have experienced numerous previous malaria attacks, infections will often present only with mild or no symptoms at all; and (4) adults from an island where he only detected Plasmodium malariae became readily infected and ill with P. falciparum malaria upon migrating to a highly endemic area of mainland New Guinea, leading him to hypothesise that immunity against one Plasmo- dium species offers little protection against the other species. He summarised his observations as follows: “One may assume an anti-malarial immunity, which under ordinary conditions is acquired only in 4–6 years and after repeated attacks, to be conferred artificially and in a short time. Seeing, however, that as yet we have no idea how to obtain the toxins necessary for the immunizing process, chances of progress in this direction are small”(Koch, 1900c). Based on Robert Koch’s seminal observations and many subsequent studies, it is accepted that NAI is the capacity of the host to respond more effectively during the second and subsequent exposures to a pathogen, as compared to the primary exposure. The quality and longevity of this adaptive response is highly variable, with some individuals acquiring long-term protection following a limited number of exposures, whereas repeated or ongoing exposure being needed to generate and sustain protective immunity in other cases. Both situations occur in malarial infection depending on the host, parasite, age, type of prior exposure and transmission intensity (related to parasite diversity). An emerging consensus indicates that the essential features of NAI are multifactorial, being primarily directed to blood-stage infection. In P. vivax malaria, the development of NAI is achieved by, or dependent on, exposure to both primary blood-stage infections and relapse infections in the blood. Although the human immune system will attack almost at any stage in the parasite life cycle in the human host, from sporozoite entry to the gamete fertilisation process in the mosquito midgut (Hollingdale et al., 1984), NAI appears to be primarily target blood-stage infections. With respect to P. vivax NAI is manifested as protection against high-density parasitaemia and uncomplicated clinical disease (fever, malaise and anaemia). This immunity has the following primary characteristics: (1) the highest degree of NAI occurs in adult residents of holoendemic areas, since this immunity is presumed to depend on cumulative exposure to multiple parasite infections (and relapses) over time, yielding a sufficiently diverse repertoire of 80 Ivo Mueller et al. strain-specific immune responses of sufficient magnitude. (2) It is generally thought that a significant degree of continuous or regular antigenic exposure, a condition referred to as premunition, is required, and in some circumstances NAI is greatly diminished on cessation of infection (transmission). (3) NAI is relatively species specific (with respect to P. vivax versus P. falciparum), and relatively strain specific (Collins et al., 2004a), although strain-specific immunity to P. vivax is more difficult to determine than with P. falciparum, because of the potential for diverse parasites resulting from the relapse of dormant liver stages representing different strains. However, there can be some degree of cross-protection among strains (Collins et al., 2004a). NAI also develops to severe malaria-related illness (manifestations that result in organ dysfunction, or severe anaemia <5 gm/dl), which appears to be acquired quickly and persists for a long time. This phenotype has been well studied for falciparum malaria, but remains poorly understood for vivax malaria. Premunition does not seem to be important in maintaining NAI to this phenotype. It seems to be more important to maintaining NAI to uncomplicated clinical malaria. NAI can also result in a reduction in transmission in sexual stages to mosquitos and/or interferes with the development of gametes in the mosquito midgut. There is still relatively little information available at the genomic and post-genomic levels for P. vivax, but such information is becoming available (Acharya et al., 2011; Bozdech et al., 2008; Carlton et al., 2008; Chen et al., 2010; Dharia et al., 2010), allowing for deeper enquiry into these questions and mechanisms of NAI to P. vivax. While there are naturally acquired immune responses against pre-erythrocytic antigens, it is unlikely that natural exposure to sporozoites is sufficiently high to induce protection against pre-erythrocytic infection. Broadly defined, premunition implies the maintenance of high levels of malaria-specific antibodies (Abs) and a high frequency of malaria-specific T cells, as a result of frequent or continual priming to malarial infections. The role of premunition in maintaining NAI too is controversial; the precise duration of time required for loss of NAI is unknown, as are the effects of age and variable transmission conditions. It is unclear whether continuous antigenic exposure impairs the ability to generate long-term immunologic memory and/or whether protection is partially maintained by activating innate immune mechanisms. Alternatively, repeated antigen exposure may be necessary to sustain high levels of cross-reactive Abs to multiple parasite stage to be fully protective. Both these hypotheses are testable and remain to be fully explored. NAI to P. vivax has been noted without premunition, such as with individuals treated for syphilis with malaria therapy (Boyd, 1947; Ciuca et al., Natural Acquisition of Immunity to Plasmodium vivax 81

1934; Collins et al., 2004a), and in low P. vivax transmission areas, such as in the Solomon Islands (Harris et al., 2010) and Amazon (Alves et al., 2002; Branch et al., 2005). Thus, NAI may develop with comparatively much lower exposure than previously appreciated. This may occur because of restricted parasite diversity. In low- or seasonal transmission settings, NAI to P. vivax has not been adequately explored, and it may involve mechanisms of immune protection distinct from those occurring in high-transmission areas. With the recent wide introduction of malaria control measures, a better understanding of NAI in intermediate- and low-transmission conditions is timely and important to understand how elimination efforts, including interventions such as universal deployment of long-lasting insecticide-treated nets (LLINs), may affect the health of local communities. On this note, it is important to recognise that LLINs can help to prevent initial infections, but not relapses. Absolute in vitro correlates of protection to malaria are unknown. Spe- cific biomarkers of protection to malaria have been proposed; however, there is a lack of consensus as to what these biomarkers are. Potential biomarkers for P. falciparum include elevated levels of serum Abs directed at merozoite antigens (particularly invasion ligands) and/or variant surface antigen (VSA) on infected erythrocytes (IEs). Potential biomarkers for P. vivax are not known, given the limited investigation in this area to date, although there is some evidence that functional Abs to PvDBP (Cole-Tobian et al., 2009; King et al., 2008) and PvMSP1 (Nogueira et al., 2006) correlate with protection. These immune responses represent associations rather than causal relations of protective immunity, but they are valuable if they can predict the level of NAI in populations before and after the implementation of malaria control measures.

2. DIFFERENTIAL ACQUISITION OF IMMUNITY TO P. VIVAX AND P. FALCIPARUM UNDER NATURAL EXPOSURE In the New Guinea island, which is highly endemic for both P. falciparum and P. vivax (Muller et al., 2003), P. vivax is the predominate source of malarial infections and disease in children younger than 2 years (Senn et al., 2012). The incidence of P. vivax malaria started to rapidly decrease from the second year of life, whereas P. falciparum incidence continued to increase until the fourth year in observational cohort studies in children aged 1–4 (Lin et al., 2010) and 5–14 years (Michon et al., 2007; Fig. 3.1A) By 5 years of age most children had acquired an almost complete immunity to clinical P. vivax yet remained at considerable risk of P. falciparum illness despite a similar burden of P. vivax blood-stage infections (Fig. 3.1B). 82 Ivo Mueller et al.

Figure 3.1 Evidence for rapid acquisition of immunity to P. vivax under natural exposure in areas with different transmission intensities. (A) Incidence of malaria in a cohort of PNG children aged 1–4 years (Lin et al., 2010). (B) Time to first PCR-positive infection and clinical episode in PNG children aged 5–14 years (Michon et al., 2007). (C) Prevalence of Plasmo- dium spp. infection among patients attending a rural PNG health centre and corresponding incidence of malaria-attributable fevers (lower panel) (Muller et al., 2009). (D) Incidence of malaria in a cohort in Thailand (light bars: P. falciparum, dark bars: P. vivax) (Phimpraphi et al., 2008). (E) Prevalence of infection by LM and PCR in a cross-sectional population survey in East Sepik Province, PNG (Mueller et al., 2009). (F) Incidence of malaria in Sri Lanka (Mendis et al., 2001). (For colour version of this figure, the reader is referred to the online version of this book). Natural Acquisition of Immunity to Plasmodium vivax 83

As a consequence of this remarkable difference in the rate of acquisition of clinical immunity, the incidence of P. vivax-attributable febrile illness in routine health surveillance is highest in children 1.0–1.9 years old, whereas that of P. falciparum peaks in children aged 2.0–3.9 years (Fig. 3.1A) (Muller et al., 2009). Although P. vivax is a very significant source of severe illness in infants (Poespoprodjo et al., 2009), the proportion of P. vivax infection presenting with severe symptoms decreases rapidly with age (Genton et al., 2008; Tjitra et al., 2008) and in children older than 1 year the incidence of severe P. vivax illness is significantly lower than that due to P. falciparum (Manning et al., 2012). Similar differences are found in the age-specific prevalence of P. vivax and P. falciparum infections: the prevalence of P. vivax by light microscopy (LM) tends to reach its peaks in children aged 4.0–6.9 years (24%), while P. falciparum prevalence is highest in children aged 7.0–9.9 years (48%) (Mueller et al., 2009). In adults, LM-positive P. vivax infections are less frequently detected (<10% prevalence) than P. falciparum infections (approximately 20%). When diagnosed by polymerase chain reaction (PCR), the prevalence of both P. vivax and P. falciparum increased, the peak prevalence shifted into older age groups (Pv: 7.0–9.9 years: 51%, Pf: 10.0–19.9 years: 74%) and infections were commonly detected even in adults (Fig. 3.1E). Interestingly, in longitudinal cohorts of children aged 1–4 and 5–14 years, the incidence of newly acquired, genetically distinct, P. vivax blood-stage infections (i.e. the molecular force-of-infection (molFOI)) was substantially higher than that of P. falciparum (Koepfli et al., submitted for publication; Michon et al., 2007; Mueller et al., 2012) despite a comparable (Michon et al., 2007) or higher entomological inoculation (Benet et al., 2004; Hii et al., 2001) rates for P. falciparum. P. falciparum molFOI increased from 1 to 4 years (Mueller et al., 2012) and remained constant thereafter (Michon et al., 2007), whereas P. vivax molFOI did not change with increasing age (Koepfli et al., submitted for publication; Michon et al., 2007). Together these epidemiological observations indicate that in New Guinea children malarial immunity is acquired much more rapidly to P. vivax than P. falciparum. After 5 years of continuous exposure, children can acquire an almost complete clinical immunity to P. vivax, which is characterized by their control of blood-stage parasite densities (often to a level only detected by PCR) rather than the acquisition of a significant immunity against infection per se. This difference in the rate of natural acquisition of immunity between P. vivax and P. falciparum was not only found in the highly endemic areas 84 Ivo Mueller et al. of New Guinea but also in other co-endemic areas of the world, including those with substantially lower transmission levels. In longitudinal studies carried out on the Western border of Thailand (Fig. 3.1D, (Lawpoolsri et al., 2010; Phimpraphi et al., 2008)), in Sri Lanka (Fig. 3.1F, (Mendis et al., 2001)) and in Vanuatu (Maitland et al., 1996), the incidence of P. vivax malaria also decreased significantly faster with age than that due to P. falciparum, whereas in a Brazilian cohort among Amazonian settlers the risk of P. vivax malaria started decreasing after 5–6 years of residence in the endemic area compared to 8–9 yrs for P. falciparum (da Silva-Nunes et al., 2008). This is also not a recent phenomenon, as it was well known in the 1930s in areas such as Greece (Balfour, 1935) and Puerto Rico (Earle, 1939) that P. vivax malaria was a disease that predominantly affected children and P. falciparum affected adolescents and adults. The age at first exposure may be an important modulator of the natural acquisition of malarial immunity. Studies involving non-immune migrants to Indonesian New Guinea who were exposed for the first time to heavy transmission indicated that adults acquired clinical immunity to malaria after relatively few P. falciparum infections, in contrast to children, who remained susceptible after comparable exposure (Baird et al., 1991, 2003). This relationship was not observed with P. vivax infections; adults did not acquire clinical immunity any more quickly than children (Baird, 1995). This suggests there may be fundamental differences in how NAI develops to Pv compared to Pf.

3. ACQUISITION OF IMMUNITY IN EXPERIMENTAL INFECTIONS – LESSONS FROM MALARIA THERAPY PATIENTS AND IRRADIATED SPOROZOITES Prior to the confirmation in 1947 that penicillin could cure syphilis, the primary treatment for neurosyphilis was fevers induced with malarial infections. Plasmodium vivax was the preferred treatment over P. falciparum because it could produce sustained fevers without requiring treatment. Because malaria therapy did not cure syphilis, but just delayed the progression of disease, individuals were often repeatedly infected. With support from the Rockefeller Foundation in the 1930s and early 1940s, a malaria research centre based at the Florida State Hospital in Tallahassee, Florida, undertook a remarkable series of experiments involving neurosyphilis patients to study P. vivax biology and immunity (see (Boyd, 1947)). Natural Acquisition of Immunity to Plasmodium vivax 85

Similar studies were being performed in the South Carolina State Hospital (Collins and Jeffery, 1999; Collins et al., 2003, Collins et al., 2004a, 2004b) and in hospitals in Europe (Ciuca et al., 1934). These experimental studies were designed to study the natural history of Pv infections in humans. Indi- viduals were infected either intravenously with Pv-IEs or by infected mosquitoes (to mimic natural infection). Malarial parasites were first detected in malaria-naïve subjects by day 11 or 12 following a mosquito inoculation of P. vivax sporozoites. The onset of fevers occurred almost simultaneously with the presence of parasites in the peripheral blood, with levels as low as 10 parasites/µl. Peak P. vivax parasitaemias generally occurred 5–7 days after the onset of symptoms, with parasite densities rarely exceeding 12,000 parasites/µl. Peak parasitemias coincided with highest fevers. As parasitaemia declined, fevers abated slightly, yet they persisted over the next 20 or more days. There was a rough correlation with parasitaemia levels and fevers yet parasites could persist in the peripheral blood at low densities for 2–3 months in the absence of fevers indicating some tolerance or immunity to disease independent of infection. In some subjects with natural infections, relapse would occur after clearance of blood-stage parasites and these induced much more attenuated clinical disease and lower parasitemias. A similar pattern of infection was observed when blood-stage parasites were directly inoculated, but the pre-patent period was shorter and depended on the inoculum. Relapses, as expected, did not occur under this experimental protocol. About 2–3 months after clinical symptoms had disappeared (although low levels of parasitaemia often persisted), the subjects were experimentally reinoculated either by mosquito infection or intravenous blood-stage challenge with the same parasite strain. Up to two-thirds of the individuals were completely immune to clinical malaria upon rechallenge. By the third exposure with the same strain, almost 100% of the individuals were clinically immune (Boyd, 1947; Ciuca et al., 1934). This acquisition of clinical immunity with repeated infection of the same P. vivax parasite strain occurred more rapidly than observed with Pf, although patients often had to be treated for Pf when they became severely ill. These experimental studies support epidemiological studies described above (Gunewardena et al., 1994; Lin et al., 2010; Maitland et al., 1996; Michon et al., 2007) that NAI develops more rapidly to Pv compared to Pf. Clinical immunity persisted for many years to the same strain. This was shown in some patients who cleared their parasitemias and were again infected 3.5 years later and again 3.5 years after that with the same strain and retained solid clinical immunity, 86 Ivo Mueller et al. although they developed low-level parasitemias. One subject had an interval of 11 years between Pv infections and still retained solid clinical immunity. It is unknown whether submicroscopic parasitemias persisted over this period (relapsing parasites rarely occur past 2 years following initial infection) and whether they were naturally exposed to P. vivax since the parasite was still endemic at the time in Eastern Europe and the United States. Thus these malaria therapy studies cannot exclude whether any persisting low-grade infection(s) (premunition) accounted for the observed long-term immunity. Since subjects quickly developed clinical immunity to the same parasite strain, they were then infected with different P. vivax strains with the aim to continue fever treatments. It became quickly apparent that immunity was strain specific. In a series of patients treated between 1940 and 1963 at the South Carolina State Hospital (see (Collins and Jeffery, 1999a, 1999b, 1999c, 1999d; Collins et al., 2004a, 2004b) for details), there were 36 patients with primary infections with the St. Elizabeth strain of P. vivax followed by reinfection with P. vivax. Of these, 14 were re-infected with the homologous strain of the parasite (Group 1) and 22 patients were re-infected with heterologous strains (17 with the South Pacific ‘Chesson’ strain, Group 2). During a secondary infection with the homologous St. Elizabeth strain the geometric mean maximum parasite count was reduced from 9101/µl to 998/µl (P < 0.001) and the geometric mean daily parasite count for the first 20 days was reduced from 924 to 16/µl/day (P < 0.001). This resulted in an 80% reduction in the incidence of fever of or above 101 °F (2.98 vs. 0.60/ person/week, rate ratio = 0.20. P = 0.002) and in a 91% reduction in fevers of or above 104 °F (1.92 vs. 0.18/person/week, rate ratio = 0.09. P = 0.003). These results show the rapid development of strain-specific immunity even after a single primary infection. By contrast, only partial protection was observed against the heterologous strain. Even though the maximum parasitaemia in 22 pairs of patients re-infected with heterologous parasites was comparable to the primary infection (8460/µl and 9196/µl), the geometric mean daily parasite count for the first 20 days of infection was significantly lower (847/µl/day vs. 336/µl/day, P = 0.002) and the incidence of fevers of or above 101 °F and 104 °F were reduced by 48% (2.08 vs. 1.07/person/ week, rate ratio = 0.52, P 0.007) and 55% (1.24 vs. 0.57/person/week, rate ratio = 0.45, P = 0.004), respectively (Collins et al., 2004a). Similar results were found in many different studies involving experimental malaria therapy patients (reviewed in (McGregor and Wilson, 1988; Taliaferro, 1949)). For example, in a very large series of Roumanian patients (with likely prior natural exposure), Ciuca et al. (1934) found that protection against P. vivax Natural Acquisition of Immunity to Plasmodium vivax 87 parasitaemia rose from 34 to 100% during five P. vivax inoculations but only from 22 to 97% during 10 homologous P. falciparum infections. Thus a single P. vivax infection was sufficient to induce a strong clinical protection during homologous re-infections and a partial protection during heterologous re-infections. However, no protection was found when patients initially infected with P. vivax were re-infected with P. falciparum (Collins and Jeffery, 1999a), indicating no evidence for cross-species protection. A more detailed description of experimental P. vivax infections in humans is found in Chapter X [Snounou]. Experimental infections produced by inoculation with malaria- IEs compared to those induced by bites of infected mosquitos induced similar patterns of immunity. This indicates that the NAI induced was directed to blood-stage infection. To assess whether pre-erythrocytic stage immunity could develop and control the onset of a blood-stage infection, five volunteers were vaccinated with irradiated P. vivax sporozoites in the 1970s (Clyde, 1990; Rieckmann, 1990). One volunteer exposed to 1979 mosquito bites remained protected (i.e. no blood-stage parasites were detected) for up to 9 months when challenged by a dose of six infective bites but not when challenged with 12 infective bites. A second volunteer, who was vaccinated with both irradiated P. vivax (539 mosquito bites) and P. falciparum sporozoites, was also protected for 8 months against both homologous and heterologous infections, but this person was re-infected when re-challenged at 11 months (Clyde, 1990). No protection was observed in the other three volunteers exposed to fewer than 200 irradiated infected mosquitoes. A similar dose-dependent protection was observed in two trials with irradiated P. vivax sporozoites in Saimiri monkeys (Collins et al., 1992) and Aotus monkeys ( Jordan-Villegas et al., 2011). These data indicate that very high numbers of sporozoites acquired at a single time are required to induce sterile pre-erythrocytic immunity. Such doses are unlikely to be reached under natural exposure, although the cumulative number of sporozoites over months and years may reach similar levels in areas of high malaria transmission. This “trickle effect” of sporozoite exposure on pre-erythrocytic stage immunity is unknown. It is difficult to extrapolate infections with irradiated sporozoite to natural infection since irradiated sporozites arrest development and can persist in the liver, which may induce a different type of immune response compared to natural exposure (Scheller and Azad, 1995). Table 3.1 summarises the primary findings from these extensive studies of malaria infections in thousands of patients. There are some important limitations to these human studies, however. It is uncertain whether the 88 Ivo Mueller et al.

Table 3.1 Features of NAI Based on Artificially Induced Infections in Humans – Immunity is directed against blood-stage infection – no evidence of pre-erythrocytic stage immunity. – A substantial degree of clinical immunity develops after a single untreated infection and complete clinical immunity after two or three infections by the same strain. – Immunity is strain specific with substantial cross-protection among strains. – Immunity is species specific; Pv does not seem to protect against Pf. It is unclear whether the reverse is true. – Clinical immunity develops to P. vivax more rapidly than to P. falciparum. – Solid clinical immunity can persist for years. It is unknown whether this requires relapsing blood-stage infection or persisting low-grade infections, but this seems unlikely. – Exposure to large numbers of irradiated sporozoites could elicit pre- erythrocyte stage immunity, but is not of long duration (<1 year). An equally large exposure to sporozoites is unlikely under natural conditions.

‘strains’ used were really just one strain. In addition, submicroscopic parasitaemia could not be detected since only blood smears were used at the time. Today, genetic methods can distinguish the presence of individual or multiple strains or submicroscopic levels of P. vivax in the blood.

4. UNIQUE BIOLOGICAL CHARACTERISTICS OF P. VIVAX THAT CONTRIBUTE TO NAI Individuals exposed to a comparable number of infections and clinical episodes of P. vivax and P. falciparum acquire clinical immunity to P. vivax more rapidly than P. falciparum (Lin et al., 2010; Maitland et al., 1996; Michon et al., 2007). Pv has several important differences in its biology compared to Pf, which may help to explain the differences in the acquisition of immunity to each of these species.

4.1. Relapses In contrast to P. falciparum, P. vivax develops latent stages in the liver referred to as hypnozoites. The hypnozoites can become activated to initiate one or more blood-stage infections up to 2 years after an initial inoculation of sporozoites by a mosquito bite (White, 2011) (and Chapter X: white). Thus, P. vivax blood-stage immunity can be boosted even when malaria transmission Natural Acquisition of Immunity to Plasmodium vivax 89 is low or absent. Initial relapses appear to be of the same original infecting strain(s) (Imwong et al., 2012), which may contribute to the rapid and persistent NAI observed in each of the malaria therapy studies or development of NAI in low-transmission areas (Craig and Kain, 1996). As individuals acquire additional sporozoite inoculations, a diverse population of hypnozoites (and resulting relapsing parasite strains) may develop (Chen et al., 2007; Imwong et al., 2007). NAI may develop quickly in PNG and other areas of the South Pacific because of the frequent periodicity of relapsing parasites, 30–45 days following the primary infection, compared to P. vivax strains from more temperate areas (White, 2011) (and Chapter X: white). Thus in Pv endemic areas where Pv strains have longer relapsing periodicity NAI would be predicted to develop more slowly. As children presenting with clinical P. vivax are treated for both blood- and liver-stage infections (i.e. with primaquine) to also eliminate relapsing parasites, it would also be predicted that NAI may be significantly delayed if relapsing parasites are important in boosting blood-stage immunity. Longitudinal cohort studies can potentially help to distinguish and characterize the acquisition of NAI in groups of individuals over time with more or less frequent relapsing patterns and with or without receiving primaquine therapy. In a recent cohort in children aged 1–5 years in PNG, relapses contributed at least 50% of the burden of P. vivax infections (Betuela et al., 2012). As a result, the number of new P. vivax blood-stage infections is 2–3 times higher than that for P. falciparum (Mueller et al., 2012), despite similar or higher P. falciparum sporozoite rates (Benet et al., 2004; Hii et al., 2001).

4.2. Force of Blood-Stage Infection The force of blood-stage infection may also be higher for P. vivax than P. falciparum as shown in the previous section in studies in PNG, resulting in more frequent exposure to novel genotypes (Koepfli et al., submitted for publication; Mueller et al., 2012). This may arise because effective population of P. vivax is often larger in a community (Ord et al., 2008) and because of activation of genetically distinct hypnozoities (Chen et al., 2007; Imwong et al., 2007, 2012).

4.3. The Infected Red Blood Cell Membrane and Variant Surface Antigens During intraerythrocytic development, both P. vivax and P. falciparum express highly polymorphic antigens on the IE surface (Bernabeu et al., 2012; del Portillo et al., 2001; Gunewardena et al., 1994; Leech et al., 1984; Marsh and 90 Ivo Mueller et al.

Howard, 1986) In the case of Pf they undergo clonal antigenic variation, and acquired Abs against these antigens typically demonstrate a high degree of strain specificity (Biggs et al., 1991). The predominant Pf VSA is erythrocyte membrane protein 1 (PfEMP1), which is encoded by a large family of highly variable var genes (Baruch, 1999; Smith et al., 2001; Su et al., 1995) and causes the adhesion of trophozoite and schizont IE to endothelial cell receptors. One member from a repertoire of about 60 encoded proteins is typically expressed at a time, in association with electron-dense parasite- induced ‘knob’ protrusions on the surface of the IEs (reviewed in (Scherf et al., 2008)). Antigenic diversity and variation of surface antigens facilitates the development of repeated infections over time, as new infections appear to exploit gaps in the repertoire of variant-specific Abs. Prospective studies in children provide strong evidence that surface antigens are targets of protective immunity by showing that Abs are associated with a reduced risk of malaria and studies suggest that increasing exposure leads to a broad repertoire of Abs that provides protection against different variants (Bull et al., 1998; Giha et al., 2000; Mackintosh et al., 2008). Abs to these IE surface antigens are thought to confer protection by inhibiting vascular adhesion and sequestration of IEs and by opsonising IEs for phagocytic clearance. Thus NAI to VSA requires repeated exposure. The role of predicted VSA at the surface of Pv IE is less clear but is exemplified by the larger vir gene family (Bernabeu et al., 2012; Carlton et al., 2008; del Portillo et al., 2001). There are 346 vir genes in the Pv genome (Sal I strain) of varying sizes and about 160 have domains consistent with the VIR proteins targeted to the erythrocyte surface. This large diversity suggests multiple functions of these proteins including, and perhaps predominantly, host immune evasion mechanisms. The Pv VIR proteins are much smaller than PfEMP1, lack comparable adhesive domains and are not clonally expressed (Fernandez-Becerra et al., 2005). VIR proteins are expressed earlier than PfEMP1 during the intraerythrocytic cycle and the timing of their expression is not consistent among isolates (Bozdech et al., 2008) although these observations may be complicated by the difficulty of growing synchronised parasites in vitro. Recent evidence suggests that certain VIR proteins that are expressed on the surface of IEs facilitate the adherence of P. vivax IE to endothelial cells possibly via ICAM-1 (Bernabeu et al., 2012; Carvalho et al., 2010), which may facilitate their sequestration in the lung (Anstey et al., 2009), and it has also been speculated that they may facilitate escape from spleen clearance (Fernandez-Becerra et al., 2009). Natural Acquisition of Immunity to Plasmodium vivax 91

By immunofluorescent assay (IFA), schizont stages of Pv were shown to be recognised by individuals with prior Pv infection and this reactivity could be isolate specific (Mendis et al., 1988). Moreover, individuals with more than three documented Pv infections compared to individuals with fewer known Pv infections had a greater number and breadth of Pv isolates suggesting acquired Ab responses to Pv IEs (Mendis et al., 1988). It appears that some of the Ab responses are directed to VIR proteins on the Pv-infected red blood cell (RBC) surface (Bernabeu et al., 2012; Carvalho et al., 2010; del Portillo et al., 2001; Fernandez-Becerra et al., 2005, 2009); however, their role in NAI is unclear. The potential role of VIR proteins in NAI may be better understood in longitudinal cohort studies by studying the degree and breadth of Ab responses to Pv isolates. Although continuous in vitro cultures of Pv are not a reality today, short- term cultures (Grimberg et al., 2007; Nichols et al., 1987; Russell et al., 2011; Udomsangpetch et al., 2007, 2008; Wertheimer and Barnwell, 1989) using fresh isolates or frozen Pv could examine the breadth and strength of Ab recognition surface of Pv-infected schizonts using flow cytometry from Pv-exposed individuals with and without NAI to Pv. A large number of proteins have been identified in association with the P. vivax IE membranes and these structures may also be targets of the host immune response (Barnwell et al., 1990). In contrast to rigid, the knobby and sticky Pf-infected red cell membrane, Pv remodels the reticulocyte host cell membrane to produce numerous caveola–vesicle complexes (CVCs) all along the surface of the infected cell (Aikawa, 1971; Aikawa et al., 1975; Akinyi et al., 2012; Suwanarusk et al., 2004) that may increase erythrocyte membrane deformability. Where the VIR proteins may be positioned relative to these abundant surface-exposed CVC structures is not known. The function of CVCs may be targets of host immune responses. For example, the recently described Plasmodium helical interspersed subtelomeric (PHIST) protein known as PvHIST/CVC-8195 (Table 3.2) is a dominant component of the CVCs (Akinyi et al., 2012; Barnwell et al., 1990). As such, it may stimulate NAI. Alternately, its release upon rupture of infected RBCs may be pro-inflammatory and enhance virulence of the infection (Akinyi et al., 2012).

4.4. Critical Red Cell Invasion Ligands In contrast to Pf, Pv selectively invades reticulocytes and requires the Duffy antigen, a chemokine receptor on erythrocytes, for successful invasion (Miller et al., 1976) (although exceptions occur (Menard et al., 92 Ivo Mueller et al. ) ; ; ) ; Han ; al., 2008 al., ; ;

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et et Soares Nogueira et Nogueira et Yang 2005, Cole-Tobian et al., 2002, 2009) 2002, et al., Cole-Tobian 2005, Barbedo et Nogueira et Nogueira Collins et et Lima-Junior Rodrigues et et Tran Akinyi et Akinyi ( References ( ( ( ( ( (Arevalo-Herrera et al., 2006, Ceravelo et al., et al., Ceravelo 2006, et al., (Arevalo-Herrera King et al 2008) 2007, (Grimberg et al, ( − + + + + − + + − Correlate with Correlate protection – studies longitudinal − + + + + + + + + + + Ab response response Ab with increase age/exposure + + Inhibition of Pv invasion/ in vitro growth + +/− Protection in Protection non-human primates 95 blood-stage antigens that elicite host immune responses and/or correlated with protection and/or correlated host immune responses elicite that blood-stage antigens vivax P.

19 (CVC) proteins inhibitory) Merozoite surface proteins Merozoite MSP1 MSP3.10 (N-terminal) MSP9.10 (N-terminal) MSP1 N-terminal MSP1 C-terminal RBP2 surface antigens Variant VIR complex Caveola–vesicle PvPHIST-81 Apical membrane protein membrane Apical AMA1 Antigen Erythrocyte-binding ligands PvDBPII (total) PvDBPII (binding Reticulocyte-binding proteins RBP1 Table 3.2 Table Natural Acquisition of Immunity to Plasmodium vivax 93

2010)). This is reminiscent of the requirement of the chemokine receptor, CCR5, for HIV invasion of T cells. There is a single known parasite ligand for the Duffy antigen called PvDBP (Adams et al., 1992; Chitnis et al., 1996). PvRBPs and possibly other molecules target invasion of the immature erythrocytes (Barnwell and Galinski, 1995; Galinski and Barn- well, 1996; Galinski et al., 1992). Although there are likely many more molecules yet to be discovered that are critical for Pv invasion of erythrocytes, some individuals may develop NAI by targeting immune responses to these critical and perhaps non-redundant pathways. This might contribute to rapid development of NAI compared to Pf with more redundant invasions pathways.

4.5. Immune Regulation The induction of potent immunosuppressive networks can impair the development of adaptive and innate immunity. Malaria infections often develop asymptomatic parasitemia, occasionally with high parasite burdens. Thus infection in the absence of inflammation (lack of costimulatory or other signals) promotes immune tolerance. It has been shown that patients with severe falciparum malaria induce a potent set of regulatory T cells whose numbers and suppressive function increase with parasite burden (Hansen and Schofield, 2010; Jangpatarapongsa et al., 2008; Minigo et al., 2009; Scholzen et al., 2009, 2010; Walther et al., 2009). Other immunoregulatory networks likely exist with heavy infections such as development of atypical and potentially suppressive B cells (Pierce, 2009; Weiss et al., 2009) and sup- pression of antigen-presenting cell functions akin to endotoxin tolerance (Boutlis et al., 2006). Immune tolerance can also result from in utero exposure to malaria or their soluble products (King et al., 2002; Mackroth et al., 2011; Malhotra et al., 2008, 2009; Metenou et al., 2007) that can persist into early childhood (Malhotra et al., 2009). Malaria in pregnancy is much less common and if present, is less likely to cause altered placental dysfunction for Pv compared to Pf (Rogerson et al., 2007; ter Kuile and Rogerson, 2008). Thus prenatal exposure to Pv antigens are less likely to occur compared to Pf; however, prenatal exposure to Pv remains poorly studied.

5. EFFECTOR MECHANISMS FOR BLOOD-STAGE IMMUNITY Adoptive transfer of serum from avian (Manwell and Goldstein, 1940), murine (Parashar et al., 1977), non-human primate (Coggeshall 94 Ivo Mueller et al. and Kumm, 1937) and human falciparum malarias with NAI (Cohen et al., 1961; McGregor, 1964) into naïve animals or humans protects against clinical malaria or significantly attenuates the severity and burden of malaria (Cohen et al., 1961; McGregor, 1964). Such experiments have established that Abs are critical for NAI to blood-stage malarial infection. Interestingly, passive transfer of 500 ml of blood from neurosyphilis subjects with solid clinical immunity to Pv failed to induce any protection against Pv disease in naïve subjects (Boyd, 1947) either at the time of infection or early in the course of clinical disease. The relative amount of transferred serum was similar to that observed with successful passive transfer of immunity observed to Plasmodium knowlesi in rhesus macaques (Coggeshall and Kumm, 1937) but with about half the amount of total Abs observed with Pf in humans (Cohen et al., 1961). Humoral immunity is likely important for NAI to Pv but cellular immunity may contribute to Pv protection more than appreciated. For example, it has been proposed that Pv IE escape splenic clearance by increasing their deformability, enabling passage through adjacent endothelial cells into the venous sinus lumen, thus avoiding destruction by splenic macrophages (Fernandez-Becerra et al., 2009). If this is the case, then immune mechanisms that may impair Pv IE deformability would facilitate their clearance by the spleen. Another proposed hypothesis is that VIR proteins on the surface of Pv IE facilitate adherence in the spleen, preventing phagocytosis by splenic macrophages. Since NAI to Pv is strain specific, this suggests that targets of NAI are likely to be highly variable among strains.

6. TARGETS OF BLOOD-STAGE IMMUNITY Although much can be inferred from the basic biology of Plasmo- dium merozoites (Fig. 3.2), there are many gaps in our knowledge of the biology of P. vivax merozoites. For example, why they selective invade host reticulocytes and details of the growth and development of the various blood-stage developmental forms (reviewed in Galinski et al., 2005). In addition, only a relatively small group of merozoite proteins have so far been identified with specific localisations and confirmed functional roles. It is important to remain cognizant of the fact that there are a large number of hypothetical proteins and that many are likely to have critical biological functions and be important for the development of NAI. More- over, the specific proteins, precise host–parasite interactions, and order of Natural Acquisition of Immunity to Plasmodium vivax 95

Figure 3.2 Potential targets and mechanisms for blood-stage immunity. Experimental systems based on different species of Plasmodium have shown that there are multiple points in the erythrocyte invasion process that could be targets of the host immune response. The exact molecules involved and sequence of events remain to be fully defined, and these may differ among species. Free merozoites, prior to invasion of RBCs, are susceptible to host immune responses. The various MSPs that cover the merozoite could function in the initial recognition of RBC host cells and be the target of opsonising Abs than can trigger cell-mediated merozoite killing or directly interfere with initial adherence of the merozoite to the RBC. The merozoite quickly reorients, and through sequential predicted interactions of the RBPs and the DBP, the parasite’s apical pole is brought into close apposition to the RBC membrane. Next, RON proteins are released from rhoptry organelles near the apical end of the merozoite and presumably inserted into the merozoite membrane. With the interaction of apical membrane protein (AMA1) a junction forms with the RBC membrane that generates an actin-dependent process that facilitates the entry of the merozoite into the RBC. A parasitophorous vacuole forms in the process. As P. vivax parasites grow, the IE membrane is modified by the development of numerous CVCs and proteins that may facilitate some level of adherence to the vascular endothelium. All these alterations represent potential targets of opsonising Abs and iRBC clearance. Since the Pv membrane is permeable to Abs (Lyon and Haynes, 1986) and proteinases are essential for the release of merozoites from the IE (Salmon et al., 2001), it is conceivable that acquired immune responses may also target these proteinases. (For colour version of this figure, the reader is referred to the online version of this book). molecular events can differ among the species of Plasmodium, and among strains within a species. In this section, key highlights of the current known targets of P. vivax blood-stage immune responses are presented. Several recent review articles also summarise what is known about the biology, immunology and vaccine progress pertinent to each of these proteins (Arevalo-Herrera et al., 96 Ivo Mueller et al.

2010; Galinski and Barnwell, 2008; Mueller et al., 2009). Post-genomic research and modern technologies have the potential to enable the rapid great expansion of our understanding of the biology of P. vivax blood- stage parasites and stimulate a greater understanding of NAI and the relative importance of immune responses to a much broader panel of antigens. Evaluating the global individual and population-based immune response using P. vivax blood-stage protein arrays is one way forward to grasp a more complete picture (Chen et al., 2010; Trieu et al., 2011). The ultimate challenge will be to decipher which of these immune responses can be confidently and reliably associated with protection of individuals and not simply represent active immune stimulation and the possible masking of protective responses. Protective immune responses are likely to be composed of functional Abs of high affinity and memory T cells that target critical portions of molecules involved in essential biological function of the parasite. The parasite is likely to have evolved mechanisms to impair development of protective immune responses such as antigenic variation, poor immunogenicity, masking of critical regions by non-essential immunogenic regions or controlled expression of these regions only at essential points in the parasite invasion or development. Therefore, a detailed understanding of the structural biology and function of these molecules are essential. So far, no robust correlates of NAI have been identified for Pf, although the levels and breadth of Ab reactivity to different variants of PfEMP1 suggest an association with NAI (Bull et al., 1998; Giha et al., 2000; Mackintosh et al., 2008; Marsh et al., 1989). Likewise, confirming reliable immune correlates of NAI for Pv will continue to pose a challenge. Plasmodium vivax merozoite proteins that have been investigated with some consistency over the past 20 years include the PvRBPs, the PvDBP, apical membrane protein (PvAMA1) and several PvMSPs. Merozoites can initially attach to RBCs at any point on their surface and then must reorient prior to invasion so that the apical pole of the parasite is juxtaposed to the RBC surface (Galinski and Barnwell, 1996; Srinivasan, P., et al., 2011 also reviewed in Chapter X and see Fig. 3.2). At least a dozen distinctive MSP (including MSP3 which constitutes a family of proteins) have been reported to cover the surface of merozoites and can contribute to the merozoite’s reversible adhesive properties. They can be abundantly expressed and as a group these proteins have been considered important vaccine candidates based on their location and extensive immune response and vaccination studies carried out for over 30 years. Natural Acquisition of Immunity to Plasmodium vivax 97

Plasmodium vivax research on MSPs has so far focused predominantly on PvMSP1, PvMSP3 and PvMSP9.

6.1. PvMSP1 This is the most abundant and best-studied malaria blood-stage antigen. The known biological and immunological processes relating to MSP1 have derived from studies of Pf and Pk. Whether these observations can be extrapolated to PvMSP1 is uncertain, yet its biology is likely to be similar among Plasmodium spp. MSP-1 undergoes two successive proteolytic cleavage events (Blackman et al., 1994). The second processing event occurs immediately before merozoite invasion of RBCs, resulting in the cleavage of the p42 fragment into p33 and p19 sub-fragments (Gerold et al., 1996). The p19 molecule remains attached to the merozoite surface through a glycosylphosphatidylinisotol (GPI) anchor and is composed of two epidermal growth factor-like domains (Morgan et al., 1999), which may have a role in the invading complex. Experimental data suggests that PvMSP1 has a similar biology and function as PfMSP1 (del Portillo et al., 1991; Han et al., 2004). Antibodies specific to PfMSP1, particularly the C-terminal region (42- and 19-kD sub-fragments) have been shown to block parasite invasion in vitro (Egan et al., 1999), induce protective immunity in animal models and functional Abs to PfMSP1 correlate with protection in human studies ( John et al., 2004). With respect to Pv, multiple studies have shown older or more heavily exposed individuals who are more likely to have NAI to show greater humoral and cellular reactivity to PvMSP1 (Bang et al., 2011; Collins et al., 1999; Egan et al., 1999; Galinski et al., 1999, 2001; John et al., 2004; Singh et al., 2009; Valderrama- Aguirre et al., 2005) and human Abs specific to PvMSP119 correlated with protection against clinical P. vivax malaria in a longitudinal study (Nogueira et al., 2006). However, vaccination of non-human primates with PvMSP119 failed to consistently induce protection. Immunisation of Aotus monkeys with a polymorphic N-terminal region of PvMSP1 afforded only partial protection to challenge with Pv (Valderrama-Aguirre et al., 2005), while immunisation with PvMSP119 failed to induce substantial protection in splenectomised Saimiri boliviensis monkeys (Collins et al., 1999). Yet, immunisation with recombinant MSP142 and MSP119 from Plasmodium cynomolgi, a simian malaria species closely related to Pv, in its natural host, Macaca sinica, was highly protective to challenge infection (160) suggesting that protection may be host specific or production of properly refolded PvMSP119 may be more difficult than other Plasmodium 98 Ivo Mueller et al.

spp. Thus the role of PvMSP1 in NAI and the value of PvMSP119 as a vaccine candidate remain uncertain.

6.2. PvMSP3 Plasmodium vivax has a family of msp3 genes (Galinski et al., 1999, 2001) with 11 members reported for the initially sequenced strain, Sal I (Carlton et al., 2008). These gene family members are positioned head to tail on chromosome 10 and gene expression studies demonstrate that all but the final member are expressed during a blood-stage infection. These data are similar to earlier studies showing the presence of an msp3 gene family in P. falciparum on chromosome 10 and the expression of all members (Singh et al., 2009). The first PfMSP3 described has been advancing as a viable vaccine candidate to clinical trials (Bang et al., 2011; Belard et al., 2011; Sirima et al., 2011). PvMSP3s can be dramatically diverse, as demonstrated for the PvMSP3α and PvMSP3β members, showing numerous point mutations as well as insertions and deletions (Rayner et al., 2002, 2004a). Basic immunological enquiry to understand the role that PvMSP3s may have in NAI has so far been focused on PvMSP3α (Lima-Junior et al., 2011, 2012). In summary, Lima-Junior et al. (2011) have reported the identification of a set of B-cell epitopes from the central α-helical region of PvMSP3α that are widely recognised in Rondonia State, in the Brazilian Amazon. Levels of total IgG and specifically subclass (IgG1 and IgG3) Abs were associated with increased exposure to the parasite Lima-Junior et al., 2011. This group has also begun to investigate the human leukocyte antigen (HLA) types associated with such responses (Lima-Junior et al., 2012). Others have confirmed the natural acquisition of Abs to a different central domain called PvMSP3α359–798, noting a predominance of IgG1 Abs followed by IgG2 and an association with anaemia (Mourao et al., 2012). A pre-clinical vaccine study showed partial efficacy upon immunisation with PvMSP3α and PvMSP3β near full-length recombinant proteins using Freunds adjuvant in S. boliviensis monkeys (Barnwell and Galinski, unpublished data); however, no efficacy was achieved with these proteins in a subsequent trial using Montanide 720 as an adjuvant ( Jiang, Barnwell, Galinski et al., unpublished data). PvMSP3α, β and γ have been renamed as PvMSP3.10, PvMSP3.3 and PvMSP3.1, respectively, based on their chromosomal positioning among all 11 gene family members. The relative importance of the different members of the PvMSP3 family needs further investigation, with regard to the biology of the parasite, diversity and NAI Natural Acquisition of Immunity to Plasmodium vivax 99 and to discern which components of this family may warrant further study as vaccine candidates. The function of these proteins is not known. Each member is characterized by an NLRNG motif near the signal peptide, a centrally located alpha helical coiled-coil domain and the absence of a membrane-anchoring mechanism (Carlton et al., 2008; Galinski et al., 1999, 2001). The expression pattern of each protein is not precisely identical and one member is in fact uniquely expressed at the apical pole of the merozoite ( Jiang et al submitted).

6.3. PvMSP9 Similar to PvMSP3 family members (Carlton et al., 2008; Galinski et al., 1999, 2001), PvMSP9 associates with the surface of the merozoite but does not have an anchoring mechanism, i.e. hydrophobic transmembrane domain or GPI anchor (Barnwell et al., 1999; V argas-Serrato et al., 2002). It has a homologue in P. falciparum known as p101 or ABRA (Kushwaha et al., 2000) and the simian malaria species P. knowlesi and P. cynomolgi (Barn- well et al., 1999; V argas-Serrato et al., 2002). Short-term cultures have been used to show that a P. vivax monoclonal Ab (mAb) and polyclonal antisera raised against the native P. cynomologi protein can inhibit merozoite invasion of reticulocytes, raising its profile as a possible vaccine candidate (Barnwell et al., 1999; Vargas-Serrato et al., 2002). NAI studies have been ongoing in Brazil and PNG, two epidemiologically distinctive regions with regard to malaria transmission (Lima-Junior et al., 2008, 2011, 2012). In the Brazil- ian Amazon, Lima-Junior and colleagues have studied humoral and cellular immune responses in a cross-sectional study involving individuals from three Rondonia communities with different exposure histories. Abs to the conserved N-terminal region and a C-terminal repeat region were higher in residents who had established in the area for the longest period of time, and the responses correlated with age. IgG1 and IgG2 subclass responses predominated and interferon (IFN)-γ and interleukin (IL)-4 responses were generated against PvMSP9 peptides (Lima-Junior et al., 2008). Promiscu- ous T-cell epitopes in PvMSP9 were shown to induce the production of these cytokines in the individuals from this study, with diverse HLA-DR backgrounds, which could prove to be advantageous for vaccine development (Lima-Junior et al., 2010). HLA-DRB1*04 carriers, frequent in the native Amazon population, produced higher levels of PvMSP9 Abs compared to PvMSP1 and PvMSP3α and the time of exposure correlated with these responses (Lima-Junior et al., 2012). A more recent prospective study 100 Ivo Mueller et al. in PNG concluded that Abs to the N-terminal region of PvMSP9 were significantly associated with a reduced risk of P. vivax malaria (unpublished observations). Continued research is underway in these endemic regions, aiming for a more comprehensive understanding of NAI to PvMSP9, other proteins and P. vivax overall. 6.4. PvAMA1 Investigations on the NAI to Apical Membrane Antigen-1 of P. vivax (PvAMA1) have followed in the footsteps of PfAMA1, which has been advancing as a vaccine candidate (Dutta et al., 2009; Remarque et al., 2008a, 2008b, 2012; Sheehy et al., 2012). This interesting protein localises to the microneme organelles of the merozoite and then becomes trans- located to the surface where it binds to the moving junction protein complex at the interface of the host erythrocyte target cell through interaction with the RON2 protein (Hossain et al., 2012; Lamarque et al., 2011; Srinivasan et al., 2011). Antibodies directed to PvAMA1 may function to prevent its adhesive interactions and pathogenic properties (see below). Lacerda Bueno et al have been investigating NAI to PvAMA1 in Manaus and Cuiabá, Brazil, and have identified a linear B-cell epitope in Domain II that is highly antigenic in natural infections. IgG and subclass Abs that correspond to this region and the whole protein were analysed (Bueno et al., 2011). The genetic diversity and immune response to PvAMA1 have also been studied in Sri Lanka (Dias et al., 2011), with a predominance of IgG1 and IgG3 responses (Wickramarachchi et al., 2006). Most individuals studied developed Ab responses to linear and conformational epitopes of Domain II. Extensive polymorphism was reported in this suggesting this region of PvAMA1 is under immune selection (Dias et al., 2011). Similar results were reported from genetic studies in India and Thailand, with the immunological implications of varying selective forces and functional constraints (Putaporntip et al., 2009; Thakur et al., 2008). However, polymorphism was not detected in the Domain II loop region that has been shown to be conserved and predicted to be functionally important in PfAMA1 (Chesne-Seck et al., 2005, Pizarro et al., 2005). Both B- and T-cell epitopes have been identified in this region of the molecule (Lal et al., 1996, Bueno et al., 2011). NAI to Domain II was also found to stand out in earlier immunogenicity studies from Brazil (Mufalo et al., 2008). Interestingly PvAMA1 itself is pro-inflammatory which may contribute to its strong immunogenicity (Bueno et al., 2008, 2009). Natural Acquisition of Immunity to Plasmodium vivax 101

6.5. PvRBPs PvRBPs may be the first, or among the first apically located proteins, to bind in an irreversible manner to reticulocyte target cells, in essence per- forming the critical functions of host cell section and commitment (Galin- ski and Barnwell, 1996; Galinski et al., 1992). At the time of their discovery, two members of the PvRBP family (PvRBP1 and PvRBP2) were identified and shown to adhere to reticulocytes in in vitro erythrocyte adhesion assays (Galinski et al., 1992). It has been proposed that the PvRBPs select the immature RBCs as host cells and then trigger the release of the PvDBPs from their micronemal location in time for the critical ‘junction formation’ step that precedes entry of the merozoite into the target host cell (Singh et al., 2005). The PvRBPs are large proteins of approximately 330 kDa. Related ‘reticulocyte binding-like’ (RBL) proteins have been identified in P. falciparum (Rayner et al., 2000; Triglia et al., 2001) (reviewed in (Gaur and Chitnis, 2011)), P. knowlesi (Meyer et al., 2009) and other non-human primate (P. cynomolgi, Plasmodium reichenowi) and rodent malaria model systems (Keen et al., 1994; Iyer et al., 2007; Okenu et al., 2005; Rayner et al., 2004b, 2004c). The pvrbp2 gene was found to be much more diverse than pvrbp1 in an initial survey of parasite isolates originating from Brazil and Thailand (Rayner et al., 2005). Polymorphisms within pvrbp1 were most abundant in the N-terminal region of this protein, within the location of a predicted RBC-binding domain (Rosas-Acosta, 1998; Urquiza et al., 2002); a similar binding region has been identified in P. falciparum (Gao et al., 2008; Gaur et al., 2007; Rodriguez et al., 2008; Triglia et al., 2011) and P. knowlesi (Semenya et al., 2012) RBL homologues. Although regarded as adhesion proteins that could potentially be inhibited by Abs, there have only been a few studies geared at understanding NAI to PvRBP1 and PvRBP2. Segments spanning these proteins have been expressed as recombinant proteins and immune responses to PvRBP1 studied in comparison to PvDBP-II (Region II) in Brazil (Tran et al., 2005). Ab responses were predominantly cytophilic IgGs and significantly correlated with exposure, and the highest levels of anti-PvRBP1 IgG were to the N-terminal region that is predicted to contain the binding domain and focal increased polymorphisms (Tran et al., 2005). As noted below with regard to the PvDBP-II-binding region, diversity of the N-terminal region of RvRBP1 may similarly result from immune pressure on this protein’s binding domain. It is also noteworthy that more rapid and robust 102 Ivo Mueller et al.

Ab responses were detected in this study for PvDBP-RII; however, the evidence suggests that anti-PvRBP1 Abs may last longer in the absence of repeat vivax infections (Tran et al., 2005). The P. vivax genome (Sal I strain) project reported the existence of additional pvrbp genes (Carlton et al., 2008). Continued sequence analysis of these genes (as many as 10 reported) from Sal I and other monkey-adapted P. vivax isolates indicates that several of them represent gene fragments or incomplete reading frames (unpublished data). Recent investigations of four P. vivax isolates from Thailand confirmed the presence of multiple pvrbp pseudogenes and a high level of diversity in the pvrbp2a and pvrbp2b members of this gene family, suggesting immune pressure on these particular family members (Kosaisavee et al., 2012). Continued investigations are important to help predict which of these proteins, and which regions of them, may be most critical for the biology of the parasite and relevant for the development of NAI.

6.6. PvDBP Plasmodium vivax DBP is a microneme protein associated with the deci- sive irreversible step of junction formation between the merozoite and the host erythrocyte receptors during invasion, giving DBP priority as a prime target for vaccine-induced Ab-mediated immunity against asexual stages of the parasite (Fig. 3.2) (Adams et al., 1990; Chitnis, 2001). The receptor for DBP is the Duffy antigen receptor for chemokines or Fy blood group antigen, which is the non-transducing chemokine receptor on the erythrocyte surface (Horuk et al., 1993; Miller et al., 1975, 1976). Fy is a minor blood group antigen that has two immunologically distinct alleles referred to as FY*A and FY*B resulting from a single point mutation. Historically, the vital need of the DBP-Fy interaction is evident from the absence of P. vivax from regions of Africa with a high prevalence of Fy negativity (Guerra et al., 2010). Recent additional evidence of the importance of the DBP-Fy interaction was the demonstration of the protective effect against clinical vivax malaria by the FY*A allele. Individuals with the Fy a+b− phenotype demonstrated a 30–80% reduced risk of clinical vivax, but not falciparum malaria, in a prospective cohort study in the Brazilian Amazon (King et al., 2011). The major finding is the FY*A allele, which is predominant in Southeast Asian and many American populations, that confers a selective advantage against vivax malaria. Naturally acquired Abs to DBP are prevalent in residents of areas where vivax malaria is endemic and these Abs can inhibit DBPII binding Natural Acquisition of Immunity to Plasmodium vivax 103 and merozoite invasion of human erythrocytes (Grimberg et al., 2007). Individuals tend to show significant quantitative and qualitative differences in their anti-DBPII serological responses that overall increase with age and exposure (Chootong et al., 2010; Cole-Tobian et al., 2002; Van - Buskirk et al., 2004; Xainli et al., 2003). Interestingly, about 8–10% of P. vivax exposed individuals acquire high titre, strain-transcending broadly neutralising Abs (Chootong et al., 2010; King et al., 2008) that are associated with 50% reduction in the risk for P. vivax infection (King et al., 2008) and disease (unpublished data) based on longitudinal cohort studies. Usually, the initial Ab response is not broadly protective, while strain tran- scendent immunity develops only after repeated exposure (Cole-Tobian et al., 2002; Xainli et al., 2003). Vaccine-induced anti-DBP Abs partially inhibit erythrocyte binding (Devi et al., 2007; Grimberg et al., 2007; Van- Buskirk et al., 2004) and invasion of host erythrocytes and induce partial protection in monkeys (Arevalo-Herrera et al., 2005). Epitope targets of neutralising Abs mapped onto DBPII crystal structure suggest a functional mechanism of anti-DBP immunity that blocks DBP dimerisation to inhibit merozoite invasion (Batchelor et al., 2011). However, PvDB- PII is highly polymorphic presumably as an immune evasion mechanism responsible for strain-specific immunity. Strain-transcending neutralising immunity is achieved when Abs target functionally conserved epitopes thereby blocking DBP dimerisation and inhibiting invasion. The challenge in developing PvDBPII-based vaccine is the induction of strain- transcending Abs. Of interest, in vitro studies suggest that naturally acquired and artificially induced Abs block binding of recombinant PvDBPII to erythrocytes expressing Fya+a+ (associated with protection) more effectively than to Fy b+b+ erythrocytes. Thus, Fy polymorphism may be an important variable suggesting that vaccine efficacy to PvDBPII-based vaccine may be more effective in people with Fya+a+ compared to Fy b+b+ genotype (King et al., 2011). Other erythrocyte polymorphisms such as Southeast Asian ovalocytosis may also affect the acquisition of PvDBPII-blocking Abs under natural exposure (Rosanas-Urgell et al., 2012).

6.7. Role of T-Cell Immune Responses in Acquired Immunity Although an increasing number of studies have begun to look at T-cell responses to P. vivax antigens, there is no study that has correlated certain P. vivax-specific T-cell responses with NAI in longitudinal cohort studies. CD4+ T cells provide help to B cells and regulate inflammatory responses 104 Ivo Mueller et al. to acute malaria infections, e.g. by induction of natural and adaptive regulatory T cells (i.e. increased IL-10 production). T cells play important roles in parasite elimination by activation of natural killer (NK) cells and monocytes that facilitate parasite elimination by direct killing or phagocytosis of opsonised merozoites and IEs in the peripheral circulation and particularly the spleen. P. vivax-specific CD4+ T cells have been shown to activate NK cells and γδ T cells involved in innate and intermediate immune mechanisms that facilitate removal of malaria-infected RBCs (Artavanis-Tsakonas et al., 2003; D’Ombrain et al., 2008), possibly by granzyme B release from NK cells causing erythrocytosis (Bottger et al., 2012). Cytokines involved in NK activation include IL-12, IL-15, IL-18 and IL-2. Thus antigen-specific CD4+ memory T cells that secrete IL-2 (i.e. (T helper) Th1-type) can enhance NK cell activation along with pro-inflammatory mediators, i.e. molecules that engage Toll-like and innate receptors on monocytes to produce IL-12. NK cells themselves are potent producers of IFN-γ and tumour necrosis factor (TNF)-α that can further amplify NK-cell-mediated and macrophage-mediated parasite elimination. Recently, an expanded absolute number of IL-17- secreting CD4+ memory T cells have been associated with P. vivax infection (Bueno et al., 2012). This is another important effector CD4+ T cell subset that further amplifies pro-inflammatory cytokine networks and facilitates recruitment of neutrophils and monocytes. Overall, subjects with putative immunity to Pv have been shown to have increased frequencies of memory CD4+ T cells ( Jangpatarapongsa et al., 2006), suggesting a possible role in NAI; however, these were not assessed for their antigen specificity or pattern of cytokine secretion. This Pv antigen- specific effector CD4+ T-cell expansion appears to be reciprocally regulated by an increase of CD4+(FoxP3+) natural regulatory T cells to modulate the inflammatory response. Following an acute P. vivax infection there is expansion of CD4+(FoxP3+) natural regulatory T cells ( Jangpa- tarapongsa et al., 2008) whose numbers positively correlated with P. vivax parasite load (Bueno et al., 2010; Goncalves et al., 2010). Interestingly, acute P. vivax infections induce threefold higher serum levels of IL-10 compared to acute P. falciparum (Goncalves et al., 2010), consistent with the more pro-inflammatory nature of P. vivax (lower pyrogenic thresh- hold of Pv compared to Pf ) (Bueno et al., 2010). Overall, these studies would predict that P. vivax antigens that correlate with NAI will show an increased ratio of P. vivax antigen-specific Th1- and Th17-specific T cells relative to regulatory T cells. Natural Acquisition of Immunity to Plasmodium vivax 105

6.8. Immunological Memory and Duration of Clinical Immunity As noted above, NAI to Pf takes longer to acquire compared to Pv in highly endemic areas, requiring multiple and often persistent infections and when individuals move away from an endemic area their immunity to severe malaria appears to persist yet immunity to uncomplicated malaria wanes after several years in a non-malaria endemic area (Castelli et al., 1999; Jelinek et al., 2002; Matteelli et al., 1999; Struik and Riley, 2004). One interpretation of these data is that malaria induces only short- term memory. The observations mostly arise from studies in sub-Saharan Africa where malaria transmission is intense. This loss of NAI is often associated with the rapid decline or loss in blood-stage specific Abs (Lang- horne et al., 2008) that may result from memory B cell (MBC) anergy or exhaustion (Pierce, 2009). Blood-stage Pf may suppress generation and maintenance of malaria MBC and long-lived plasma cells by virtue of their high Ag loads that elicit systemic pro-inflammatory responses, e.g. increased TNF-α and IFN-γ (Hemmer et al., 1991; Karunaweera et al., 1992; Rudin et al., 1997), which are eventually downregulated (Weiss et al., 2010) (possibly explaining, in part, why many malaria infected children in endemic areas are asymptomatic). Whether this is also true for Pv is unknown. Highly immunogenic P. vivax-specific Abs such as AMA1 persist for years after known malaria infection (Braga et al., 1998; Bueno et al., 2009); however, Abs to less-immunogenic antigens such as the PvDBP can be transient (Ceravolo et al., 2005, 2008, 2009). Memory T and B cells to both P. vivax pre-erythrocytic stage antigens have also been shown to persist for years in areas of low transmission (e.g. Brazil, Thailand) (Bilsborough et al., 1993; Wipasa et al., 2010; Zevering et al., 1994) where individuals are frequently observed with asymptomatic parasitaemia (Alves et al., 2002; Branch et al., 2005; V inetz and Gilman, 2002) indicating development of NAI. Thus, it appears that long-term immunological memory frequently develops in areas of low P. vivax transmission associated with NAI. Whether the presence of Pv-specific Abs and T and B cells are associated with long-term protection against clinical P. vivax infections is unknown and difficult to assess because of infrequent P. vivax infection in low-transmissions settings. In areas of higher P. vivax transmission such as in PNG, the persistence of immunological memory and association of NAI has not yet been rigorously investigated. The recent interventions to reduce both Pf and Pv transmission in PNG afford the opportunity to examine this in detail. 106 Ivo Mueller et al.

7. IMMUNE RESPONSES TO MALARIA PRE-ERYTHROCYTIC STAGES In humans, malarial infection is initiated by the bite of an infected Anopheles mosquito that injects sporozoites into the host’s bloodstream. Although mosquitoes from highly endemic areas are estimated to carry up to 104 sporozoites in their salivary glands, it has been calculated that during a blood meal an infected mosquito inoculates a median number of only 15 parasites into the host (Rosenberg et al., 1990). After inoculation, the sporozoites move through dermal cells and enter into the bloodstream and within 1-hour’s time, they go in the liver and subsequently invade hepatocytes, where they differentiate into mature liver schizonts. However, it is uncertain as to how many of the infective forms actually reach the liver and successfully develop into mature schizonts, remain in the skin (Sidjanski and Vanderberg, 1997) or are removed by the lymph node via lymphatic circulation, where antigen-specific T-cell responses are initiated (Amino et al., 2008; Mota et al., 2001). It has been recently shown that malarial parasites successfully develop to schizonts in the skin (Guilbride et al., 2012; V oza et al., 2012), but it is not yet clear if this affects blood infection and malaria immunity (Hafalla et al., 2011). It has been demonstrated that hepatocyte invasion is mediated through a specific interaction between hepatocyte receptors and binding domains present in at least two sporozoite surface proteins (SSPs) (Frevert et al., 2008), the circumsporozoite (CS) protein (Cerami et al., 1992) and the SSP2/TRAP (Hedstrom et al., 1990; Sultan et al., 1997), thus initiating parasite entry into the liver cell. This interaction occurs through the formation of ionic bonds between the negatively charged chains of heparan sulphate proteoglycan molecules present on the basolateral domain of hepatocytes and the positively charged and hydrophobic residues of the region II-plus of the CS and SSP2/TRAP proteins (Frevert et al., 1993; Gantt et al., 1997; Sinnis et al., 1994). Once inside the cell, the parasite undergoes asexual division or exoerythrocytic schizogony. Maturation from malaria trophozoites to schizonts and subsequent merozoite release is a process that can take as little as 2 days in rodent malarias to as long as 5–8 days in human malaria species. Plasmo- dium vivax schizonts each produce approximately 10,000 daughter parasites with P. falciparum resulting in an estimated 40,000 merozoites per liver schizont (Wernsdorfer and McGregor, 1988). In P. vivax and Plasmodium ovale, Natural Acquisition of Immunity to Plasmodium vivax 107 liver parasites may transform into hypnozoites, a latent form that can persist in the liver for several years after initial infection, thus producing relapsing malaria episodes. Hypnozoites were first described in 1985 by Kroto- ski (Krotoski, 1985), who revealed the presence of uninucleated bodies of 4.5–6.6 nm inside liver parenchyma cells. The periodicity of the relapses appears to depend on the geographic origin of the parasite strain; parasites from tropical areas emerge more quickly than those from temperate zones (Shute et al., 1976). At the pre-erythrocytic stages, i.e. sporozoite and liver stages, immune responses against Plasmodium parasites are mediated by both humoral and cellular mechanisms (Fig. 3.3). Abs play a central role in protection through different mechanisms including blockage of sporozoite invasion of liver cells by specific neutralising Abs that induce what is known as the ‘circumsporozoite precipitation (CSP) reaction’ (Vanderberg et al., 1969). This reaction has been consistently shown to be induced by Abs specific to the CS protein which is abundantly expressed on the sporozoite surface. Abs are also able to induce opsonisation (Schwenk et al., 2003) and Ab-dependent T-cell-mediated cytotoxicity (Mazier et al., 1990), as well as inhibition of intra-hepatic development (Hollingdale et al., 1984; Mellouk et al., 1990). Cell-mediated mechanisms appear to be particularly important for malaria pre-erythrocytic immunity. First, innate mechanisms involve both monocytes/macrophages, dendritic cells and polymorphonuclear leu- kocytes that have the capacity to eliminate pre-erythrocytic parasites by phagocytosis (Ferrante et al., 1990; Hafalla et al., 2011). As mentioned above, it has been demonstrated that macrophages in the presence of hyperimmune sera can efficiently phagocytise and eliminate sporozoites (Danforth et al., 1980). In addition, infiltration of cells such as macrophages and neutrophils is noted around intra-hepatic parasite forms (Faure et al., 1995; Khan and Vanderberg, 1992). However, the protective role of Küpffer cells (KC), which are liver-specific tissue macrophages, is as yet not clear (Hafalla et al., 2011). Some ino viv studies suggest that these cells contribute to the sporozoite invasion process (Baer et al., 2007; Meis et al., 1982), may also modulate cytokine profile and induce apoptosis in KC (Klotz and Frevert, 2008), while other studies indicate that ino viv depletion of KC increases the number of intra-hepatic forms (Vreden et al., 1993). Second, cytolytic activity mediated by antigen-specific CD4+ and CD8+ T cells is believed to play a prominent role in liver-stage malaria defence mechanisms. Peripheral blood lymphocytes of human immune donors from endemic areas recognise multiple Th (Tse, Radtke et al. 2011) and cytotoxic lymphocyte 108 Ivo Mueller et al.

Figure 3.3 Immune response against malaria pre-erythrocytic stages (sporozoites and liver stages). DC, dendritic cells; Th, CD4+ T-helper cells; Tc, CD8+ T-cytotoxic cells; MHC, major histompatibility complex; NK, natural killer cells; IFN-γ, Interferon-gamma; NO, nitric oxide; ROI, reactive oxygen intermediates. (For colour version of this figure, the reader is referred to the online version of this book). Natural Acquisition of Immunity to Plasmodium vivax 109

(CTL) epitopes both in sporozoite and liver-stage antigens in a genetically restricted manner (Aidoo et al., 1995; Malik et al., 1991; Nardin and Nussenzweig, 1993). The role of these two lymphocyte subsets has been actively studied particularly in the rodent system (Del Giudice et al., 1986; Nardin and Nussenzweig, 1993; Romero et al., 1989; Weiss et al., 1988). Because of technical limitations, the protective role of T-cells (Th and CTL) in humans has been more difficult to document unequivocally. In rodents, it has been shown that in vivo depletion of CD8+ T lymphocytes completely abolishes malaria immunity (Schofield et al., 1987; Weiss et al., 1988). In addition, passive transfer of cytotoxic T-cell clones recognising the Plasmodium berghei CS protein completely protects mice against sporozoite challenge (Romero et al., 1989). Further- more, murine CD8+ T cells have been shown to contribute to protective immune mechanisms against malaria liver stages (Overstreet et al., 2008; Renia, 2008; Rodrigues et al., 1991; Weiss et al., 1988). Likewise, passive transfer and cell subset depletion studies have indicated the protective role of CD4+ T cells clones in rodents. Passive transfer of cytolytic CD4+ T cell clones has shown to induce protection in a parasite-stage-specific manner (Tsuji et al., 1990) regardless of the Th1 or Th2 phenotype (Renia et al., 1993; Takita-Sonoda et al., 1996). Moreover, a number of soluble immune mediators have been shown to be capable of blocking liver schizogony, e.g. cytokines IL-12 (Sedegah et al., 1994), IFN-γ (Ferreira et al., 1986; Mellouk et al., 1987; Schofield et al., 1987; Tsuji et al., 1995), TNF-α (Korner et al., 2010; Nussler et al., 1991), IL-1 (Mellouk et al., 1987) and IL-6 (Nussler et al., 1991; Pied et al., 1991). Additionally, reac- − tive oxygen and nitrogen intermediates, O2 (Allison and Eugui, 1983) and nitric oxide (Mellouk et al., 1994; Nussler et al., 1991, 1993; Seguin et al., 1994), respectively, significantly contribute to protection. Other proteins described as participating in the immune responses are hemopexin, α1-anti-trypsin, α2-macroglobulin (Pied et al., 1995) and C-reactive protein (Pied et al., 1989). Several studies have indicated an important protective role for IFN-γ in both human and non-human primates, either as a critical immune mediator or as a valuable surrogate marker of protection (Arevalo-Herrera et al., 2010, 2011; Doolan and Martinez-Alier, 2006; Herrera et al., 2007; John et al., 2004; Jordan-Villegas et al., 2011; Perlaza et al., 2008, 2011). 110 Ivo Mueller et al.

In humans, most work has involved P. falciparum where CD4+ T cells have been shown to be correlated with protection from natural infection and disease in West Africa (Reece et al., 2004). CS-specific cytolytic CD4+ T cells derived from individuals vaccinated with irradiated sporozoites (Moreno et al., 1991) or PfCS peptides (Calvo-Calle et al., 2005) and CD8+ T cells appear to contribute significantly to protection at the pre-erythrocytic level (Bettiol et al., 2010; Doolan and Martinez-Alier, 2006; Tsuji and Zavala, 2003). In P. falciparum, several CD4+ and CD8+ T-cell epitopes have been identified in pre-erythrocytic antigens, i.e. CS, LSA-1, LSA-3, TRAP, using classical techniques (Cockburn et al., 2010; Joshi et al., 2000; Over- street et al., 2008). Furthermore, immunisation of both Aotus monkeys and chimpanzees with the P. falciparum LSA-3 liver-stage antigen formulated in different adjuvants has been shown to induce significant IFN-? production in protected as compared to non-protected monkeys (Perlaza et al., 2008). The absence of P. vivax parasite cultivation methods has limited studies on this Plasmodium species. This is reflected in the number of P. vivax parasite antigens identified thus far. Blood samples of individuals from malaria- endemic areas or from volunteers participating in P. vivax vaccine studies in Colombia have been used to characterize immune responses to naturally and artificially acquired P. vivax CS (Arevalo-Herrera et al., 2002; Fleischhauer et al., 1999; Herrera et al., 1994). Studies have shown that similar CS epitopes are recognized by individuals from endemic areas and by non-human primates and human volunteers immunised with long synthetic peptides (Arevalo-Herrera et al., 2011; Herrera et al., 2005). Volunteers participating in phase I vaccine clinical trials mounted significant CD4+ and CD8+ T cell responses with high levels of IFN-? upon vaccination (Herrera et al., 2007, 2011), together with production of Abs capable of efficiently blocking parasite invasion in vitro. A fragment of another pre-erythrocytic antigen PvTRAP (amino acids 209–256) containing the region II motif involved in parasite binding to hepatocytes has been shown to induce partial protection of Aotus primates against experimental P. vivax sporozoite challenge (Castel- lanos et al., 2007). Although not considered pre-erythrocytic per se, MSP-1 and possibly other malaria antigens expressed in hepatic schizonts are likely to be involved in pre-erythrocytic immunity (Suhrbier et al., 1989). In recent years with the advent of genomic and proteomic technologies, significant progress has been achieved in the discovery of human parasite antigens associated with the induction of immune responses to target T- and B-cell epitopes of malaria antigens (Doolan, 2011). Using sera from naturally and experimentally malaria-exposed individuals, 5% of the P. falciparum Natural Acquisition of Immunity to Plasmodium vivax 111 genome was screened using protein microarrays (Doolan et al., 2008). A similar strategy has been used to analyse P. vivax gene sequences (Dharia et al., 2010). In conclusion, a number of studies have suggested the importance of the induction of protective Ab- and T-cell-specific responses against the pre- erythrocytic forms of malaria parasite. In terms of the prevention and/or reduction of the clinical manifestation of infection, both Abs and cytokines, particularly IFN-γ, play a critical role in protection. Nonetheless, more studies are warranted for better understanding of the immune mechanisms involved in protection against malaria. Development and use of additional innovative genomic approaches to vaccine design are also needed.

8. SEXUAL STAGE PARASITES AND TRANSMISSION- BLOCKING IMMUNITY The target stages of the TBI are the sexual stages of the Plasmodium life cycle. The sexual stage is initiated in the vertebrate host bloodstream as male and female gametocytes. After the ingestion of the infected blood by Anopheles mosquito, male and female gametes emerge and fertilise to form zygotes in the mosquito midgut. The zygotes transform into motile ookinetes, which traverse the epithelium of the mosquito midgut to further develop into oocysts (Tsuboi et al., 2003). TBI has initially been reported with sexual stage parasites in non-human malaria models (Carter and Chen, 1976; Gwadz, 1976; Gwadz and Green, 1978; Mendis and Targett, 1979), and is mainly mediated by Abs that prevent fertilisation and destroy the gametes and newly fertilised zygotes by acting against the surface proteins of the sexual stage parasites within the midgut of a blood-fed mosquito. After the ingestion of the blood, Abs against the gametes act to prevent their fertilisation and formation of ookinetes (Carter, 2001). Individuals in malaria endemic areas that have been repeatedly exposed to the parasite infection acquire specific immune responses against blood- stage parasite capable of controlling parasite burden and the clinical symptoms. In addition, sera from individuals in malaria endemic areas are able to block malaria transmission (Arevalo-Herrera et al., 2005; Gamage-Mendis et al., 1992; Mendis et al., 1987). While ingesting the blood meal from the host, the mosquito also ingests host Abs specific to sexual stage antigens. These Abs interfere with the development of the parasite life cycle in the mosquito midgut (Peiris et al., 1988). 112 Ivo Mueller et al.

8.1. Evidences of Naturally Acquired TBI of P. falciparum TBI appears to depend on the malaria transmission intensity, suggesting that the generation of TBI requires prolonged exposure to multiple malaria inoculations (Bousema et al., 2010; Premawansa et al., 1994). TBI also correlates with the period of exposure to gametocytes and the number of gametocytes developed during the infections; sera of individuals with high Ab concentrations are more efficient at blocking parasite transmission to the mosquito (Boudin et al., 2004; Bousema et al., 2011). Sera of humans exposed to P. falciparum contain IgG Abs that recognise sexual-stage antigens on the surface of gametocytes and gametes. Pfs230 and Pfs48/45 were identified as antigens responsible for the TBI (Carter, 2001; Healer et al., 1999; Roeffen et al., 1994). The levels of Abs against Pfs230 and Pfs48/45 could be boosted by exposure to gametocytes in further infections (Bousema et al., 2010). Recently, Sutherland has summarised the efforts to identify novel sexual-stage antigens conferring TBI against P. falciparum (Sutherland, 2009).

8.2. Evidences of Naturally Acquired TBI of P. vivax There is a big difference in the timing of the gametocyte appearance between P. falciparum and P. vivax. In the case of P. falciparum, gametocytes appear a few weeks after the onset of febrile illness. In contrast, gametocytes appear almost at the same time as the asexual parasitaemia in P. vivax (Bousema and Drakeley, 2011). Although previous studies on malaria TBI have mostly focused on P. falciparum and have been carried out in Africa, so far, only a small number of studies have been taken up to understand the TBI against P. vivax in both Asia and Latin America. Pioneering work conducted on P. vivax in Sri Lanka has demonstrated the development of TBI as a result of naturally acquired malaria infection. The sera of approximately 48% of patients significantly suppressed gametocyte infectivity (Gamage-Mendis et al., 1992; Peiris et al., 1988). The efforts (Premawansa et al., 1990; Snewin et al., 1995) hitherto to identify the definitive target antigens that can elicit TBI against P. vivax by production of anti-gamete mAbs and screening genomic expression library of P. vivax by the TB mAbs were unsuccessful. There are only a few studies in Thailand that suggest that serum factors play a key role in the ability of P. vivax gametocytes to infect mosquitoes. Sattabongkot et al. (2003) compared the infectivity of P. vivax-infected blood containing the patient’s own sera with that of blood in which the Natural Acquisition of Immunity to Plasmodium vivax 113 patients’ sera had been replaced with malaria-naïve sera. They found that the patients’ own sera significantly reduced mosquito infection rates as well as oocyst density. Although they did not specifically identify host factors that were responsible for reductions in mosquito infectivity, it seems likely that TB Abs may cause a reduction in infectivity. All patients evaluated in this study were symptomatic adults, suggesting that TB Ab levels may have been relatively low. In addition, Coleman et al. (2004) reported that the blood obtained from individuals in a highly endemic village in western Thailand exhibited reduced infection in mosquitoes. Naturally acquired TBI to P. vivax was also studied in patients from the southern coast of Mexico. Anopheles albimanus mosquitoes were fed with patients’ IEs in the presence of autologous or malaria-naïve serum. Patients from both primary and secondary infection had TBI, although the quality and quantity of the blocking activity was significantly higher in the secondary infection groups (Ramsey et al., 1996). P. vivax TB activity was also assessed in sera from acutely infected patients from a malaria-endemic area in Colombia. The reduction in the number of oocysts in An. albimanus mosquitoes artificially fed with blood from these patients were measured in comparison with parasitised erythrocytes mixed with malaria-naïve serum as negative control. One-third (36.4%) showed full TB activity (≥90% inhibition) when mixed with autologous sera and 29.6% showed partial activity (50–89%). The TB activity correlated with Ab titre by an immunofluorescent Ab test and decreased with the serial dilution of the sera (Arevalo-Herrera et al., 2005).

8.3. Towards the Discovery of the Target Molecules against TBI of P. vivax Although a few definitive antigens that elicit TBI against P. falciparum have been identified (Bousema et al., 2010, 2011), candidate antigens that induce TBI against P. vivax have not been identified yet (Tsuboi et al., 2003). Recently, we have established an efficient recombinant malaria protein expression system using the wheat germ cell-free system (Tsuboi et al., 2008, 2010a, 2010b). Using this protein expression system, we were able to express 89 blood-stage proteins of P. vivax as a protein array, screen them by the human immune sera and successfully identify novel blood-stage antigens (Chen et al., 2010). Similarly, in the near future, we will express gametocyte-stage proteins of P. vivax as a protein array to screen them by the serum samples having TBI, to discover novel TB vaccine candidates against P. vivax. 114 Ivo Mueller et al.

9. CONCLUSIONS Although we have only just begun to understand some of the major processes involved in NAI to P. vivax, the results to date support the premise that developing a multistage P. vivax vaccine may be feasible and is worth pursuing. The more rapid development of NAI to P. vivax even indicates that a highly efficacious P. vivax vaccine may be easier to achieve that a vaccine to P. falciparum. This might be especially true if there are fewer redundant pathways for erythrocyte invasion. A pre-erythrocytic-stage vaccine may be of particular value because each infective mosquito bite has the potential to generate multiple blood-stage infections because some sporozoites became latent. An ideal P. vivax vaccine should target not only blood stage but also pre-erythrocitic and transmission stages. While the rationale for continued P. vivax vaccine development efforts is strong, a lot of work remains to identify the optimal combination of antigens to be included in such a vaccine. While the lack of continuous long-term P. vivax culture greatly complicates both the screening and development of novel vaccine candidate, important insights can be gained by conducting further in-depth studies of the acquisition of immunity in naturally exposed population that combine appropriate epidemiological design, state-of-the-art immunology and novel genomic and proteomic technologies. In addition, a more thorough use of the existing non-human primate models as well as experimental infection in human volunteers will be essential for advancing our understanding of the basic processes and specific antigens involved in the establishment of NAI to P. vivax.

10. FUTURE DIRECTIONS

1. Compare Ab responses from putatively immune and susceptible individuals using protein arrays representing Pv blood-stage antigens in longitudinal cohort studies with the aim to identify new targets of blood-stage immune responses. 2. Better characterize surface-expressed proteins on Pv-IEs and relate how these proteins are involved in stimulating NAI, and, potentially, immune evasion strategies. 3. Study the role of host cellular immunity and protection against blood- stage Pv infection, including the role of opsonizsng Abs and the spleen. Natural Acquisition of Immunity to Plasmodium vivax 115

4. Continue to define the biology of molecules involved in the invasion of and release from erythrocytes. 5. Identify the conserved binding motifs in critical Pv invasion molecules such as RBPs and DBPs as potential blood-stage antigens. 6. Determine the interaction of erythrocyte polymorphisms related to susceptibility to Pv infection (e.g. Duffy antigen polymorphisms or South- east Asian ovalocytosis) and how these modify development of NAI. 7. Characterize the pre-erythrocytic-stage antigens and immune responses in humans associated with protection following immunisation with irradiated sporozoites. 8. To further identify antigens associated with protection against TB NAI, especially molecules expressed on gametocytes in the peripheral circulation. Immunisation with such molecules would facilitate natural boosting by blood-stage infections to sustain high Ab levels required for TBI.

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G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

Rosalind E. Howes*,1, Katherine E. Battle*, Ari W. Satyagraha†, J. Kevin Baird‡,§, Simon I. Hay* *Department of Zoology, University of Oxford, Oxford, UK †Eijkman Institute for Molecular Biology, Jakarta, Indonesia ‡Eijkman-Oxford Clinical Research Unit, Jakarta, Indonesia §Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK 1Corresponding author: E-mail: [email protected]

Contents 1. Introduction 135 2. Historical Overview 136 2.1. Favism 136 2.2. The Path to Primaquine 137 2.3. Primaquine Tolerability and Safety 140 3. Glucose-6-Phosphate Dehydrogenase Deficiency: The Enzyme and Its Gene 143 3.1. G6PD Genetics and Inheritance 143 3.2. The G6PD Enzyme 146 3.3. The Pentose Phosphate Pathway as an Anti-Oxidative Defence 147 3.4. Clinical Manifestations of G6PD Deficiency 148 4. Diagnosing G6PD Deficiency 150 4.1. Phenotypic Diagnostic Tests 150 4.2. Molecular Diagnostic Tests 152 4.3. The Case for a New Diagnostic for Safe P. vivax Radical Cure 153 5. Mapping the Spatial Distribution of G6PD Deficiency 155 5.1. G6PD Deficiency Prevalence Mapping 155 5.1.1. Generating a Map: the Evidence-Base 156 5.1.2. Generating a Map: the G6PD Mapping Model 156 5.1.3. G6PD Deficiency Prevalence Map: an Overview 157 5.2. G6PD Deficient Population Estimates 161 5.3. G6PD Deficiency Mutation Mapping 163 6. Spatial Co-occurrence of G6PD Deficiency with P. vivax Endemicity 164 6.1. G6PD Deficiency in Asia 166 6.2. G6PD Deficiency in Asia-Pacific 167 6.3. G6PD Deficiency in the Americas 168 6.4. G6PD Deficiency in Africa, Yemen and Saudi Arabia (Africa+) 169

7. Evolutionary Drivers of the Distribution of G6PD Deficiency 170 7.1. Evidence of a Selective Advantage 171 7.1.1. Epidemiological Evidence 171 7.1.2. In vitro Evidence 171 7.1.3. Case-Control In vivo Evidence 172 7.2. Neglect of the Selective Role of P. vivax as a Driver of G6PD Deficiency 173 8. Primaquine, P. vivax and G6PD Deficiency 174 8.1. Mechanism of Primaquine-Induced Haemolysis 175 8.1.1. Primaquine and its Metabolites 175 8.1.2. A Role for Oxidative Stress 176 8.1.3. A Role for Methaemoglobin 177 8.1.4. A Role for Altered Redox Equilibrium 177 8.1.5. Significance of Primaquine-Induced Haemolysis 178 8.2. Factors Affecting Haemolytic Risk 179 8.2.1. Dose Dependency 179 8.2.2. Variant Dependency 180 8.2.3. Red Blood Cell Age Dependency 183 8.2.4. Sex Dependency 184 8.3. Predicting Haemolytic Risk 184 9. Towards a Risk Framework for P. vivax Relapse Treatment 185 9.1. Assessing National-Level Haemolytic Risk of Primaquine Therapy 185 9.1.1. Proposed Framework for Ranking National-Level Risk from G6PD Deficiency 185 9.1.2. Important Limitations to Predicting National-Level Haemolytic Risk 186 9.2. Assessing Haemolytic Risk at the Level of the Individual 187 10. Conclusions 190 Acknowledgements 191 References 192 Abstract Glucose-6-phosphate dehydrogenase (G6PD) is a potentially pathogenic inherited enzyme abnormality and, similar to other human red blood cell polymorphisms, is particularly prevalent in historically malaria endemic countries. The spatial extent of Plasmodium vivax malaria overlaps widely with that of G6PD deficiency; unfortunately the only drug licensed for the radical cure and relapse prevention of P. vivax, primaquine, can trigger severe haemolytic anaemia in G6PD deficient individuals. This chapter reviews the past and current data on this unique pharmacogenetic association, which is becoming increasingly important as several nations now consider strate- gieso t eliminate malaria transmission rather than control its clinical burden. G6PD deficiency is a highly variable disorder, in terms of spatial heterogeneity in prevalence and molecular variants, as well as its interactions with P. vivax and primaquine. Consideration of factors including aspects of basic physiology, diagnosis, and clinical triggers of primaquine-induced haemolysis is required to assess the risks and benefits of applying primaquine in various geographic and demographic settings. Given that haemolytically toxic antirelapse drugs will likely be the only therapeutic options for the coming decade, it is clear that we need to understand in depth G6PD deficiency and G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 135

primaquine-induced haemolysis to determine safe and effective therapeutic strategies to overcome this hurdle and achieve malaria elimination.

1. INTRODUCTION Glucose-6-phosphate dehydrogenase (G6PD) is a ubiquitously expre ssed enzyme that has a housekeeping role in all cells, and is particularly critical to the integrity and functioning of red blood cells (RBCs). The G6PD gene has many mutant alleles which entail a decrease in enzyme activity, expressing the G6PD deficient phenotype. This trait is widespread in many human populations in whom several of the underlying mutant alleles are present at variable polymorphic frequencies. G6PD deficiency selectively affects RBCs for two reasons. First, most known mutations cause a decreased stability of the enzyme, and since these cells do not have the ability to synthesise proteins, the enzyme level decreases as cells age during their 120 days lifespan in circulation. Second, RBCs are exquisitely susceptible to oxidative stress from exogenous oxidizing agents in the blood as well as the oxygen radicals continuously generated as haemoglobin cycles between its deoxygenated and oxygenated forms. When G6PD activity is deficient, they have a diminished ability to withstand stress, and therefore risk destruction (haemolysis). Fortunately, the large majority of G6PD deficient subjects have no clinical manifestations and the condition remains asymptomatic until they are exposed to a haemolytic trigger. For centuries, the most common known trigger of haemolysis has been fava beans, and favism remains a public health problem in areas where these are a common food item and G6PD deficiency is prevalent. However, a haemolysing trigger of great contemporary public health significance is the antimalarial primaquine, a key drug for malaria control as the only licenced treatment against (i) the relapsing liver stages of Plasmodium vivax – hypnozoites – which become dormant in infected hepatocytes and subsequently reactivate blood-stage infections, and (ii) the sexual blood stages of all species of Plasmodium. Since its introduction, primaquine has emerged as a major drug trigger of haemolysis in G6PD deficient individuals, making this a paradigm of pharmacogenetics. This chapter focuses on the use of primaquine as a hypnozoitocide in P. vivax malaria. The complex problem of its use as a gametocytocide in Plasmodium falciparum malaria is not further considered here: detailed reviews of the effectiveness and safety of single-dose transmission-blocking primaquine have recently been carried out for the WHO Primaquine Evidence Review group (Recht et al., 2012) and in a Cochrane Review (Graves et al., 2012). 136 R. E. Howes et al.

The widespread prevalence of G6PD deficiency across populations in malaria endemic areas has hindered the use of this drug, despite its uniquely useful range of therapeutic properties. One aim of studying G6PD deficiency is to increase access to primaquine. Optimising primaquine use involves delivery of the drug in such a way as to maintain its therapeutic activity against parasites whilst reducing risk for G6PD deficient individuals. Here, we review the knowledge base and interactions between the different component parts of this relationship (the human G6PD gene, the parasite and the drug). All of these elements must be considered when weighing the risks and benefits of putting this valuable drug to work. We examine these factors in the historical context of their development – the discovery of G6PD deficiency having been tied to the early research into primaquine, and consider the important knowledge gaps which remain despite six decades of continuous use of this drug. Extensive work was conducted in the mid- twentieth century to improve the chemotherapeutic options for treating P. vivax, particularly against its relapsing form. This chapter opens by revisit- ing those early experiments, asserting why G6PD deficiency seems to be an unavoidably major hurdle in hypnozoitocidal therapeutics. We examine what is known about G6PD – the enzyme, its mutations and population genetics. We then consider how this problem is being or could be managed in the context of contemporary targets for malaria elimination. This leads us to suggest steps to be taken to move forward into a new era when G6PD deficiency no longer seriously impedes treatment of P. vivax infection in individual patients and when primaquine can be used to its full potential as an essential tool for eliminating reservoirs of P. vivax.

2. HISTORICAL OVERVIEW 2.1. Favism Awareness of the symptoms associated with G6PD deficiency was well established long before the underlying mechanisms were understood (Beutler, 2008). The earliest suspected reports of G6PD deficiency are from Pythagoras forbidding his students to eat fava beans (Vicia faba). His strong aversion to these commonly eaten beans must mean that favism had already been recognised as a dangerous disease; and since G6PD deficiency is common in Greece, it is possible that he or some of his followers may have suffered from favism, the haemolytic condition triggered by ingesting these beans (Simoons, 1998). In more recent times there has been a vast literature on favism (Fermi and Martinetti, 1905; Luisada, 1940; Meloni et al., 1983). Fava G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 137 beans are unique among other beans because they contain high concentrations of two glucosides, vicine and divicine; and their respective aglycones, convicine and isouramil, are powerful triggers of oxidative stress that causes the characteristic haemolytic attacks (Chevion et al., 1982; Luzzatto, 2009).

2.2. The Path to Primaquine Although methylene blue was the first synthetic compound used to successfully treat acute malaria, it was never developed or distributed. The first such drug, structurally derived from methylene blue, was the 8-aminoquinoline pamaquine (Fig. 4.1) which became widely used, and quickly feared. While Mühlens (1926) was correct in asserting in 1926 that his laboratory’s production of pamaquine (marketed as Plasmochin) was of “huge importance” and would have “immeasurable effect on malarial countries” (trans.), his clinical trials (conducted among malaria-infected individuals of European origin) did not support his assurances about its safety. The only side-effects reported in his initial paper were some cases of cyanosis in lips, gums, tongues and fingernails, which he did not consider prohibitive to the drug’s widespread use (in retrospect, this was probably cyanosis caused by methaemoglobinaemia). At least 250 case reports of toxic reactions to pamaquine were subsequently published; these were cases of acute haemolytic anaemia (AHA), some resulting in death (Beutler, 1959; Hardgrove and Applebaum, 1946). In 1938, the League of Nations recommended against use of this drug. In 1941, no synthetic drug effectively competed with quinine for the treatment of malaria, and quinine could not protect against relapsing malaria. The Dutch operated a global cartel on the trade of quinine, with 95% of production coming from cinchona tree plantations on Java in the

Figure 4.1 Chemical structure of key 8-aminoquinolines: pamaquine, primaquine and tafenoquine. 138 R. E. Howes et al.

Netherlands East Indies. The fall of those holdings to the Imperial Japa- nese armed forces in early 1942 forced the Allies to use the few inferior synthetic drugs available, principally atabrine (also called mepacrine or quinacrine) and pamaquine (Elyazar et al., 2011). The embattled Ameri- cans holding out on the Bataan peninsula and Corregidor Island near Manila ( January to April 1942) suffered terribly from malaria. Condon- Rall (1992) encapsulated the significance of this, stating that the medical disaster that developed among the US troops in the Philippines was “a symptom as much as a cause of the American general military defeat”. In the region of New Guinea, 1598 American soldiers died of wounds sustained in battle, whereas 6292 perished with a diagnosis of malaria ( Joy, 1999). Similar figures were reported among Australian forces, who suffered 21,600 malaria casualties during the same campaigns. At Guadalcanal in the Solo- mon Islands during 1942, the US Army Americal division suffered malaria attack rates of 1.3/person–year (despite atabrine prophylaxis). More tell- ing, however, was what happened to this division when they were evacu- ated to nonmalarious Fiji for rest and recuperation: the malaria attack rate was 3.7/person–year, virtually all of it relapses of P. vivax (Downs et al., 1947). The US Navy estimated that 79% of the 113,774 recorded cases of malaria were relapses ( Joy, 1999). Despite the great demand for antirelapse therapy, in 1943 the US Sur- geon General withdrew pamaquine for prevention of relapses (Office of the Surgeon General, 1943) due to its toxicity, which was highly significant in some individuals. Acute haemolytic attacks could be triggered by the use of daily pamaquine dosing (30 mg1), with associated jaundice, dark urine and weakness due to severe anaemia (Earle et al., 1948). Furthermore, when used with atabrine, its plasma levels increased 10-fold causing serious toxicity problems (Baird, 2011). This unexpected drug–drug interaction effectively removed the only therapeutic option to the serious threat of malaria relapse. The US government responded to this problem by launching one of the largest biomedical research endeavours up to that time. Created in 1943, the Board for the Coordination of Malaria Studies oversaw basic and clinical research on over 14,000 compounds for antimalarial activity (Condon-Rall, 1994). Beginning in 1944, academic clinical investigators and US armed forces clinicians set up the capacity to study induced malaria in volunteers at a number of prisons in the United States (Coatney et al., 1948), and the penitentiary at Stateville, Illinois specialised in the

1 All doses in this review are subscribed for adults weighing 40–70 kg, mean of 60 kg. G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 139 evaluation of therapies against relapse (Comfort, 2009). They leveraged a strain of P. vivax taken from an American soldier infected in New Guinea in 1944, called the Chesson strain (Alving et al., 1948; Craige et al., 1947). In the context of the clinical trials, this strain offered the advantage of relatively frequent, rapid, and multiple relapses compared with Korean or North American strains, though also required higher drug dosages to achieve wholly efficacious radical cure. A Stateville Penitentiary study protocol from 1948 states that approximately 500 volunteers were involved (Alving et al., 1948) although a recent review of the overall project suggested that thousands of inmates were ultimately inoculated and included in the experiments (Comfort, 2009). The very modern and controlled prison environment provided a con- venient setting for running multiple complex protocols involving many different compounds (Alving et al., 1948), especially 8-aminoquinolines (Craige et al., 1948; Earle et al., 1948; Jones et al., 1948). The stage was thus set for careful clinical observation of the AHA induced by this class of compounds. The vetting of 24 candidate 8-aminoquinolines, a family of drugs whose antirelapse efficacy had been demonstrated by pamaquine, for safety, tolerability, and efficacy in subjects not sensitive to haemolysis had been completed by about 1949. Subsequently, the best candidate, primaquine (Edgcomb et al., 1950; Elderfield et al., 1955), was widely used in American soldiers in the Korean War (1950–1953). It is thus important to understand that the 8-aminoquinolines were not evaluated for optimum safety in G6PD deficient subjects. Instead, the US Army programme later strived to evaluate safety of primaquine alone in vulnerable subjects. Studies were conducted with pamaquine to investigate the predisposing conditions which made some individuals particularly vulnerable to haemolysis. These suggested that pamaquine sensitivity was racially correlated, being more common in subjects of African origin (6 of 76) than Caucasians (1 of 87) (Earle et al., 1948). They also found that haemolysis was unlikely to be associated with the plasma pamaquine levels. These observations led investigators to suspect a “predisposing factor” in certain individuals, triggered by the drug acting as a “precipitating factor” (Earle et al., 1948); although they found race to be the most significant predictor of haemolysis when considered alongside a range of haematological and exogenous factors, its genetic basis remained only a possibility. Indeed, it required 10 more years of investigation to zero in on that cause. Carson et al. (1956), working at the Stateville Penitentiary, described G6PD deficiency as the basis of “primaquine sensitivity” in 1956. 140 R. E. Howes et al.

The newly qualified clinician, Ernest Beutler (1928–2008), went to Stat- eville and participated in the ground-breaking work characterising G6PD deficiency. The remainder of his prolific and distinguished professional life substantially advanced understanding of this important disorder (Beutler, 2009). A Chicago University database archives approximately 150 publications arising from the Stateville Penitentiary Malaria Treatment Trials (http:// www.lib.uchicago.edu/e/collections/sci/malaria.html). In this next section, we review those studies relating to G6PD deficiency and tolerance to effective primaquine dosing. While these studies have been held as a prime example of unethical human experimentation (Harcourt, 2011), their legacy still forms the foundation of P. vivax radical cure today.

2.3. Primaquine Tolerability and Safety The early experimental clinical studies in nondeficient individuals established that a total primaquine dose of approximately 200 mg was needed to achieve P. vivax radical cure (Alving et al., 1953; Coatney et al., 1953; Edgcomb et al., 1950). The next step was establishing an efficacious dosing regimen with acceptable safety profiles. Most et al. (1946) described a 14-day regimen of quinine and pamaquine very early in the 8- aminoquinoline clinical development programme, which they considered to be the optimum compromise between safety (3- to 7-day dosing came with high risk of intolerability or toxicity) and practicality (dosing up to 21 days risked poor compliance). The 14 days of daily 15 mg dosing was subsequently applied to all candidate 8-aminoquinolines for the simple reason that their therapeutic indexes were essentially similar to pamaquine. The course of haemolysis in G6PD deficient subjects, with the onset of symptoms after the third dose, permitted withdrawal of the treatment after relatively little exposure to the drug. Higher daily doses over a shorter duration, although equally efficacious, were considered too risky for use in unscreened patients. The 14-day regimen emerged before G6PD deficiency was known as the basis of the 8-aminoquinolines’ most severe toxicity. The compromise thus effectively included unscreened G6PD deficient patients. Indeed, the US Army would not screen its troops for G6PD deficiency until 2005. They considered the recommended dosing regimens not threatening, largely because withdrawal of therapy after low exposures to drug was at least possible, and even 14 days of treatment did not appear seriously harmful among the African American troops exposed to that dosage. G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 141

Primaquine sensitivity was nonetheless a major concern during the development of primaquine (Editorial, 1952, 1955) (though did not preclude its licencing by the US Food and Drug Association [FDA] in 1952) and extensive studies were undertaken at Stateville to understand this problem. Prima- quine toxicity was investigated among both African American (Hockwald et al., 1952) and Caucasian–American prisoner volunteers (Clayman et al., 1952), comparing the toxicity of primaquine with pamaquine, and assessing the influence of co-administration with quinine. Follow-on experiments were carried out on sensitive individuals who had undergone haemolysis to investigate the severity and toxicity of multiple-drug treatments and regimens on the same individuals. Although all cases of severe haemolysis recovered without transfusion after cessation of treatment, the higher dose of 30 mg primaquine daily was deemed too dangerous for administration without close supervision: of 110 African American volunteers given 30 mg primaquine daily over 14 days, five developed severe anaemia with severity comparable with that following equivalent dosage of pamaquine, and there were 17 cases of mild anaemia. Reducing the schedule to 15 mg daily doses did not trigger any cases of severe anaemia, thus this was deemed a safe daily dose; nevertheless, 12 patients still developed mild anaemia. The relatively safe 15 mg dose among this population was corroborated by other large- scale studies (Alving et al., 1952; Hockwald et al., 1952). Parallel toxicity studies were conducted among Caucasian volunteers. As would be expected, abdominal complaints among others were also reported for high-dose regimens (60, 120, 240 mg daily); no symptoms were reported with 15 mg daily regimens (n = 699 Caucasian volunteers at Stateville Penitentiary), and only mild side-effects noted with the 30 mg dose. Severe haemolysis was not encountered among this group of patients, even at doses as high as 240 mg daily – this clearly contrasts with the threshold of haemolytic susceptibility in the African American volunteers. Haemolysis in primaquine sensitive African American subjects was noticed to be self-limiting (Dern et al., 1954a). Radiochromium labelling (51Cr) used to mark sensitive and nonsensitive RBCs showed that cells maintained the same haemolytic predisposition to risk regardless of the status of the host they were transfused into (Dern et al., 1954b). Time-series data then characterised the course of the haemolysis and found a self-limiting pattern: in spite of continued drug administration throughout the initial acute haemolysis (which lasted about a week, at which point up to half the original cell population had haemolysed with the 30 mg/day dosage), a marked recovery and then re-establishment of equilibrium of haemoglobin 142 R. E. Howes et al. concentrations was observed. In one experiment, G6PD deficient subjects were given 30 mg primaquine daily for 4 months (Kellermeyer et al., 1961). Following trough haematocrits around day 7–10, these levels returned to normal within a week or so despite continued daily dosing with primaquine. This cycle of haemoglobin levels mirrored the percentage of reticulocytes in the blood (Dern et al., 1954a). 59Fe-labelling studies of RBCs demonstrated that only older RBCs (63–76 days old vs. 8–21 days old) were susceptible – this age-dependent phenomenon led the investigators to astutely surmise the involvement of an enzyme deficiency (Beutler et al., 1954). The self-limiting nature of primaquine-induced haemolysis in Afri- can American volunteers, however, cannot be generalised to all G6PD deficient patients globally. Differences were noted between individuals originating from different areas. Studies of a severe G6PD variant (Medi- terranean variant) in the 1960s showed even the youngest reticulocytes to be vulnerable to primaquine (Piomelli et al., 1968). In other words, continued daily dosing would result in progressively steeper losses of RBC and, presumably, death if not discontinued. The existence of these highly vulnerable variants, in contrast to the relatively nonthreatening African type (A- variant), imposes risk of fatal outcomes with the unbridled application of primaquine among populations where the character of locally prevalent G6PD variants is not known. The safety of primaquine hinges on G6PD status and primaquine sensitivity phenotype of the variant involved. The impediments imposed by G6PD deficiency and primaquine toxicity in these patients still severely limit the effectiveness of this singularly important drug. Growing acknowledgement of this problem today, especially in the context of emergent malaria elimination strategies, has recently activated research endeavours on this old and persistent problem. The disap- pointing search for superior alternative drugs, both in scope and outcome, that essentially ended around 1980 is detailed in Chapter 4 of V olume 80. Only one plausible successor to primaquine exists today, tafenoquine (Fig. 4.1), a GlaxoSmithKline (GSK) - Medicines for Malaria Venture (MMV) partnership drug currently in Phase IIb/III trials originally discovered and developed by the US Army (Crockett and Kain, 2007; Shanks et al., 2001). However, also being an 8-aminoquinoline, tafenoquine presents similar haemolytic challenges as primaquine to G6PD deficient individuals, and this complicates the path to licencing: tafenoquine has already been in development since the 1980s. Chapter 4, Volume 80 also lays out alternative G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 143 and unexplored approaches to mitigating 8-aminoquinoline toxicity. The safe exclusion of patients from harm caused by this drug by the diagnosis of G6PD deficiency currently requires technical capacities nearly wholly absent where most malaria patients live, although work is ongoing to develop a practical point-of-care kit. Greater understanding of the geographic distribution of G6PD deficiency in relation to endemic malaria will inform decisions on primaquine therapeutics policy and practice. The remainder of this chapter details the science and technology underpinning all of these important endeavours.

3. GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY: THE ENZYME AND ITS GENE The G6PD enzyme plays a critical role in maintaining RBC integrity through catalysing a key step in the cell’s metabolic production of reducing equivalents that maintain reduction–oxidation (redox) equilibrium of the cytoplasm. This protects the cell from oxidative attack by radicals derived from oxygen and organic compounds such as drugs and their metabolites. In spite of its vital function, the G6PD enzyme is highly variable, both biochemically and genetically. Detailed reviews of G6PD genetics, biochemistry and clinical characteristics have been previously published (Beu- tler, 1994, 1996; Cappellini and Fiorelli, 2008; Luzzatto, 2006, 2009, 2010; Mason et al., 2007; Mehta et al., 2000; WHO Working Group, 1989).

3.1. G6PD Genetics and Inheritance The advent of molecular diagnostics following the successful mapping of the G6PD gene’s 13 exons (Martini et al., 1986) which span 18.5 kb, and the gene’s cloning and sequencing in 1986 (Persico et al., 1986; Takizawa et al., 1986) started to uncover the genetic basis to the enzyme’s great variability (Vulliamy et al., 1988) (Fig. 4.2). This Mendelian X-linked gene is one of the most highly polymorphic of the human genome with at least 186 mutations having been described (Minucci et al., 2012). That said, not all mutations are polymorphic and of public health significance, but many instead appear only sporadically within populations: almost half (66 of 140 mutations reviewed in 2005 by Mason and V ulliamy) are associated with the most severe clinical phenotypes and are very rare. Most mutations are single point substitutions (121 of 140 (Beutler and Vulliamy, 2002; Mason and Vulliamy, 2005)) leading to amino acid substitutions. The absence of more severe mutations reflects the enzyme’s 144 R. E. Howes et al.

Figure 4.2 Diversity of mutations in the G6PD gene and enzyme. Panel A shows the distribution of common mutations along the G6PD gene coding sequence. Exons are shown as open numbered boxes. Open circles are mutations causing Class II and III variants; filled circles are Class I variants; filled squares are small deletions; the cross represents a nonsense mutation; “f” shows a splice site mutation. (Figure from Cappellini and Fiorelli (2008), reprinted with permission from Elsevier; figure originally modified from Luzzatto and Notaro (2001)). Panel B shows the distribution of amino acid substitutions across the enzyme’s tetrameric structure (each identical monomer subunit is labelled A–D), numbered according to the affected amino acids. The diamonds indicate polymorphic or sporadic mutations, and their colour shows the associated clinical phenotype. The grey shadowed areas cover the two dimer interfaces. Across this region, a molecule of structural NADP per monomer is buried which stabilises the monomers and the associations between them. Each mutation is shown in only one monomer, but would be present in all four. (Figure from Mason et al. (2007), reprinted with permission from Elsevier). The positions of a few common mutations (A-, Mediterranean, Seattle, Union) are shown both in the gene (Panel A) and the enzyme (Panel B). (For a colour version of this figure, the reader is referred to the online version of this book). G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 145 housekeeping function which requires some residual activity for cell survival. Knockout studies in mice found G6PD-null mutations to be lethal (Longo et al., 2002) and a high degree of evolutionary conservation of certain regions of the gene was identified by comparing the position of mutations across 42 different organisms, pinpointing certain regions of the gene as highly conserved, and hence essential for enzyme function and cell survival (Notaro et al., 2000). All known mutations have been found to affect the coding regions of the gene and none described in the regulatory regions (Beutler and Vulliamy, 2002; Fig. 4.2), suggesting that reduced enzyme activity levels are associated with enzyme instability, rather than deficiencies in gene expression. The G6PD gene’s position on the X chromosome has important implications for its population genetics. Unlike in males, for whom the G6PD phenotype was early-on observed to be binary with individuals being either deficient or nondeficient depending upon which allele was inherited (Beutler et al., 1955), the gene’s X-linked inheritance means that deficiency in females is more complex. Females inherit two copies of the X chromosome and therefore have two populations of RBCs, each expressing one of the two G6PD alleles they carry. If females inherit two identical alleles (both either normal or deficient), their phenotype and clinical symptoms will be identical to those of hemizygous males. Heterozygous females, however, inherit one wild-type and one deficient allele but display a mosaic effect of expression as only one X chromosome is expressed in each cell. One population of cells will express the normal allele and the other population the deficiency (Beutler et al., 1962). The ratio of normal to deficient cells is variable, due to the phenomenon of Lyonization (Lyon, 1961). Lyoniza- tion is a random process and the resulting proportions of normal and deficient cells may deviate significantly from the expected 50:50 ratio (Beutler, 1994), leading some heterozygotes to have virtually normal expression, and others with expression levels comparable with female homozygotes (i.e. entirely deficient). Heterozygotes may therefore express a spectrum of phenotypes; making appropriate diagnoses with standard binary methods much harder than for deficient males, as many heterozygotes will be phenotypically normal. At the population level, G6PD deficiency is more commonly expressed in males, though in populations with high frequencies of deficiency, homozygotic inheritance can be common, and the prevalence of affected heterozygotes may also be of public health concern. More details about the population genetics of the G6PD gene are discussed by Hedrick (2011). 146 R. E. Howes et al.

3.2. The G6PD Enzyme The G6PD enzyme consists of either dimer or tetramer forms of a protein subunit consisting of 514 amino acids. Each subunit binds to an NADP+ molecule for its structural stability, which are positioned close to the interface where the two subunits of each dimer bind (Au et al., 2000; Fig. 4.2). The majority of mutations disrupt the enzyme structural stability and thus reduce its overall activity. T he effect of each mutation on enzyme structure and function depends on the location of the substituted amino acid. For example, many of the most severe mutations map to exon 10 (Mehta et al., 2000) which encodes the binding interface of the subunits and therefore disrupt its qua- ternary structure and stability. These mutations cause the most severe clinical symptoms and as such do not reach polymorphic frequencies; instead they usually result from independent spontaneous mutations (Fiorelli et al., 2000). Mutations which do not cause such severe reductions in enzyme activity are widely distributed across the gene’s coding region and throughout the enzyme structure (Fig. 4.2), and have been found to reduce the efficacy of protein folding, for example (Gomez-Gallego et al. (1996)). The residual enzyme activity of G6PD variants ranges from <1% to 100%. As with all enzymes, G6PD activity decreases with cell age: it is estimated that in normal blood, reticulocytes have about five times higher activity levels than the oldest 10% of RBCs (Luzzatto, 2006). The oldest cells are therefore most vulnerable to oxidative stress. In individuals with intrinsically reduced G6PD enzyme activity due to genetic mutations, the ageing process is effectively sped up, with larger proportions of cells having lower enzyme levels and being at increased risk of oxidative damage. This has implications for the clinical severity of the mutations, as discussed below. The properties of these enzyme variants correspond to a broad spectrum of enzyme biochemical phenotypes, i.e. electrophoretic properties, heat stability and enzyme kinetics. WHO guidelines (WHO Working Group, 1989) for standardised biochemical characterisation of the enzyme led to 387 variants of G6PD being described by 1990 (Beutler, 1990), though many of these would later prove to be genetic duplicates. Few variants have been fully characterised, but a handful of those which are common enough to be of major public health significance have been well researched. The residual enzyme activity of these variants affects cell primaquine susceptibility. Though it is widely accepted, these must be inversely correlated; little evidence informs that presumption (Baird and Surjadjaja, 2011). Three such variants have formed the basis to current drug recommendations, which are further discussed in Section 8.2 (p. 179). G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 147

All the earliest evidence about the haemolytic risk of G6PD deficiency pertained to the African A- variant (G202A/A376G), due to the racial background of the “primaquine sensitive” patients studied in the 1950s Stateville primaquine experiments (Section 2, p. 136). Although rare as a genetic variant for having a double-point mutation, this type of deficiency is very common among individuals of sub-Saharan African origin. The A- variant characteristically expresses residual enzyme activity about 10% of normal levels (Beutler, 1991). It was studies with this variant which led to the discovery of G6PD deficiency (Carson et al., 1956). The Mahidol variant (G487A) is the predominant allele among many G6PD deficient populations of Myanmar and is also common among Thais (Section 5.3, p. 163 and Fig. 4.6A). Enzyme activity is reduced to 5–32% of normal levels (Louicharoen et al., 2009). Finally, the Mediterranean variant (C563T) was originally known for its association with the clinical pathology of favism, and causes some of the most severely deficient phenotypes (Beutler and Duparc, 2007). This variant usually expresses <1% enzyme activity, with undetectable enzyme levels in older erythrocytes (Piomelli et al., 1968). Despite expressing such low levels of enzyme activity, carriers of this mutation are nevertheless asymptomatic until exposed to haemolytic triggers (Beutler, 1991) (Section 3.4, p. 148).

3.3. The Pentose Phosphate Pathway as an Anti-Oxidative Defence G6PD enzyme activity is necessary for RBC survival as it catalyses the only metabolic pathway capable of generating reducing power to these cells lacking mitochondria (Pandolfi et al., 1995). Reducing power, supplied in the form of NADPH, is necessary as an electron donor, i.e. chemical reduction, for detoxifying oxidative challenges to cells. The metabolic reactions concerned are part of the pentose phosphate pathway (PPP, also called the hexose monophosphate shunt), the first and rate-limiting step of which is catalysed by the G6PD enzyme: the oxidation of glucose-6-phosphate into 6-phosphoglucono-δ-lactone, which simultaneously reduces NADP to NADPH. The electron of NADPH passes to abundant glutathione dimers (GSSG) via another enzyme, glutathione reductase. Reduced glutathione monomers (GSH) represent the primary defence against hydrogen perox- ides, organic peroxidises, and free radicals. When G6PD functions normally, the drain of electrons from the NADPH pool caused by oxidative challenge within the cell prompts the PPP to accelerate according to need, i.e. maintaining an NADP–NADPH equilibrium that strongly favours NADPH. 148 R. E. Howes et al.

This in turn maintains the oxidised–reduced glutathione (GSSG–2GSH) equilibrium strongly in the direction of the reduced state, i.e. 1:500 at steady state (Greene, 1993). However, in cells that have a mutant and defective G6PD gene, the PPP may, depending upon the extent of the enzyme activity defect, function at near-maximum rate even at steady-state redox equilibrium. When oxidative challenge occurs and the equilibrium of NADP to NADPH shifts to the oxidised direction, the PPP is intrinsically unable to accelerate rapidly enough to force the equilibrium in favour of NADPH. This effectively stymies the flow of electrons to GSH, and that equilibrium shifts in favour of GSSG. The oxidants consuming these reducing equivalents, in turn, overwhelm the ability of the cell to provide them and damage may then occur. Visible evidence of such occurs in the form of Heinz bodies in the RBC membrane that attend acute primaquine-induced haemolytic anaemia (Greene, 1993). Heinz bodies cause the membrane to become rigid, and thus decrease the cells’ lifespans. The mechanism of primaquine-induced haemolysis remains uncertain, but will be discussed in more detail later in this chapter (Section 8.1, p. 175).

3.4. Clinical Manifestations of G6PD Deficiency In discussing the clinical manifestations, it is important to note that the majority of G6PD deficient individuals are asymptomatic most of the time. The public health importance of this condition comes from the sheer numbers affected and at potential risk of developing clinical symptoms: 400 million globally (Cappellini and Fiorelli, 2008), or 350 million within malaria endemic countries (Howes et al., 2012). Symptoms are induced when cells are exposed to exogenous oxidative stresses against which they cannot defend themselves. The severity of the clinical symptoms and the subsequent treatment required depends upon the degree of enzyme deficiency (which is variant-dependent), the nature and total dose of the oxidative agent, the time course of exposure, the presence of additional oxidative stresses and pre-existing factors such as age, haemoglobin concentration and concurrent infection (Cappellini and Fiorelli, 2008). The relative contribution of each to determining the severity of the response is not fully known but is further discussed in Section 8.2, p. 179). The most clinically serious symptom of G6PD deficiency is neonatal jaundice (NNJ), which peaks 2 to 3 days after birth (Luzzatto, 2010). This is highly variable in severity but can lead to kernicterus (Beutler, 2008) and permanent neurological damage or death if left untreated (Doxiadis and G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 149

Valaes, 1964; Luzzatto, 2006). Not all neonates with NNJ are G6PD deficient, but this congenital condition greatly increases the risks, and in some countries is the most common cause of NNJ (Luzzatto, 2010). AHA is the most common manifestation of the deficiency, and may be triggered by a range of exogenous agents causing intravascular haemolysis and jaundice, and may include haemoglobinuria (dark urine) (Luzzatto, 2009). The most severe outcome of AHA is acute renal failure (Cappel- lini and Fiorelli, 2008). The longest-known of these triggers are fava beans: “favism” can be very severe or even life-threatening if left untreated without transfusion (Beutler, 2008; Luisada, 1941); favism is most common in children. Infection is another important trigger of AHA (Burka et al., 1966), with severe pathology having been previously attributed to hepatitis viruses A and B, cytomegalovirus, pneumonia, and typhoid fever (Cappellini and Fiorelli, 2008). Finally, a number of haemolysis-inducing drugs have also been identified as triggers of AHA (Youngster et al., 2010); in the present context of P. vivax malaria, the most pertinent is primaquine. The exceptions to G6PD deficiency being asymptomatic until triggered by certain exogenous triggers are those sporadically emerging, highly unstable variants expressing very low residual enzyme activity. These variants never reach polymorphic frequencies due to their severe pathology, which is characterised as chronic nonspherocytic haemolytic anaemia (CNSHA). While individuals with these mutations make up only a very small minority of the population affected by G6PD deficiency (almost always males), they are the most clinically severe and may be transfusion-dependent (Luzzatto, 2010). In addition to susceptibility to all the aforementioned triggers of AHA, the very low residual enzyme levels mean that cells cannot even protect themselves against oxygen radicals continuously generated by the on-going process of haemoglobin de-oxygenation. CNSHA is therefore a lifelong condition, with haemolysis ongoing even in steady state. Based on these pathologies, G6PD alleles can be categorised into three types: (1) those sporadic severe variants associated with chronic symptoms, (2) polymorphic types which are typically asymptomatic but susceptible to trigger-induced acute haemolytic episodes, and (3) those with normal activity (Table 4.1). Previous classifications have included additional subdivisions of the polymorphic variants into “mild” and “severe” types (WHO Work- ing Group, 1989; Yoshida et al., 1971). However, as suggested by Luzzatto, the distinctions between these further classes are blurred and are no longer useful (Luzzatto, 2009). As such, we distinguish only three variant types (Table 4.1). 150 R. E. Howes et al.

Table 4.1 G6PD Variant Types and Their Key Characteristics Residual Type Enzyme Activity Prevalence Clinical Significance 1 <10% Sporadic, never Severe and chronic: CNSHA polymorphic 2 1–50% Polymorphic Asymptomatic until triggered: risk of NNJ, AHA, favism 3 Normal (>50%) Polymorphic (wild-type) None

In the present context of P. vivax therapy, the generally asymptomatic polymorphic variants vulnerable to AHA (type 2 variants) are the primary threat to safe therapy. These variants are the subject of this review. Prima- quine-induced haemolysis is further discussed later in this chapter (Sec- tion 8.1, p. 175), but can require transfusion even after relatively low doses (Shekalaghe et al., 2010). Identifying this risk is therefore essential.

4. DIAGNOSING G6PD DEFICIENCY Given the absence of a universally safe drug and the potential severity of primaquine-induced AHA, widespread safe radical treatment of P. vivax is contingent upon reliable diagnosis of G6PD deficiency. There are two types of tests for diagnosing G6PD deficiency: biochemical enzyme activity tests and molecular DNA-based methods. These are suited to different situations, depending upon the type of diagnosis required and the laboratory capacities available. We discuss here those currently available and consider their limitations in respect to the heterogeneity of this condition, then discuss on-going developments towards improving these methods.

4.1. Phenotypic Diagnostic Tests Most tests for G6PD deficiency consider the biochemical phenotype – qualitative or quantitative measures of residual enzyme activity. These tend to use dyes or fluorescence markers serving as direct or proxy indicators of enzyme activity representing the rate of NADP reduction to NADPH (Beutler, 1994); qualitative assessments generally allow classification as normal, intermediate, or deficient, while quantitative tests employ spectropho- tometry to determine exact measures of enzyme activity. One of the earliest screening methods was developed by Motulsky and Campbell–Kraut, using the rate of brilliant cresyl blue decolourisation: if decolourisation had not occurred within a predetermined timeframe (commonly 180 min), the G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 151 sample was deemed to be G6PD deficient. Developments to this method have included Brewer’s methaemoglobin (metHb) reduction test (Brewer et al., 1962), Bernstein’s DPIP method (2,6-dicholorophenol indophenol dye test (Bernstein, 1962)), Beutler’s fluorescent spot test (FST) (Beutler and Mitchell, 1968; which is the WHO recommended method (Beutler et al., 1979)), and most recently the WST-8/1-methoxy phenazine methosulfate (PMS) method (Tantular and Kawamoto, 2003) created to facilitate field use of the diagnostic. Applying these diagnostic tests in large-scale population screening is typically feasible in the context of research endeavours. However, such testing is very often impracticable for routine care in those settings where most malaria patients live. Most methods still depend on a cold chain and specialised laboratory equipment. Even the FST, perhaps the most widely used test, requires cold storage of reagents, micropipetters, a water bath and a UV light source. Further practical limitations include the time-delay on obtaining the results, which may take several hours, and the difficulties of reading the results – naked eye judgements have been found to be subjective with some of the colour changes, which are also influenced by room temperature and humidity. Furthermore, although standardised protocols for using the most common methods were published in 1979, tests are invariably modified by users (for example, in terms of the cut-off times imposed) and adapted to local conditions and survey constraints. This diagnostic variation, therefore, hinders comparative analyses between surveys by adding a level of uncertainty into the results. Furthermore, anaemia is an important potentially confounding factor increasing the probability of false-positive diagnoses. This condition reduces the overall number of RBCs, and therefore the level of G6PD enzyme per volume of blood. Conversely, increased proportions of reticulocytes following malaria infection can lead to false negatives as reticulocytes have the highest G6PD activity levels. The influence of malaria parasites on qualitative tests has not been evaluated. The most important limitation to these qualitative tests, however, is their relatively poor ability to diagnose female G6PD deficient heterozygotes. As previously explained (Section 3.2, p. 146), heterozygotes express a mosaic of two RBC populations: cells expressing the normal G6PD gene and cells with the deficiency. The deficient cells carry exactly the same haemolytic risk as those of homo- or hemizygotic individuals. Heterozygote diagnostic outcome is dependent upon: (i) the diagnostic test’s threshold for determining deficiency (the longer the delay, the larger the proportion of samples that will appear “normal”); (ii) the mutation and the level of residual 152 R. E. Howes et al. enzyme activity expressed; and (iii) the ratio of normal to deficient cells – determined by Lyonization, as previously described. These issues present serious problems to heterozygote diagnosis, as the normal enzyme expression in one population of cells can mask serious deficiency in others. A heterozygote may have, for example, 70% of her RBCs expressing very low enzyme activity, and therefore exquisitely sensitive to primaquine and vulnerable to harm, but she may test as “normal”. Although some biochemical tests have been found to be better suited to detecting heterozygosity, including G6PD/6-phosphogluconate- dehydrogenase (6PDG) and G6PD/pyruvate kinase (PK) ratio analysis, and the cytochemical G6PD straining assay (Minucci et al., 2009; Peters and Van Noorden, 2009), these are highly technically challenging and therefore impractical for large-scale, field-based population surveys, and rarely used. Molecular methods, on the other hand, provide an unambiguous diagnosis of genetic heterozygotes. A recently described flow cytometric assay (Shah et al., 2012) appears much more practical, albeit still limited to the setting of relatively sophisticated laboratories. Haunting all of these methods is the important question of what represents an acceptable level of enzyme activity with respect to risk of primaquine-induced haemolysis. In other words, at what level of residual activity does each test classify patients as normal, and does this genuinely exclude all at risk of harm? This is the issue of sensitivity. Specificity poses the converse problem by excluding patients who could safely receive effective treatment of their infection. A clinically appropriate sensitivity/specificity balance must be incorporated into the diagnostics. Unfortunately, very little evidence regarding primaquine sensitivity phenotypes informs this decision across the broad spectrum of mutant enzymes.

4.2. Molecular Diagnostic Tests A seemingly more clear-cut diagnostic approach examines the gene itself. Molecular methods use variant-specific primers to identify the presence or absence of specific mutations. These direct methods overcome uncertainties associated with variable enzyme activity cut-offs, anaemia, reagent breakdown and subjective classifications. Molecular diagnoses allow insight into the severity of the condition for those mutations for which residual enzyme level phenotypes and primaquine sensitivity phenotypes are known. Female heterozygotes will not be dangerously misclassified as G6PD normal. Irrespective of these benefits, the high-end laboratory requirements and time lags for results leave these methods impracticable for population G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 153 screening or routine care in their current form. Even if these limitations could be overcome, deeper limitations remain. Although heterozygosity can be diagnosed, the state of Lyonization, and thus the phenotype remains uncertain, potentially putting individuals with these intermediate genotypes at risk. For instance, a study in Tanzania reported a case of severe haemolysis (defined as <5 g/dl) in a heterozygote A- variant female child following a single dose of primaquine (45 mg dose) (Shekalaghe et al., 2010). Further, although the enzyme activity of three major variants has been characterised (Mediterranean, A-, Mahidol (Baird and Surjadjaja, 2011)), the clinical character of the vast majority remain unknown; in these cases, molecular diagnoses cannot inform clinical severity. Even in the well-studied A- genotype, enzyme activity assays in Uganda identified surprising variation between individuals of the same genotypes (Johnson et al., 2009). Finally, molecular methods look for specific mutations, which is usually only a subset of all described mutations, and hence cannot reliably diagnose all cases of potentially clinically significant deficiency. For example, the Tanzanian study previously referred to used primers to identify the common A (A376G) and A- (G202A) mutations, but then reported instances of haemolysis in apparent wild-type B individuals (Shekalaghe et al., 2010). It seems plausible, given greater heterogeneity in the pool of G6PD deficient variants reported from individuals in The Gambia (Clark et al., 2009) and Senegal (De Araujo et al., 2006), that diversity in other populations of sub-Saharan Africa has also been underestimated and that some of the Tanzanian wild-type individuals who haemolysed were in fact deficient, but that the primers employed were not comprehensive enough to identify all cases of phenotypic deficiency.

4.3. The Case for a New Diagnostic for Safe P. vivax Radical Cure Given the limitations to existing diagnostic methods, it is evident that there is currently no well-adapted point-of-care G6PD test for use prior to primaquine treatment in the field. A new, practical and standardised kit is required to ensure safe use of primaquine – this will require the test not only to identify an enzyme deficiency, but to give a binary assessment of whether a given dosage of primaquine can or cannot be safely administered. An important prerequisite step will be determining the position of this binary cut-off against the spectrum of haemolytic outcomes, which are known to range from negligible to highly severe. Although the mechanisms whereby primaquine triggers haemolysis remain uncertain (Section 8.1, p. 175), and the factors determining the 154 R. E. Howes et al. severity of haemolysis not conclusively established, is it likely that this test will assess residual enzyme activity levels. Given this, environmental conditions are of major concern as these will strongly influence the diagnostic outcome as enzyme activity is heavily dependent on ambient temperature which fluctuates significantly over diurnal and seasonal cycles. Total RBC count – as influenced by anaemia and the blood volume used – will also impact the diagnosis. In terms of practicality, the test needs to be inex- pensive, provide a result rapidly, and be easily used and interpreted with minimal training (Asia Pacific Malaria Elimination Network (APMEN), 2012). Although a binary qualitative test would be easiest to implement, the heterogeneity of this disorder may require quantitative diagnoses; this consideration is particularly relevant to diagnosing heterozygotes. Two visual, qualitative rapid diagnostic test (RDT) kits have been described. The BinaxNOW® G6PD assay (Binax, Inc., Maine, USA) was found to have sensitivity and specificity of 0.98 and 0.98, respectively with a cut-off of 4.0 U/g Hb (Tinley et al., 2010)2; this accuracy may not be sufficient for mass screening; furthermore, at around $25 per test, the cost of the test would be prohibitively high for mass screening or routine clinical use where malaria is endemic. However, this test’s major shortcoming is that it must be used within a temperature range of 18–25 °C, and is thus further unsuited to most field-based settings where P. vivax is prevalent. The Care- Start™ G6PD screening test (AccessBio, New Jersey, USA), another qualitative phenotypic test, is similar to a malaria RDT in appearance and use (Kim et al., 2011), and has been demonstrated to be robust after prolonged storage at high temperatures (up to 45 °C for 90 days). Using a cut-off of 3.5 U/g Hb with ethylenediaminetetraacetic acid (EDTA) blood samples, the test had sensitivity of 0.68 and specificity of 1 (n = 903). The test was able to identify deficient cases up to 2.7 U/g Hb, corresponding to 22% residual enzyme activity (Kim et al., 2011). However, 13 of 903 individuals with very low G6PD activity (<2 U/g Hb) were misdiagnosed as G6PD normal; these false negative cases would therefore be at high risk of harm if administered primaquine. This evaluation study followed the manufacturer recommendations to classify any test showing even slight colour change as “normal”. In practice a far more conservative approach would be taken which would increase this test’s sensitivity and decrease its specificity. The discrepancy between the thresholds used by these two binary tests (4.0 U/g

2 These sensitivity and specificity values were from heparinized blood samples; EDTA blood samples had 0.98 sensitivity and 0.97 specificity. Samples included 50 individuals with G6PD activity ≤4.0 U/g Hb (Trinity Assay) and 196 individuals with G6PD activity >4.0 U/g Hb. G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 155

Hb for BinaxNOW vs. 2.7 U/g Hb for AccessBio) demonstrates the uncertainty around what exactly determines an intolerance to primaquine and an acceptable level of haemolysis. These basic questions need to be answered before an apparent arbitrary threshold is set. So, while these tests show tan- talizing promise, further development is required for their widespread use to enable more aggressive application of primaquine with the confidence of patients’ safety.

5. MAPPING THE SPATIAL DISTRIBUTION OF G6PD DEFICIENCY Maps provide an important evidence-base for assessing disease burden, for public health decision-making and for efficient resource-allocation through optimal targeting of interventions (Cromley, 2003; Hay and Snow, 2006; McLafferty, 2003), not least in relation to disorders as spatially and genetically heterogeneous as G6PD deficiency. The prevalence of G6PD deficiency has been mapped among populations in malaria endemic countries (Howes et al., 2012) to allow identification of where this disorder may be a problem for malaria treatment options. From this modelled map, sex-specific population estimates of deficient individuals were derived and aggregated to national and regional scales. Reported occurrences of the underlying G6PD gene variants have also been assembled (Howes et al., in preparation). The characteristics of the underlying variants are what will determine the severity of potential primaquine-induced haemolysis; while the prevalence map indicates how common the deficient phenotype is. Assembly of the evidence- bases of both types of population data, and the methodological steps involved in generating the maps are summarised here. The following Section 6 (p. 165) then explores in more detail what these maps indicate about the spatial characteristics of G6PD deficiency in relation to the endemicity of P. vivax.

5.1. G6PD Deficiency Prevalence Mapping Various attempts to represent the spatial distribution of G6PD deficiency prevalence have been made. The most recent WHO map dates from 1989, and presented only national-level summaries of available data (Luzzatto and Notaro, 2001; WHO Working Group, 1989). A similar national-level map published by Nhkoma et al. in 2009 updated this effort, but still presented only national summaries, masking any subnational spatial variation in prevalence. Subnational variation was possible to discern from the impressive com- pilation of human gene maps in Cavalli-Sforza’s History and Geography of 156 R. E. Howes et al.

Human Genes (Cavalli-Sforza et al., 1994), however the statistical mapping methods used were rudimentary, and modern geostatistics have advanced significantly since their publication. A main limitation to all of these maps is that they give no measure of uncertainty in their predictions, either at the national-level or in a spatially specific manner. Modern geostatistics allow more comprehensive approaches to mapping, particularly in respect to sum- marising relative confidence in the predictions (Patil et al., 2011). We focus here on the recently developed G6PD deficiency map by Howes et al. 2012, an effort of the Malaria Atlas Project (MAP), which attempted to address some of the limiting factors associated with existing maps. This map was intended to represent the prevalence of clinically significant deficiency, as diagnosed by phenotypic tests, in malaria endemic countries. The evidence-base of surveys and all maps are available for down- load from the MAP website (www.map.ox.ac.uk).

5.1.1. Generating a Map: the Evidence-Base A total of 1734 spatially unique surveys were included in the map evidence- base which met four criteria: (i) Community representativeness: all potentially biased samples were excluded: patients (including malaria cases), all related individuals, and all samples which selected individuals according to ethnicity; (ii) Spatial specificity: surveys had to be geographically specific and possible to geoposition accurately; (iii) Gender specificity: to allow the model to represent the X-linked inheritance mechanism of the G6PD gene, data had to be reported according to sex; (iv) Phenotypic diagnosis: only surveys which had used phenotypic diagnostic methods were included.

5.1.2. Generating a Map: the G6PD Mapping Model Modelling a continuous map of the prevalence of G6PD deficiency had to account for several difficulties: heterogeneity in the data set (both in terms of variable G6PD deficiency prevalence found at nearby locations, and in terms of the uneven distribution of the surveys across the map), the relative reliability of the data set (from highly ranging sample sizes), and the difficulties of predicting deficiency in heterozygotes due to the gene’s X-linked inheritance (Section 4.1, p. 150). A final requirement of the model was that predictions were supported by uncertainty metrics (Patil et al., 2011). A Bayesian geostatistical model was developed to cope with separate input data according to sex and the different outputs required. The core assumption of geostatistics is that of spatial-autocorrelation: namely that populations closer in space would be more similar than populations further apart. While there were some exceptions, the raw data supported G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 157 this assumption. Populations were also assumed to be in Hardy–Weinberg equilibrium (Hardy, 1908; Weinberg, 1908).

5.1.3. G6PD Deficiency Prevalence Map: an Overview G6PD deficiency is widespread across malaria endemic regions (Fig. 4.3). The modelled prevalence map of G6PD deficiency represents the allele frequency of phenotypic deficiency, equivalent to the prevalence of deficiency in males. At the continental scale, frequency of the deficiency is highest among the populations of sub-Saharan Africa, where prevalence peaks

Figure 4.3 The prevalence of G6PD deficiency in malaria endemic countries. The prevalence is the allele frequency, which corresponds to the frequency of deficiency in males. Panels A–D correspond to Asia, Asia-Pacific, the Americas, and Africa+ regions, respectively. (The figure is adapted from Howes et al. (2012)). (For a colour version of this figure, the reader is referred to the online version of this book). 158 R. E. Howes et al.

Figure 4.3—cont’d G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 159 around 30% in several areas, but is also absent from parts of southern Africa and communities in the Horn of Africa. Prevalence of G6PD deficiency is less common across the Americas, being concentrated among populations in coastal regions. While prevalence is generally lower among Asian populations than sub-Saharan Africans, the condition is widespread across Asia and particularly patchy and heterogenous in some areas. The associated uncertainty map of the predictions is shown in Fig. 4.4; and the allele frequency map must be considered alongside these metrics

Figure 4.4 Uncertainty in the prevalence map. Uncertainty is quantified by the interquartile range of the model prediction and is closely associated with the proximity of population surveys, which are shown by the black dots. Panels A–D correspond to Asia, Asia-Pacific, the Americas, and Africa+ regions, respectively. (The figure is adapted from Howes et al. (2012)). (For a colour version of this figure, the reader is referred to the online version of this book). 160 R. E. Howes et al.

Figure 4.4—cont’d G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 161 of model confidence, as areas rich in data and with more homogenous frequencies of G6PD deficiency will be easier to model than areas where surveys are scarcely distributed or where available data indicated the underlying prevalence of deficiency to be heterogeneous. The G6PD deficiency prevalence map’s prediction confidence is quantified in the uncertainty map by the interquartile range (IQR, the 50% confidence interval) around the model prediction. Thus, a smaller IQR is indicative of a more reliable prediction. Areas of greatest uncertainty are those which would most benefit from new population surveys. Some potential sources of variability, however, are not accounted for by this measure, such as the underlying heterogeneity in the local population (which will not be represented if no surveys are available from that region) and the variation introduced by the diagnostic methods used. These limitations are discussed in greater detail in the original publication of this map (Howes et al., 2012). Regional prevalence of the deficiency is discussed in detail in relation to P. vivax endemicity in Section 6 (p. 164). Geographic information systems (GIS) grids and high-resolution images of these maps, as well as the input evidence-base of surveys are freely available via the MAP website (www. map.ox.ac.uk).

5.2. G6PD Deficient Population Estimates Estimates of the population of G6PD deficient individuals needed to account for high-resolution patterns of population density as well as short-scale variation in G6PD deficiency prevalence to ensure that population density was reflected in the overall prevalence estimate. Howes and colleagues (2012) derived these population estimates in a Bayesian framework so as to generate uncertainty metrics around the population estimates (Patil et al., 2011). The aggregated national-level numbers of G6PD deficient individuals are mapped in Fig. 4.5. An overall allele frequency of deficiency of 8.0% (IQR: 7.4–8.8) was predicted across all malaria endemic countries. This corresponded to 220 million affected males (IQR: 203–241) and an estimated 133 million females (IQR: 122–148). The model results indicate that a median estimate of 26% of expected genetic heterozygotes were diagnosed as being phenotypically deficient based on the raw survey data. Within the subset of 35 countries targeting malaria elimination, where primaquine treatment would be particularly beneficial, overall prevalence was lower with a predicted allele frequency of 5.3% (IQR: 4.4–6.7), meaning that in 2010 an estimated 61 million males (IQR: 51–77) and 35 million females (IQR: 29–46) were predicted to be phenotypically G6PD deficient in countries eliminating malaria. 162 R. E. Howes et al. Howes et al. (2012)) . et al. Howes from (Figure National allele frequency of G6PD deficiency.

Figure 4.5 Figure G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 163

Although many of the highest frequencies of deficiency were predicted from sub-Saharan Africa, the very high population densities across Asia meant that the overall population burden was largely focussed there. China and India, for instance, were estimated to be home to 41.3% of all G6PD- deficient males across the whole malaria endemic region globally. The high allele frequencies seen in Africa meant that 28.0% of the overall deficient population was in sub-Saharan Africa, while only 4.5% of the global population burden was in the Americas, and 67.5% across the whole of Asia. National-level estimates for all malaria endemic countries were provided in the original publication (Howes et al., 2012).

5.3. G6PD Deficiency Mutation Mapping To understand the clinical characteristics of G6PD deficiency in different regions, it is also necessary to map the underlying G6PD mutations. A simple map of key genetic variants was published in 2001 (Luzzatto and Notaro, 2001), and a new database of G6PD variants has since been assembled to update this effort (Howes et al., 2012; Howes et al., in preparation). Surveys were collated which provided measures of the proportions of each variant among G6PD deficient individuals in different areas. Exclusion of population samples from hospitalised or exclusively symptomatic patients avoided a bias towards higher proportions of the more severe variants, which might otherwise have been preferentially identified. Striking patterns emerged across malaria endemic regions (Fig. 4.6). Genetic heterogeneity was found to be relatively low across populations of the Americas and West Asia, where the A- and Mediterranean variants predominated, respectively. Further east, genetic diversity increased among Indian populations where the Orissa variant, barely reported outside India, predominated among certain communities in east, central India. Populations in countries east of India carried a completely different set of mutations, and genetic diversity of the G6PD gene was far greater. Although a handful of variants were found to be more commonly reported from specific populations (such as the Mahidol variant across Myanmar and Thailand; Viangchan variant across Mekong region; Kaiping variant among Chinese populations and the Vanua Lava variant in central and eastern Indonesia), genetic diversity was high among these populations. The proportion of “Unidentified” variants was also greatest among Asian populations, emphasising the inadequacy of molecular methods for diagnosing phenotypic deficiency: a limited number of primers cannot reliably identify all possible cases of deficiency. 164 R. E. Howes et al.

6. SPATIAL CO-OCCURRENCE OF G6PD DEFICIENCY WITH P. VIVAX ENDEMICITY We now discuss the spatial epidemiology of G6PD deficiency, its prevalence and genetic variants, in relation to its public health significance in the context of P. vivax transmission. The geographical limits of P. vivax

Figure 4.6 Proportions of G6PD variants among phenotypically deficient community samples. Panels A to D correspond to Asia, Asia-Pacific, the Americas and the Africa+ regions. Pie charts show the local proportions of each variant, sized according to the survey sample sizes and plotted on a logarithmic scale. The individuals from whom these data originate are known to be phenotypically deficient. All samples are therefore deficient, and the “Other/Unidentified” category represents G6PD variants which were too rarely reported to be individually represented in the map, or which could not be identified. (For a colour version of this figure, the reader is referred to the online version of this book). G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 165

Figure 4.6—cont’d 166 R. E. Howes et al.

Figure 4.7 Global endemicity of Plasmodium vivax endemicity. Assembly of this map is described in Chapter 1 of Volume 80. (Figure from Gething et al. (2012)). (For a colour version of this figure, the reader is referred to the online version of this book). transmission and its endemicity within those limits have been described in detail in the opening chapter of volume 80 and are shown in Fig. 4.7. The map shows P. vivax endemicity, as quantified by community parasite rate in the 1–99 year age range (PvPR1–99), with grey areas representing unstable transmission where <1 case per 10,000 population occurs per year (Chapter 1 of V olume 80; and Gething et al., 2012).

6.1. G6PD Deficiency in Asia The majority of the global population at risk of P. vivax transmission is on the Asian continent (Gething et al., 2012), defined as stretching from Turkey, south to Vietnam and east to the People’s Republic of Korea. The prevalence and genetic variants of G6PD deficiency among these populations were found to be heterogeneous, as was the transmission endemicity of P. vivax. While P. vivax endemicity is generally very low in Asian countries west of India, being either at no risk or having small patches of low endemicity in areas of unstable transmission, several of these countries had relatively high prevalence of G6PD deficiency compared with other parts of Asia. For instance, the national allele frequency of deficiency across Iran was predicted to be 11.8% (IQR: 9.9–14.1), and Azerbaijan had a national estimate of 10.2% (IQR: 8.9–11.7); both countries had only limited areas of very low/unstable P. vivax transmission. However, G6PD prevalence model uncertainty was high in this region, peaking (up to 37% IQR) in an area devoid of data across southern Pakistan. This high uncertainty emphasises the need for additional community surveys to support mapping of G6PD deficiency in this apparently high prevalence region. The underlying G6PD mutation among these populations was reportedly G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 167 the Mediterranean variant, which was identified in >70% of deficient individuals in all surveys between Turkey and Pakistan (Fig. 4.6A). Further east, G6PD deficiency was present but at relatively low prevalence (2–5%) across much of the Indian subcontinent, increasing in the eastern states of Chhattisgarh, Orissa and Jharkhand where prevalence reached 5–25%; the common variants in this area were the Mediterranean, Kerala-Kalyan and Orissa variants, the latter being highly restricted in its spatial extent to east India. These areas coincided with high P. vivax endemicity, which reached >7% PvPR1–99 in the neighbouring province of Andhra Pradesh. Another high prevalence area of G6PD deficiency in Asia (approximately 20%) was around the northern Lao–Thai border, an area largely P. vivax free, with only small patches of unstable transmission. Lower G6PD deficiency frequencies were predicted along coastal Myanmar (2–3%), an area of high (>7%) P. vivax endemicity. High frequencies of the two diseases, however, occurred simultaneously in southern Thailand where endemicity was >7% and G6PD deficiency prevalence 5–9%. Prevalence of both disorders was therefore variable with often highly focal P. vivax transmission across this region. In terms of the G6PD genetic mutations, there was a stark change in variants and greatly increased diversity across countries east of India. G6PD Mahidol was either universal or very common among communities in Myan- mar, remaining prevalent among Thai populations, in whom the Viangchan was also frequently reported. No single variant predominated among Chinese populations, instead the Kaiping, Canton and Gaohe variants were all common. A high proportion of samples were also rare or could not be identified using standard molecular primers, an indicator of high genetic heterogeneity.

6.2. G6PD Deficiency in Asia-Pacific Some of the largest continuous regions of high P. vivax endemicity globally were predicted across the Asia-Pacific region, where endemicity reached >7% across much of Papua New Guinea, the Solomon Islands and Vanuatu. Coinciding with this, G6PD deficiency was also prevalent on these islands, peaking at 23% prevalence on the southern reaches of Santa Isabel and Gua- dalcanal islands (where the Union G6PD variant was the main cause of deficiency, Fig. 4.6B). Both diseases were highly heterogeneous across Indo- nesia, by far the most populous nation in this region. Both G6PD deficiency prevalence and P. vivax endemicity were particularly high across the central Nusa Tenggara islands, such as on Flores where endemicity was over 7% and G6PD deficiency approximately 10%. A dearth of G6PD population surveys on Sulawesi introduced relatively high uncertainty to the map; in contrast, 168 R. E. Howes et al. numerous parasite rate surveys indicated that P. vivax transmission was mostly unstable in this area. Additional G6PD surveys would be particularly valuable among the south-eastern populations of Sulawesi, where endemicity reached 5%, and G6PD deficiency was predicted at 8% due to the high frequencies observed on nearby islands. G6PD deficiency was heterogeneous across the region. Relatively high genetic diversity was reported with multiple variants commonly co-existing in high proportions alongside important frequencies of unidentified variants. For instance across Papua New Guinea, frequencies of 1% were found along the southern coast which rose to 15% along the East Sepik northern coast. The Vanua Lava G6PD variant was commonly reported from populations in both Indonesia and Papua New Guinea, but was not reported from anywhere outside this region. A neonatal screening programme for G6PD deficiency exists across the Philippines which contributed high density of prevalence data (n = 636 data points) indicating a spatially variable national prevalence of 2 to 3% (population-adjusted national allele frequency estimate is 2.5% [IQR: 2.4 to 2.5] (Howes et al., 2012)). Across the Philippines, stable P. vivax transmission was only found on islands at the northern and southern ends of the country, peaking in northern Luzon at around 5% endemicity, where G6PD deficiency ranged in prevalence between 1 and 4%.

6.3. G6PD Deficiency in the Americas The lowest predictions of G6PD deficiency prevalence globally were across the Americas, where 40.8% of the land area had median prevalence predictions of ≤1%. Prevalence ranged from 0% across parts of Mexico, Peru, Bolivia and Argentina, to a national allele frequency prediction of 8.6% in Venezuela. Surveys were relatively scarce across large parts of the continent, particularly in Venezuela which led to high uncertainty around the national allele frequency estimate (IQR: 4.0–18.0). Plasmodium vivax endemicity in this area of high G6PD deficiency prevalence was patchy, with areas of 3 to 4% PvPR1–99 interspersed with large expanses of unstable transmission. Deficiency prevalence was also predicted to rise in the central and southern coastal provinces of Bra- zil, such as around the cities of São Paulo and Porto Alegre where P. vivax was absent. The A- variant of G6PD was predominant, causing >80% of deficiency cases across coastal communities of Brazil and Central America, and explaining 62% of deficient cases in a survey in central Mexico (Fig. 4.6C). Plasmodium vivax endemicity peaked in two areas of the Americas: Honduras and Nicaragua in Central America, and Amazonas province in northwest Brazil (Fig. 4.7); areas where G6PD deficiency prevalence was relatively low compared with other parts of the continent. Parasite rates of 6 G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 169 to 7% coincided with G6PD deficiency allele frequency national estimates of 2.9% (IQR: 1.5–5.8) in Honduras and 1.5% (IQR: 0.6–3.6) in Nica- ragua. Very few G6PD surveys were found from Amazonian communities, but parts of this region where P. vivax endemicity exceeded 7% were predicted to have G6PD deficiency prevalence of up to 3%. Additional G6PD deficiency surveys in both these areas of high P. vivax endemicity would be valuable, particularly focussed in areas of high population density.

6.4. G6PD Deficiency in Africa, Yemen and Saudi Arabia (Africa+) Plasmodium vivax epidemiology in the Africa+ region differs starkly from other malaria endemic areas due to the high prevalence of Duffy negativity among these populations (Howes et al., 2011) which depresses transmission to unstable levels across most of the continent (Chapter 2 of this volume). Therefore, in spite of its high prevalence, G6PD deficiency in Africa+ has relatively little bearing on the global picture of P. vivax therapy. The only two areas of stable P. vivax transmission in Africa+ are Ethiopia and close surrounds, and Mada- gascar. G6PD deficiency prevalence in the Horn of Africa is low, with national allele frequency in Ethiopia estimated at 1% (IQR: 0.7–1.5); though stable, the coincident parasite endemicity is equivalently low, with most areas being at 1% PvPR1–99, interspersed with patches of 2% endemicity. The highest single prediction of G6PD deficiency prevalence globally is on the Persian Gulf coast of Saudi Arabia where P. vivax is absent. Plasmodium vivax transmission is negligible across this whole peninsula, with only a narrow strip of unstable transmission along the west coast of Saudi Arabia and Yemen. Endemicity on Madagascar is more significant, reaching 2 to 3% PvPR1–99 in the inland and central coastal regions. Only a single-community G6PD survey was available so although the national allele frequency estimate is high at 19.4%, additional surveys would be needed to reduce uncertainty in this estimate (IQR: 11.5–30.3). Although G6PD deficiency does not present a hurdle to P. vivax radical cure in most parts of Africa+, the main potential application of primaquine across this region is for blocking transmission of P. falciparum (Eziefula et al., 2012; Bousema and Drakeley, 2011). Prevalence estimates of G6PD deficiency in Africa+ are the highest globally, with 14 countries predicted to have national allele frequencies >15%, all across sub-Saharan Africa from Ghana (19.6% [IQR: 14.2–27.0]) in the west across to Mozambique in the east (21.1% [IQR: 14.7–29.8]). Several areas were predicted to have particularly high prevalence (approximately 30%), including the coastal areas of West Africa (Ghana to Nigeria), the mouth of the Congo river (western Congo, Democratic Republic of Congo and Angola) and west Sudan. Subnational 170 R. E. Howes et al. heterogeneity was important in some areas, for example ranging from 30% around Ibadan to 2% in northwest Nigeria. Prevalence of the deficiency decreased at the continental extremities: in the western Sahel, southern Africa and the Horn of Africa. Prediction uncertainty across the continent was heterogeneous, being very high in areas lacking data such as central Africa between the Democratic Republic of Congo and Madagascar, and the Sudan–Chad border. Additional G6PD community surveys are imperative to reduce the map’s high uncertainty, and caution with primaquine administration must reflect the high prevalence of the condition. There is a notable absence of G6PD variant surveys from Africa (Fig. 4.6D), which may be in part associated with the presumption of low G6PD genetic heterogeneity among those populations. As a consequence, many of the community G6PD surveys conducted do not use phenotypic diagnostics to identify deficient individuals and instead use only molecular methods to detect a narrow range of variants. These data cannot therefore inform the relative prevalence of variants among deficient individuals. Available surveys indicated that the A- variant was predominant across deficient individuals in sub-Saharan Africa (Burkina Faso and the Comores), with the exception of a Sudanese study which identified a greater diversity, including the Mediterra- nean variant (Saha and Samuel, 1991). The Mediterranean variant was common (>50%) in two investigations of deficient individuals in Saudi Arabia.

7. EVOLUTIONARY DRIVERS OF THE DISTRIBUTION OF G6PD DEFICIENCY The widespread distribution and frequent high prevalence of G6PD deficiency—a genetic disorder associated with important clinical costs— presents an evolutionary paradox. The spatial overlap between G6PD deficiency and the precontrol distribution of malaria (Lysenko’s map of precontrol malaria is reprinted in Chapter 1 of Volume 80) was first remarked upon by Motulsky (1960) and Allison (1960) shortly after the description of G6PD deficiency and led Allison and Clyde (1961) to propose Haldane’s Malaria Hypothesis (Haldane, 1949) as an explanation for the natural selection of this deleterious condition. This idea, which had recently been substantiated by empirical evidence for the sickle-cell mutation (Allison, 1954), implied that the deleterious G6PD deficiency condition carried, at least in some genotypes (homo- or heterozygotes), a selective survival advantage over normal enzyme levels against malaria morbidity or mortality: a possible case of bal- ancing selection (Haldane, 1949). This has attracted much attention over the past half century with evidence from epidemiological observations, in vitro G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 171 laboratory findings studies and in vivo clinical studies strongly supporting the hypothesis that this common genetic trait has been selected for by malaria through conferring some degree of resistance against the severity of the infectious disease. A number of excellent reviews of this body of work have been published (Greene, 1993; Hedrick, 2011; Kwiatkowski, 2005; Luzzatto, 1979, 2004; Ruwende and Hill, 1998; Tripathy and Reddy, 2007).

7.1. Evidence of a Selective Advantage 7.1.1. Epidemiological Evidence The most convincing epidemiological evidence of selection by malaria is the sheer diversity of variants of the G6PD gene, many of which have reached polymorphic frequencies in genetically isolated populations suggesting the independent selection of each variant: an apparent case of convergent evolution by a common agent of selection, perhaps. Given that all polymorphic variants are found in historically malaria endemic regions, it would appear that these two diseases, infectious and congenital, are somehow associated. Micro-mapping studies across altitudinal or climatic gradients of malaria endemicity also suggest that the prevalence of this disorder is the result of selection by malaria, rather than random drift or selection (Luzzatto, 1979; Ruwende and Hill, 1998). A recent study from Sumba island in Indonesia provides an impressive example of G6PD deficiency frequencies correlating with a strong malaria endemicity gradient over short spatial distances (Satyagraha, unpublished data). G6PD deficiency prevalence ranged from 2.2 to 3.8% in Central Sumba where malaria endemicity was low, to as high as 11% in highly endemic malaria hotspots to the west and southwest of the island where P. falciparum was found year-round together with seasonable P. vivax endemicity. While these observations provide no evidence of causality, the spatial association between these two diseases is evident.

7.1.2. In vitro Evidence In vitro studies have demonstrated unambiguously that parasitaemia is less successful in G6PD deficient cells than in wild-type cells (Cappellini and Fiorelli, 2008; Roth et al., 1983). The clearest demonstration of this was by Luzzatto et al. (1969), who compared parasitaemia in both cell types in heterozygotes (studying heterozygotes overcame the potentially confounding effect of different levels of acquired immunity which would exist between different individuals). Cells with normal enzyme levels were 2–80 times more likely to be infected than deficient cells. Cappadoro et al. (1998) examined P. falciparum intracellular development and identified selective early-stage phagocytosis of infected G6PD deficient cells as a possible 172 R. E. Howes et al. protective mechanism. Although they found no significant difference in the growth and development of P. falciparum parasites between normal and deficient cells (Mediterranean variant), infected deficient cells were 2.3 times more intensely phagocytosed when parasites reached the early stages of the schizogonic developmental cycle, early in the erythrocytic stages of infection.

7.1.3. Case-Control In vivo Evidence Large-scale case-control studies look for a protective effect against malaria at the population level. Specifically, this in vivo evidence is drawn on to identify which G6PD deficient genotypes the selective advantage is conferred upon: hemi- and homozygotes, or heterozygotes? While studies all concur in identifying a selective advantage associated with G6PD deficiency, the particular genotypes benefitting vary between studies, with data seemingly supporting all scenarios. For instance, a study in southwest Nigeria reported significantly lower parasitaemia in heterozygous females but not hemizygous males (Bienzle et al., 1972). Subsequent large case-control studies used clinical symptoms rather than parasitaemia as the indicator of protection, and found robust evidence for a protective role of the G6PD A- variant (G202A) in hemizygous males and homozygous females in sub-Saharan Africa (Guindo et al., 2007; Ruwende et al., 1995); the protection extended to heterozygous femals remained contentious. Ruwende et al. (1995) surveyed children in The Gambia and Kenya and found a 46% reduction in risk from severe malaria in A- heterozygotes, similar to the 58% protection estimated against severe malaria in hemizygotes. This study compared mild or severe malaria against community controls who were asymptomatic or parasite-free. A comparable case-control study in most respects was subsequently conducted in Malian children (Guindo et al., 2007), except that the control samples used were of uncomplicated malaria cases, rather than asymptomatic/malaria-free cases, a reflection of the local hyperendemic malaria transmission. This study found no protection conferred by this same A- mutation against heterozygous females (n = 221), in spite of a very similar positive result for hemizygotes. As well as the different indicators of parasitic protection (parasitaemia vs. clinical symptoms) and the different control groups (asymptomatic/malaria free vs. mild parasitaemia), further difficulty in comparing case-control studies may be introduced by the method used to diagnose the enzyme deficiency. Johnson et al. (2009) demonstrated in Uganda how differences between phenotypic and genotypic diagnostics can affect the apparent susceptibility to malaria of different G6PD statuses, with significant protection G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 173 from uncomplicated malaria only identified in phenotypically deficient females.

7.2. Neglect of the Selective Role of P. vivax as a Driver of G6PD Deficiency Most early studies have focussed on the protective role of G6PD deficiency on malaria in Africa, and thus considered only a narrow representation of the G6PD gene’s overall genetic variation and clearly neglected a potential role for P. vivax, despite this parasite having a wider transmission range than P. falciparum (Guerra et al., 2010) and causing significant morbidity and mortality (Chapter 3 of V olume 80 and Price et al., 2007). An important life cycle difference between these parasites is P. vivax’s preference for infecting reticulocytes (Anstey et al., 2009; Kitchen, 1938), which could confer a much greater fitness cost on the host by hindering regeneration of the erythrocyte pool. From the host perspective, G6PD enzyme activity levels in reticulocytes are at their highest. If a deficiency in enzyme activity can convey a protective advantage against severe clinical symptoms, then the deficiency would need to be particularly severe to be expressed in the reticulocyte stages. In theory, therefore, P. vivax could be exerting much stronger selection pressures on the host than P. falciparum, selecting more severe variants of the G6PD gene; the generally asymptomatic nature of these mutations would mean that the fitness cost of severe deficiency would not always be felt. Indeed, G6PD Mediterranean, one of the most severe polymorphic variants (<1% residual activity), has been found to offer significant protection against symptomatic P. vivax among male and female Afghan refugees in Pakistan (Leslie et al., 2010). Males and homozygous females were more significantly protected than heterozygotes in whom only weak protection was found. This study offered no comparison with protection against P. falciparum, however, as it accounted for only 5% of infections in the study area. Another study, in an area of co-endemic P. falciparum and P. vivax malaria in Thailand (Louicharoen et al., 2009) found evidence of P. vivax having been the selective agent of the G6PD Mahidol variant, which is common across parts of southeast Asia (Section 6.1, p. 166). In this very comprehensive study, an evolutionary approach using extensive single-nucleotide analysis (SNP) around the G6PD gene locus showed high homogeneity between haplotypes of the Mahidol mutation (G487A), indicating that the mutation had undergone recent and strong positive selection, thus suggestive of conferring a strong advantage to human survival. Clini- cal studies indicated that this survival advantage was conferred as protection against P. vivax parasitaemia, having no effect on P. falciparum parasitaemia. 174 R. E. Howes et al.

Moving forward, a number of fascinating questions remain unanswered. For instance, the large number of G6PD mutations which have reached polymorphic frequencies is of interest, as is the apparent lower diversity among African populations than others. Estimates of the ages of some of these mutations propose relatively recent origins, which may explain part of this diversity. Estimates range from 1000 to 6357 years for the A- variant (Sabeti et al., 2002; Slatkin, 2008; Tishkoff et al., 2001), 3330 years for the Mediterranean variant (Tishkoff et al., 2001), and 1575 years for the Mahi- dol variant (Louicharoen et al., 2009). Further study of the ages of a range of variants would allow a comprehensive picture of the evolutionary history of this condition, and its association with the spread of human Plasmodium infections. Studies should consider both the role of P. vivax as well as P. falciparum to allow the relative selection pressure of the two parasites to be determined with respect to specific variants. The role of P. vivax as a selective agent of human polymorphisms is further discussed in Chapter 2 of this volume, particularly in reference to the protective role of the Duffy negativity blood group.

8. PRIMAQUINE, P. VIVAX AND G6PD DEFICIENCY Primaquine has a vital and unique role in the malaria elimination toolkit, fulfilling three critical functions: first, it is the only licenced radical cure of P. vivax; second, primaquine is the only drug active against mature, infectious P. falciparum gametocytes making it vital for blocking transmission; and third, in areas of emerging drug resistance, primaquine is being used in containment programmes to prevent the spread of artemisinin resistant P. falciparum strains (WHO, 2011b). These invaluable properties make understanding the triangle of interplaying aspects determining primaquine- induced haemolytic risk crucial: the human enzyme, the drug and the parasite. The relationship between P. vivax and primaquine is considered in detail in Chapter 4 of V olume 80, and not revisited in detail again here. We consider here means of safely administering the 200 mg total dose of primaquine required for P. vivax radical cure (Alving et al., 1953; Baird and Hoffman, 2004; Baird and Rieckmann, 2003; Coatney et al., 1953; Edgcomb et al., 1950). First, we review the molecular mechanisms by which primaquine triggers haemolysis, second how haemolytic risk varies according to primaquine dosing regimens, and third, how different G6PD variants modulate haemolytic risk and severity. Understanding primaquine’s pharmacological properties and biochemical effects on the cell is necessary for the development of safer regimens, and alternative safer drugs. G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 175

8.1. Mechanism of Primaquine-Induced Haemolysis It is well established that primaquine-induced haemolysis does not occur in individuals with normal levels of G6PD activity (Baird et al., 2001; Bunnag et al., 1994; Edgcomb et al., 1950). Furthermore, haemolytic risk is greatest in the oldest RBCs, corresponding to an increasing risk as enzyme activity decays over time (Beutler, 1994; Beutler et al., 1954). These indications suggest that primaquine-induced haemolysis is directly associated with the consequences of reduced G6PD enzyme activity. However, the mechanism by which this occurs remains uncertain, and the instability and diversity of primaquine metabolites make studying this system exceedingly difficult. Despite having been in circulation since the 1950s when clinical use of primaquine began, relatively little work has been done to elucidate the precise molecular events leading to primaquine-induced AHA. Understanding these may be critical in rationally disassociating the haemolysing toxicity of the drug from its broad-acting therapeutic properties (Pybus et al., 2012) in developing superior therapies. Although the focus here of primaquine side-effects is the haemolytic risk to G6PD deficient individuals, it should also be noted that primaquine can cause a number of other side-effects, previously reviewed (Baird and Hoffman, 2004; Hill et al., 2006). For instance, abdominal pain is a common dose-dependent side-effect (Edgcomb et al., 1950; Hill et al., 2006), which can be prevented through simply taking the pills with food (Clayman et al., 1952). Primaquine also routinely causes a relatively mild, although occasionally symptomatic methaemoglobinaemia (typically about 6% for as long as dosing lasts), further discussed below.

8.1.1. Primaquine and its Metabolites Primaquine is rapidly excreted, with an elimination half-life of about 4 h (Carson et al., 1981; Greaves et al., 1980), and metabolises into a complex array of a dozen or so distinct moieties. One metabolite is carboxy- primaquine, which has been detected in plasma within 30 min of dosing, reaching plasma concentrations 10-fold higher than primaquine (Mihaly et al., 1985). Carboxy-primaquine, however, appears physiologically inert, both with respect to toxicity and therapeutic activity. On the other hand, some moieties are highly unstable and oxidatively volatile. For instance, 5-hydroxy-6-methoxy-8-aminoquinoline (5H6MQ) was 2500 times more potent than primaquine in stimulating the PPP in normal RBCs (Baird et al., 1986b). It is likely to be one or several metabolic products of primaquine which is the active agent against the parasites, rather than the parent compound itself (Beutler, 1969; Carson et al., 1981; Fletcher et al., 1988). 176 R. E. Howes et al.

The molecular events triggering haemolysis remain unproven, though several mechanisms of action have been proposed, including the build-up of methaemoglobin (metHb) causing damage to the cell membrane, and oxidative damage provoked by primaquine metabolites. The relative significance of these pathways remains unclear. In vitro haemotoxicity assays have demonstrated that it is cytochrome P450-linked pathways which metabolise the breakdown of primaquine by redox reactions into its haemotoxic metabolites, including the formation of metHb and the generation of reactive oxygen intermediates (Ganesan et al., 2009), in a dose-dependent response (Ganesan et al., 2012); the assay found no haemotoxic effect of primaquine when exposed to cells in the absence of these cytochromes. Further study with isoenzyme activity screening and steady state kinetic data has identified two cytochrome P450 enzymes (MAO-A and 2D6) to be strongly involved in primaquine metabolism, and reported that the metabolites generated by these enzymes made it likely that the drug’s toxicity and efficacy were enabled by the same single-cytochrome pathway, making it unlikely that these two properties of the drug could be separated (Pybus et al., 2012). Further study is required to support these findings and determine the relative influence of inhibitors and inducers of these specific cytochrome enzymes to test their individual effect on the drug’s efficacy and toxicity. Similarly, while a number of primaquine metabolites have been identified, none have been definitively associated with activity against the Plasmodium parasite (Baird and Hoffman, 2004; Myint et al., 2011).

8.1.2. A Role for Oxidative Stress Oxidative stress caused by primaquine metabolites has been long-held as the favoured hypothesis for explaining the drug’s toxicity. Reduced levels of G6PD enzyme activity leave the cell with diminished anti-oxidant reserves, namely NADPH and reduced glutathione (Flanagan et al., 1958), due to the constrained rate of the PPP, as already discussed. Possible oxidative agents which have been proposed include free radicals, such as activated oxygen, and hydroxylated metabolites of primaquine which auto-oxidise into qui- noneimine products, superoxides, hydroxyl radicals and hydrogen peroxide (Brueckner et al., 2001; Fletcher et al., 1988); with oxidative metabolites generated by the cytochrome P450 enzymes previously described (Ganesan et al., 2009, 2012; Pybus et al., 2012). Oxidised glutathione, which accu- mulates during oxidative stress, has been found to be a strong intracellular mediator activating membrane cation channels (Koliwad et al., 1996). The opening of these Ca2+-permeable channels can trigger cell death (apoptosis) during oxidative stress (Lang et al., 2003, 2006), although a recent study G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 177 by Ganesan et al. (2012) did not find this mechanism to be triggered by the primaquine-induced haemolytic pathway. Another suggested mechanism whereby depletion of reduced glutathione can lead to cell death was described by Bowman and colleagues, who demonstrated that primaquine- associated oxidative stress (triggered in their experiments by exposure to 5-hydroxyprimaquine) induced oxidative injury to the erythrocyte cytoskeleton (Bowman et al., 2005b), accelerating the process of cell phagocytosis (Bowman et al., 2005a).

8.1.3. A Role for Methaemoglobin The role of metHb accumulation has been proposed to be highly significant, with oxidised primaquine derivatives, such as quinones or iminoquinones (including 5-hydroxyprimaquine) found strongly associated with the formation of metHb (Link et al., 1985). Oxidative stress from primaquine can lead to the oxidation of haemoglobin iron (Fe2+ → Fe3+) and the build- up of metHb. This resulting condition, methaemoglobinaemia, is usually asymptomatic and self-limiting if only low levels accumulate (Brueckner et al., 2001; Fernando et al., 2011). At higher proportions, however, methaemoglobinaemia can result in tissue hypoxia, hypoxemia and cyanosis due to metHb’s low affinity for oxygen (Hill et al., 2006; Percy et al., 2005). However, exacerbated methaemoglobinaemia is not a feature of clinical acute haemolytic anaemia in G6PD deficient patients.

8.1.4. A Role for Altered Redox Equilibrium Other experimental evidence points away from damage mediated through oxidative degradation of the RBC cytosol or membrane. Baird and colleagues observed that the increased PPP activity stimulated by primaquine metabolites occurred independently of glutathione redox activity. They observed that when sodium nitrite was applied to the system, glutathione redox stimulated the PPP. When the dose of sodium nitrite exceeded the ability of the PPP to maintain steady state redox equilibrium, a proteolytic system that degraded irreversibly oxidised proteins became active (Baird et al., 1986b). In contrast, doses of primaquine metabolites that also satu- rated PPP activity came with no such proteolytic activity, and the drugs were not mediating oxidative damage like sodium nitrite. It appeared that the drugs themselves drove NADPH depletion and the PPP response to that disturbed equilibrium (Baird et al., 1986a). In other words, redox equilibrium between a reduced and oxidised species of primaquine would, in a G6PD deficient cell, strongly favour the oxidised species, without 178 R. E. Howes et al. necessarily prompting a broad oxidative degradation of cytosol proteins (Brueckner et al., 2001). The oxidised species could be the agent of haemolysis. Brueckner et al. (2001) proposed a mechanism similar to that demonstrated for the well-characterised molecular events in phenylhydra- zine-induced haemolysis in rats. They postulated that the covalent linkage of a metabolite to the haem moiety of haemoglobin could force the molecule from its globin fold by simple hydrophobic force into the lipid bilayer of the RBC membrane. Any mechanism of haemolysis must ultimately be reconciled with what is highly likely to be a very brief and quantitatively insubstantial oxidative challenge to the RBC by primaquine. The daily 15 or 30 mg dose may be appreciated as an exceedingly small quantity of molecules relative to its distribution in tissue, rapid metabolism to a dominant and inert species, and very rapid elimination. The oxidative challenge must be very insubstantial and fleeting compared with, for example, nitrite poisoning. Although a mild methaemoglobinaemia typically occurs with normal primaquine dosing (Carmona- Fonseca et al., 2009), it does not appear linked to depletion of GSH via a generalised oxidative attack of RBC proteins. The damage being done, whatever it may be, seems irreversible during the intervals between dosing. The damage appears cumulative across doses, with haemolysis not commencing in earnest until after the third or fourth dose. These features appear incompat- ible with a general oxidative stress and would point to an irreversible capture of the harmful primaquine species, or the damage done by it, in the RBC. An accumulation of displaced haem molecules in the RBC membrane, manifest as Heinz bodies, is compatible with all of these features.

8.1.5. Significance of Primaquine-Induced Haemolysis Whatever the mechanism, be it the build-up of metHb, direct oxidative damage, the balance of redox equilibrium, or any other pathway, the physiological damage caused to the cells leads to intravascular haemolysis, making acute haemolytic anaemia the main clinical symptom. Freely circulating haemoglobin from the haemolysed cells causes the most severe and potentially lethal conditions, including haemoglobinuria and acute renal failure (Burgoine et al., 2010). Understanding the mechanisms by which primaquine induces haemolysis is critical to developing safer administration of primaquine. In the same way that knowledge of the dose-dependency effect of primaquine (described in the next section) has allowed treatment regimens to be extended over a number of weeks to reduce their toxicity, further manipulations to improve safe dosing schedules would G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 179 likely be possible given a better understanding of its mode of action. For example, primaquine interaction with co-administered schizon- ticides has been suggested to affect its toxicity (Myint et al., 2011). If confirmed, this could prove a relatively straightforward solution towards safer therapy. Improvements to diagnostic methods by ensuring that their target corresponds to the predisposing factor to haemolysis might also be possible with an improved understanding of the predictors of haemolytic susceptibility. Another main motivation for studying the biochemical processes of primaquine action is pharmaceutical: the potential for disassociating the drug’s therapeutic properties from its toxicity. If feasible, this could lead to safe and effective killing of hypnozoites in P. vivax therapy and control.

8.2. Factors Affecting Haemolytic Risk While the biochemical mechanisms of primaquine-induced haemolysis remain uncertain, it is clear that the risk and severity of haemolysis is highly variable, affected by both exogenous and endogenous factors (Beutler, 1994). Generalisations about haemolytic risk and its clinical severity are frequently cited based on the binary categorisations of “mild” and “severe” enzyme deficiency (Hill et al., 2006; WHO, 2010, 2011a). In reality, a spectrum of clinical severity exists, ranging from asymptomatic to lethal, which is determined by several factors including the primaquine dose and the genetic and biochemical determinants of enzyme activity levels. Many studies investigating haemolytic risk aggregated all phenotypically “deficient” individuals, sometimes speci- fying “mild” or “severe” deficiency (Hill et al., 2006; Recht et al., 2012); here we consider reports relating to specific genetic variants. The primaquine sensitivity phenotypes of three have been investigated in detail, and we discuss these here.

8.2.1. Dose Dependency The dose dependency of the haemolytic effect of primaquine was noted from the first clinical investigations into primaquine, with observations that toxicity appeared to be cumulative: “in some instances symptoms began late in the course of drug administration and continued for several days after its discontinuance” (Edgcomb et al., 1950). The pharmacokinetics of primaquine, however, are unaffected by dose, and its efficacy is contingent upon the total dose administered, rather than the total amount per dose or the timescale over which it is administered (Mihaly et al., 1985). A meta-analysis by Schmidt and colleagues of the early data (1946–1975) on dosing regimens in Rhesus monkeys found that the success of radical 180 R. E. Howes et al. cure is determined by the overall total primaquine dose, independent from the timescale over which it is administered: 1-, 3-, 7- and 14-day regimens were not found to differ in their therapeutic success (Schmidt et al., 1977). This pharmacokinetic property has the advantageous benefit of permitting extended dosing schedules which reduce the risks of haemolysis without impacting on its effectiveness.

8.2.2. Variant Dependency The diversity of G6PD genetic variants leads to a spectrum of residual activity levels and associated clinical severity (Section 3, p. 143). We discuss here how drug regimens which can be tolerated by some G6PD deficient individuals were developed in relation to studies on certain key G6PD variants, and as a corollary, how haemolytic risk is variant-dependent. (a) A- variant. The early Stateville studies into “primaquine sensitivity” were all based on the A- variant, due to the origins of the individual studied. The vulnerability presented by this variant to primaquine-induced haemolysis was recently re-affirmed by a Brazilian study which resulted in severe haemolysis in three G6PD deficient P. vivax patients treated with 30 mg daily doses of primaquine over 5 or 7 days (Silva et al., 2004). The Stateville studies found that daily dosing of the 30 mg regimen led to haemolytic symptoms in affected individuals appearing on the second or third day, peaking 4–7 days after starting treatment, continuing for just over a week overall (Clyde, 1981; Hill et al., 2006; Hockwald et al., 1952). Hae- molysis ended within a few days of ceasing treatment, after which haemoglobin levels recovered; recovery even occurred with continued treatment as haemolysis was compensated by erythropoiesis and the regeneration of reticulocytes which have inherently higher G6PD levels. The drug’s total dose effect has allowed regimens with an acceptable level of toxicity for G6PD deficient individuals to be developed. Studies by Alving et al. found that “intermittent” weekly 45 mg doses of primaquine over 8 weeks was effective (treating 90% of P. vivax Chesson strain infections; n = 40) and did not produce any clinically significant haemolysis (the number of G6PD deficient individuals was not specified) (Alving et al., 1960). Subsequently, Brewer and Zarafonetis (1967), studying individuals of African origin, confirmed that twice-weekly administration of 45 mg primaquine triggered more haemolysis than once-weekly dosing in deficient individuals; no haemolysis was reported from individuals with no deficiency. A review by Clyde (1981) determined that daily 15 mg primaquine doses for 14 days would be safely tolerated, with any side-effects remaining largely G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 181 asymptomatic; but recommended the extended 8-weekly 45 mg dosing schedule. As such, current WHO recommendations for adults with “mild” G6PD deficiency are for 8-weekly 45 mg doses of primaquine (Hill et al., 2006; WHO, 2010, 2011a). As well as averting the severity of haemolysis, another benefit of extending the dosing schedule is the opportunity for affected G6PD deficient patients to discontinue treatment before the severity of haemolysis becomes too serious. In these “mild” cases, haemolysis is self-limiting and ends a few days after stopping treatment. However, a review by the American Centers for Disease Control and Prevention (CDC) recommends great caution when administering primaquine to individuals with any degree of G6PD deficiency, and emphasises that very careful risk assessments and strict medical supervision are essential (Hill et al., 2006). The authors note that this weekly dosing schedule for G6PD deficient individuals has not been approved by the FDA. Indeed, case reports of severe adverse effects to these “safe” regimens exist, including even severe reactions in individuals once deemed to be at especially low risk. For instance, a heterozygote with the A- variant (G202A mutation) was reported to suffer a clinically severe haemoglobin drop to <5 g/dl following a single 45 mg dose of primaquine (Shekalaghe et al., 2010). A possible reason for this unexpected outcome may stem from a misdiagnosis, as only one locus in the gene was tested in the mutation analysis and other more severe variants would not have been identified. Recent studies of primaquine in individuals with the A- variant are few (Eziefula et al., 2012) and focus on the drug’s application to P. falciparum gametocyte clearance – single dose of 0.75 mg/kg primaquine in combination with artemisinin combination therapy (ACT) (Shekalaghe et al., 2007, 2010). However, a clinical trial in several African countries of individuals with the A- variant with another drug-trigger of AHA in G6PD deficient individuals, dapsone, resulted in high rates of haemolysis, with 10.9% of hemi- and homozygotes requiring transfusion following 150 mg/day dosing over 3 days.3 Authors analysing these outcomes conclude that, contrary to current perception, the A- variant cannot be considered “mild” (Pamba et al., 2012). (b) Mahidol variant. This is an important variant among populations from Myanmar and Thailand, and is reported to cause residual activity to drop to

3 Direct comparison on the haemolysing effect of dapsone in relation to primaquine was investigated during the Stateville trials, when a G6PD deficient African male was exposed on different occasions to daily doses of 100 mg dapsone for 21 days, and 30 mg of daily primaquine doses for 18 days (Degowin et al., 1966). The haemolytic effect was less marked with dapsone. 182 R. E. Howes et al.

5–32% of normal levels (Louicharoen et al., 2009). A study investigating the effects of primaquine has been conducted in a relatively small number of P. vivax-positive G6PD deficient individuals (n = 22) in Thailand who were given 15 mg primaquine for 14 days with standard chloroquine treatment (Buchachart et al., 2001). No serious adverse effects were subsequently reported during the 28-day follow-up period, though a 24.5% (±13.9) drop in haematocrit was observed in the G6PD deficient patients with an equivalent only 1.2% (±14.4) drop in G6PD normal individuals. No patient required transfusion. This study concludes that standard primaquine therapy would be safe in Thailand, even for those G6PD deficient (Buchachart et al., 2001). Although these individuals were only diagnosed as deficient phenotypically, the authors reported the Mahidol variant predominant in the study area. Polymerase chain reaction (PCR) diagnosed individuals from a similar study in Thailand were all diagnosed as carrying Mahidol variant; no serious adverse effects were reported from this study of 14-day primaquine therapy (Takeuchi et al., 2010). A similar attitude is presented from an economic perspective by Wilairatana et al. (2010) who determined that in the absence of widely available G6PD testing at the malaria clinic level, only patients who develop black urine or anaemia should be tested for G6PD deficiency, and that “mild-moderate” deficient cases should be pre- scribed the 8-weekly dose. A study from Myanmar where 22 G6PD deficient individuals were given the 8-weekly 45 mg primaquine dosage also reported no severe adverse effects (Myat Phone et al., 1994), and similarly concludes that weekly dosing is safe for G6PD deficient individuals in Myanmar. The authors of these studies therefore encourage blind primaquine therapy in this region, where G6PD deficiency allele frequency is high: estimated to be 13.6% (IQR: 11.9–15.5) in Thailand and 6.1% (IQR: 4.1–9.3) across Myanmar (Howes et al., 2012). The WHO specifies that primaquine should be used for P. vivax radical cure in this region, but that prior G6PD screening is necessary (WHO, 2010). Given the difficulties of field-based G6PD diagnosis, it is uncertain whether clinicians actually risk administering primaquine ( John et al., 2012). The evidence-base for these recommendations, however, is relatively meagre (Recht et al., 2012). The heterogeneity of local variants reported from this region (Fig. 4.6A) is also an important reason to exercise great caution, even if a handful of studies have reported no serious adverse reactions to primaquine, these studies may not have included patients with severe G6PD variants. For instance, a more severe variant, Viangchan, is also common in Thailand, and along the Cambodian and Laos borders. G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 183

In spite of these risks, the use of primaquine in mass drug administration has been investigated in Cambodia for P. falciparum transmission control. Very low dosing (9 mg/10 days for 6 months; n = 6040) administered alongside an ACT without G6PD testing (despite local prevalence being 18.6% in males) was not found to cause any severe adverse effects (Song et al., 2010). Importantly, however, no active monitoring was in place to detect these. This very low dosing, therefore, was deemed safe for P. falciparum transmission control; however, this dosing bears little relation to P. vivax radical cure, and would not be logis- tically feasible as a standard therapy due to its very resource-intensive dosing. (c) Mediterranean variant. Originally known for its association with the clinical pathology of favism, the Mediterranean variant causes one of the most severely deficient phenotypes, reducing enzyme activity to <1% of normal levels (Beutler and Duparc, 2007). Serious haemolysis is caused by 15 mg daily or 45 mg weekly courses of primaquine (Clyde, 1981) and, unlike with the A- variant, haemolysis in G6PD Mediterranean individuals is not self-limiting. A review by Clyde concluded that individuals affected by the Mediterranean variant should be administered supervised weekly doses of 30 mg for 15 weeks (Clyde, 1981). WHO guidelines today, however, state that no primaquine should be given to individuals with so severe a deficiency (WHO, 2010, 2011a).

8.2.3. Red Blood Cell Age Dependency Senescent RBCs are most likely to succumb to haemolytic challenges (Beutler et al., 1954). Wild-type erythrocytes appear to have a large surplus of potential G6PD activity, allowing the PPP to be significantly upregulated when exposed to oxidative stress (Salvador and Savageau, 2003). This enzyme activity decays naturally with RBC age (as erythrocytes lack nuclei and therefore have no mechanism for regenerating enzyme levels), correlating with susceptibility to haemolytic risk. This decay was found to be exponential, with a half-life of 62 days for wild-type enzyme and 13 days for A- enzyme (Piomelli et al., 1968). The more severe variant, Mediterranean, had such a rapid decline that no enzyme activity could be detected in mature erythrocytes. Cells with the A- genetic variant were demonstrated to be insensitive to primaquine when 8–21 days old, but were rapidly destroyed 55 days later (corresponding to slightly older than half their normal lifespans) when re-exposed to primaquine (Beutler et al., 1954). In wild-type individuals natural enzyme decay does not reach levels which put the individual at clinical risk; in G6PD deficient cells, however, the ageing process being so much more marked than in normal 184 R. E. Howes et al. cells, means that even moderately deficient cells will have enzyme activity levels that drop to clinically at-risk levels. If erythropoiesis can replace haemolysed cells, then the clinical effect will be negligible and the haemolysis self-limiting once all susceptible cells have been destroyed (Alving et al., 1960; Dern et al., 1954a).

8.2.4. Sex Dependency Haemolytic risk is also sex-dependent. Although primaquine’s fundamental pharmacokinetics are not affected by gender (Cuong et al., 2006; Elmes et al., 2006), the majority of affected females are heterogeneous and therefore present less commonly with severe symptoms than males. In areas of high prevalence, however, homozygote inheritance of deficiency can be common (Howes et al., 2012), and heterozygous females can also suffer severe haemolysis (Pamba et al., 2012; Shekalaghe et al., 2010). The population of RBCs carrying the deficiency are at equal haemolytic risk as homozygous or hemizygous cells. The relative proportion of wild-type and deficient cells will have a major influence in determining the overall clinical severity of haemolytic stress at the individual level. Interactions with other genetic blood disorders may also exacerbate the effects of the deficiency in females (Chopra, 1968). 8.3. Predicting Haemolytic Risk The core motivation for understanding G6PD deficiency in the context of P. vivax control is in being able to ascertain haemolytic risk and prevent adverse drug events from primaquine therapy. However, while much research has been done and the complexity of interacting factors has been clearly demonstrated, key knowledge gaps still hinder reliable predictions of haemolytic risk. The main assumption made when assessing haemolytic risk is that enzyme activity levels can be a reliable indicator. Although this would appear to be generally true, exceptions exist, such as an Iranian boy with 19.5% residual activity requiring a transfusion after a single 45 mg dose of primaquine (Ziai et al., 1967), and a heterozygote Tan- zanian experiencing severe adverse reaction also following a single dose (Shekalaghe et al., 2010); a detailed record of adverse drug events has been compiled (Recht et al., 2012). While these cases are exceptions, from a clinical perspective, interpretation of diagnostic outcomes must allow for the uncertainties in these relationships. The practical implications of these considerations and difficulties in assessing haemolytic risk are discussed in the next section. G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 185

9. TOWARDS A RISK FRAMEWORK FOR P. VIVAX RELAPSE TREATMENT In this final section, we consider how the current state of understanding about G6PD deficiency and primaquine may be practically applied to promoting safe radical cure of P. vivax. G6PD deficiency is widespread, predicted in all malaria endemic countries, with an overall estimated allele frequency of 8.0% (IQR: 7.4–8.8), as discussed in Section 5.2 (p. 161). Given its potential clinical severity, primaquine cannot be administered without careful prior assessment of risk. This haemolytic risk may be considered at two scales: (i) large regional scales for public health perspectives, and (ii) directly by clinicians in relation to individual-level treatment decisions. Once risk has been satisfactorily judged, primaquine can be administered, or withheld, accordingly. Important limitations hinder risk assessment at both scales, however, and we discuss necessary developments towards overcoming these and improving safe access to P. vivax radical cure.

9.1. Assessing National-Level Haemolytic Risk of Primaquine Therapy Knowledge of the spatial characteristics of G6PD deficiency can be cou- pled with information about clinical phenotypes to allow comparisons of haemolytic risk between regions at scales of public health significance. It is important to note that assessment of risk at such large spatial scales cannot inform risk at the level of the individual, and can never replace the need for careful oversight of primaquine therapy by clinicians (Hill et al., 2006), even in areas considered to be at low risk from a public health perspective. A simple national-level framework assessing large-scale risk has been proposed (Howes et al., 2012), but important limitations to the data informing this analysis make it only a coarse-scaled and crude framework: one which must be refined as our understanding of clinical risk improves.

9.1.1. Proposed Framework for Ranking National-Level Risk from G6PD Deficiency Current WHO treatment guidelines consider haemolytic risk to differ between “mild” and “severe” classes of deficiency. “Mild to moderate” cases of deficiency may be treated with 8 weekly 45 mg doses, while “severely” deficient individuals should not be administered any primaquine 186 R. E. Howes et al.

(W HO, 2010, 2011a). Consequently, relative haemolytic risk from primaquine due to G6PD deficiency at the population level may be considered to be contingent upon two factors: (i) the overall prevalence of deficiency within that population, and (ii) the relative composition of mild and severe genetic G6PD variants reported from that population. The simple framework suggested by Howes et al. (2012) scores the national prevalence of deficiency based on the national allele frequency estimates described in Section 5.2 (p. 161), and assigns variant severity scores using a database of documented reports of G6PD variants to assess the relative proportion of Class II and Class III variants nationally (WHO-endorsed subdivisions of the Type 2 category of variants; Table 4.1 and WHO Working Group, 1989). An overall risk score was obtained for each country by multiplying the prevalence score by the variant severity score to give six categories of risk across a spectrum from “rare and mild” G6PD deficiency to “common and severe” G6PD deficiency (Fig. 4.8). Overall, this simple risk analysis ranked the highest level of G6PD deficiency risk as being in the Asia and Asia–Pacific regions where severe variants were reported and population prevalence of deficiency was common (>1% allele frequency). High prevalence of deficiency caused by predominantly Class III variants across sub-Saharan African countries led to moderate levels of risk in this region. Risk scores in the Americas ranged from low to moderate. National-level scores are mapped in Fig. 4.8; further details about the methodology employed are given in the original publication (Howes et al., 2012).

9.1.2. Important Limitations to Predicting National-Level Haemolytic Risk Both the underlying assumptions and the value of the described risk stratification are questionable. It is nonetheless useful to attempt to do so with regard to identifying weaknesses and the knowledge gaps that cause them. The analysis’ main assumption is that the severity of haemolysis can be predicted from the genetic variant and that haemolytic severity is adequately represented by the subjective categorisations of Classes II and III. Further, this assumes that primaquine sensitivity phenotypes inversely correlate with residual enzyme activity (categorised here as Classes II and III). Given that the mechanism of primaquine-induced haemolysis remains uncertain (Section 8.1, p. 175) only isolated case reports exist to support this assumption. Although the data from the three variants described in Section 8.2 (p. 179) would appear to support this correlation, the evidence underpinning this relationship across all variants is not robust, and G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 187 extrapolating risk to all variants based on their WHO classifications is therefore very uncertain. Furthermore, the evidence used to determine the classifications of variants into their corresponding WHO Class (tabu- lated by Beutler, 1993; Kwok et al., 2002; Luzzatto et al., 2001; Minucci et al., 2012; UCL Bioinformatics Group website; Vulliamy et al., 1997; Yoshida et al., 1971) is weak in many cases, being determined from small numbers of samples with variable laboratory techniques (Recht et al., 2012). Given this uncertainty in the risk analysis, only a very coarse classification with three scores of variant severity was used. Finally, the data set informing the national classifications of variant severity is often poor, with data reported from only 54 of 90 malaria endemic countries, meaning that severity scores had to be inferred for many of them. If a robust understanding of the relationship between genetic variants and their primaquine sensitivity phenotypes could be incorporated in this analysis with a more complete background picture of the spatial heterogeneity of the G6PD variants and their prevalence, the conclusions drawn would be much more valuable. Given these gaps in our current understanding, the main robust predictor of haemolysis is the phenotypic deficiency in G6PD enzyme activity. The modelled prevalence map may therefore be the most detailed, reliable, and appropriate risk assessment of overall G6PD deficiency-associated harm – be it moderate or severe – which is relevant to informing public health policy for P. vivax radical cure.

9.2. Assessing Haemolytic Risk at the Level of the Individual Given the numerous testing kits which have been developed to diagnose G6PD deficiency (Section 4, p. 150), assessing haemolytic risk at the level of the individual ought to be straightforward. However, logistical constraints hinder the widespread use of these diagnostic methods in the rural communities where P. vivax is most common and point-of-care testing is most needed. Further, it would appear that many of the available diagnostic methods have not been calibrated to any predetermined and clinically relevant measures of risk, and are seemingly arbitrary in their classifications of deficiency. For instance, the threshold for distinguishing deficient from nondeficient cases ranges from 10% to 60% residual enzyme activity. These assessments of haemolytic risk also assume that enzyme activity is a suitable indicator of primaquine-induced haemolysis. Their poor suitability to diagnosing deficiency in females (Section 4.1, p. 150) is a further hindrance to their suitability for discerning haemolytic risk. No molecular methods are currently suited to field-based settings. 188 R. E. Howes et al.

Although WHO treatment recommendations distinguish “mild” from “severe” deficiency, no details are provided to indicate what these categories correspond to in terms of enzyme activity or associated degree of haemolysis. Given the difficulties with diagnosing these severity types, and the largely unknown clinical risk which “mild” deficient individuals would be exposed to, it has been argued that any G6PD deficient phenotype, regardless of severity, should suffice as a contraindication for primaquine therapy (Baird and Surjadjaja, 2011). Distinguishing the degree of severity is not possible with most diagnostic tests, including the binary point-of-care tests currently in development (Section 4.3, p. 153). Attempting to account for the severity of deficiency in a framework determining safe primaquine therapy may therefore be an unnecessary complication at this stage, and instead it may be more appropriate to re-assign the WHO treatment guidelines with binary options, removing the additional subjective mild/severe distinction. Anecdotally, the importance of simplicity and great caution in determining drug guidelines is well illustrated by a recent case report of a Burmese P. vivax patient, undiagnosed as G6PD deficient, misunderstand- ing his primaquine regimen guidelines due to linguistic barriers, and taking almost his full course of primaquine (165 mg) in one go, resulting in severe haemoglobinuria (Burgoine et al., 2010). Risk assessment with a potentially harmful drug must be especially cautious. Until the evidence underpinning the mechanism of haemolytic risk (Sec- tion 8, p. 174) allows predictability of the haemolytic outcome of different variants with specific primaquine dosages, simple and safe guidelines based on practicable diagnoses appear the most appropriate end-points of haemolytic risk assessments. A better understanding of the mechanism of drug-induced

Figure 4.8 National risk index from G6PD deficiency. Two aspects of G6PD deficiency epidemiology were used to define the national risk of G6PD deficiency: (1) the national prevalence of deficiency was stratified into three classes (≤1%; >1–10%; >10%) based on the national allele frequency estimates described in Section 5.2 (Fig. 4.5); (2) the severity of local variants was classified at the national level based on a literature search of reports of occurrences of G6PD variants. Variants were classified into mild and severe types, in accordance with the WHO-endorsed classification (WHO Working Group, 1989). The relative proportion of reported Class II and Class III variants was used to categorise the severity of variants nationally (Class III variants only; minority of Class II variants, ≤1/3; Class II variants common, >1/3), as shown in Panel A. The two scores were multiplied to given an overall risk score (Panel B and C). Uncertainty in the two scores was scored and ranked between countries (Panel D and E). (Figure from Howes et al. (2012)). (For a colour version of this figure, the reader is referred to the online version of this book). G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 189 190 R. E. Howes et al. haemolysis might allow diagnostic methods to be engineered as predictors of haemolysis, rather than a direct indicator of enzyme activity (which may not prove to be the only determinant of haemolytic risk). The heterogeneity of genetic variants (Fig. 4.6A,B), particularly across areas where P. vivax radical cure is most relevant (Fig. 4.7), supports the potential benefits of improving the diagnostic resolution and tailoring treatments to different variants. How- ever, until the underlying haemolytic mechanisms can be understood, the short-term primary goal must be refinement of binary diagnostics. For individuals diagnosed as G6PD normal, a different set of considerations apply to optimising primaquine treatment options. For instance, there is pressure to reduce treatment duration from 14 days to a 7-day regimen with equivalent efficacy so as to improve compliance (Fernando et al., 2011; Krudsood et al., 2008; Schmidt et al., 1977; Takeuchi et al., 2010). Prerequisite to promoting this higher dosage schedule is a high sensitivity diagnostic of haemolytic risk for G6PD deficiency.

10. CONCLUSIONS The aim of studying G6PD deficiency in the context of P. vivax therapy and malaria elimination is to support safe use of 8-aminoquinoline drugs for radical cure that will enable access to effective, life-saving therapy. At this pivotal time in malaria control, when important progress is being made and vital funding cuts imposed (Garrett, 2012), it is more imperative than ever to maximise efficient use of available tools. As discussed in Chapter 6 of V olume 80, the relapsing P. vivax hypnozoite reservoir makes this parasite life-form the major challenge to patient health and malaria elimination programmes. The only licenced drug active against these parasites is primaquine. However, widespread G6PD deficiency (estimated allele frequency of 5.3% [IQR: 4.4–6.7] in declared malaria eliminating countries, Howes et al. (2012)) and poor associated diagnostics result in primaquine being under-used. While great caution must be taken in dealing with a potentially haemolysing drug, informed caution may increase access to this drug. Although a good deal is known about the molecular characteristics of the G6PD enzyme and its clinical manifestations as favism and NNJ, for instance, the disorder’s interactions with P. vivax and primaquine remain much less well understood. This is likely to be a symptom of the higher priority which has been placed in recent decades on therapy against the asexual blood stages of P. falciparum (Baird, 2010), to the neglect of most other therapeutic targets, such as P. vivax radical cure. As such, a recurring G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy 191 theme emerging from many sections of this review is a lack of data, tools and understanding. Prioritising these gaps to increase access to safe primaquine can be considered in three steps, though fundamental to all of these is an understanding of how residual enzyme activity levels and genetic variants interact with the underlying mechanisms triggering haemolysis, thereby providing evidence that can guide rationally developed and practical solutions to the problem. 1. Improving point-of-care assessment of haemolytic risk through development of a highly sensitive, practical diagnostic which can be considered a conservative indicator of tolerance of the standard 14-day regimen. It may be necessary to develop separate methods to adequately diagnose deficient males and females (Peters and Van Noorden, 2009; Shah et al., 2012). Adequate diagnostics would greatly increase access to primaquine and could be available in the near future. 2. Extending access to primaquine by identifying dosing regimens of reduced toxicity for G6PD deficient individuals by leveraging unexplored synergies with other drugs. 3. Developing alternative therapies (likely non-8-aminoquinolines) which present no risk to G6PD deficient individuals. The operational inadequacy and potentially mortal threat posed by primaquine due to G6PD deficiency, renders it unfit for purpose in endemic zones in its current form. The dawning realization that acute P. vivax malaria is associated with significant burdens of severe illness and death in endemic zones and no longer misclassified as benign (Price et al., 2007) may finally crystalise the determination of the scientific community to address this 60-year-old problem. There may be no higher priority for malaria research than the triangular G6PD-primaquine-P. vivax problem. If elimination is to be the focus, perspectives on future malaria therapy need to shift away from treatment of symptomatic parasitaemia towards comprehensive treatment for all parasites and multiple life stages (Baird, 2012). Given their unique therapeutic action, the importance of overcoming the dangers of the 8-aminoquinolines cannot be over-emphasised towards meeting this target.

ACKNOWLEDGEMENTS The authors are particularly grateful to Lucio Luzzatto and Pete Zimmerman for valuable comments on the manuscript, and to Jennie Charlton and David Pigott for proof- reading. This work was supported by a Wellcome Trust Biomedical Resources Grant (#085406), which funded R.E.H.; S.I.H. is funded by a Senior Research Fellowship from the Wellcome Trust (#095066) that supports K.E.B. also. A.W.S. is supported by grant #107-13 from the Asia Pacific Malaria Elimination Network (APMEN). J.K.B. is supported 192 R. E. Howes et al. by grant #B9RJIXO of the Wellcome Trust. This work forms part of the output of the Malaria Atlas Project (MAP, http://www.map.ox.ac.uk/), principally funded by the Wellcome Trust, UK.

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Genomics, Population Genetics and Evolutionary History of Plasmodium vivax

Jane M. Carlton*,1, Aparup Das†, Ananias A. Escalante‡ *Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA †Evolutionary Genomics and Bioinformatics Laboratory, Division of Genomics and Bioinformatics, National Institute of Malaria Research (ICMR), Dwarka, New Delhi, India ‡Center for Evolutionary Medicine and Informatics, The Biodesign Institute, Arizona State University, Tempe, AZ, USA 1Corresponding author: E-mail: [email protected]

Contents 1. The Importance of Studying Plasmodium Diversity 204 2. The Evolutionary History of P. vivax 204 3. The P. vivax Genome and Comparative Genomics 207 4. P. vivax Global Genetic Diversity and Population Structure 213 5. P. vivax Population Genetics in India 215 6. Conclusion 218 Acknowledgements 218 References 219

Abstract Plasmodium vivax is part of a highly diverse clade that includes several Plasmodium species found in nonhuman primates from Southeast Asia. The diversity of primate malarias in Asia is staggering; nevertheless, their origin was relatively recent in the evolution of Plasmodium. We discuss how humans acquired the lineage leading to P. vivax from a nonhuman primate determined by the complex geological processes that took place in Southeast Asia during the last few million years. We conclude that widespread population genomic investigations are needed in order to understand the demographic processes involved in the expansion of P. vivax in the human populations. India represents one of the few countries with widespread vivax malaria. Earlier studies have indicated high genetic polymorphism at antigenic loci and no evidence for geographic structuring. However, new studies using genetic markers in selectively neutral genetic regions indicate that Indian P. vivax presents complex evolutionary history but possesses features consistent with being part of the ancestral distribution range of this species. Such studies are possible due to the availability of the first P. vivax genome sequences. Next generation sequencing technologies are now paving the way for the sequencing of more P. vivax genomes that will dramatically increase our understanding of the unique biology of this species.

1.HE T IMPORTANCE OF STUDYING PLASMODIUM DIVERSITY As parasite diversity is one of the fundamental factors governing malaria transmission and immunity knowledge of parasite population genetic structure is necessary to understand the epidemiology, distribution and transmission dynamics of natural malaria parasite populations. This is essential because population genetic structure can predict how fast phenotypes of interest, such as novel antigenic variants, particular relapsing patterns, or drug resistance, arise and spread in natural populations (Brito and Ferreira, 2011). Plasmodium vivax is a major public health challenge for Central and South America, the Middle East, Central, South and South- east Asia, Oceania and East Africa, where 2.85 billion people are currently at risk of infection and 70–80 million clinical cases are reported each year (Guerra et al., 2010; Muller et al., 2009). The emergence of drug-resistant strains and severe (sometimes fatal) disease (Kochar et al., 2009) challenges the traditional view of vivax malaria as a benign infection (Price et al., 2009). Much about the biology, epidemiology and pathogenesis of P. vivax that is essential for control programs remains unknown, although it is now understood that global P. vivax populations are highly genetically diverse, and that this diversity varies greatly according to geographic regions. It has thus become apparent that in order to achieve malaria control and elimination targets, population genetic surveys are vital to map the diversity and structure of local populations and to estimate the likelihood of success and measure the outcome of malaria intervention methods (Arnott et al., 2012).

2. THE EVOLUTIONARY HISTORY OF P. VIVAX Early inferences about the origin of P. vivax were based on limited evidence. An Asian origin was proposed due to the diversity of primate malarias in Southeast Asia and their phenotypic similarity to human malaria parasite species including P. vivax (Coatney et al., 1971). Alternatively, an African origin was proposed based on the fixation of the FY*BES allele (ES is an abbreviation of erythrocyte silent or ‘Duffy negative’) from the Duffy blood group that provides resistance to vivax malaria (Miller et al., 1976), and the existence of Plasmodium schwetzi, a putatively vivax-like parasite in chimpanzees (Carter, 2003; Livingstone, 1984). Genomics, Population Genetics and Evolutionary History of Plasmodium vivax 205

Nowadays, molecular phylogenies support an Asian origin for P. vivax as the result of a host switch from a nonhuman primate (Cornejo and Escalante, 2006; Escalante et al., 2005; Mu et al., 2005). Figure 5.1 depicts a Bayesian phylogeny that includes only those Plasmodium lineages found in Asian primates. The phylogeny includes many species found in Cercopithe- cidae (mostly macaques), P. vivax, the gibbon parasite Plasmodium hylobati, and a recently rediscovered lineage of Plasmodium in orangutan [9]. As the phylogeny shows, there is evidence of several host switches including one that allowed the colonization of humans by the P. vivax lineage (Escalante et al., 2005; Mu et al., 2005). Indeed, the diversity of nonhuman primate malarias in Southeast Asia appears to be the result of an explosive adaptive radiation that took place between 4 to 8 million years ago (Mu et al., 2005; Pacheco et al., 2011, 2012). It is worth noting that the Asian origin model for P. vivax is not based on the number of Plasmodium species parasitic to primates in Southeast Asia (Coatney et al., 1971), but on the fact that malaria parasites found in Cercopithecidae are basal in a monophyletic group that includes P. vivax (Escalante et al., 2005). The closest known species related to P. vivax is Plasmodium cynomolgi, and these two parasites could have diverged as early as 1.3 Mya ago depending upon the molecular clock method used and the assumptions applied (Pacheco et al., 2011, 2012). Whereas phylogenetic evidence supports an Asian origin as the most parsi- monious explanation, it is worth noting that the fixation of the FY*BES allele in Africa does not preclude a more recent introduction of P. vivax from Asia. Natural selection, in order to act, requires population sizes that are more likely in agriculture-based societies (Hedrick, 2011). This seems to be supported by the time estimates for the fixation of the FY*BES allele, timeframes that are all consistent with a relatively late introduction of P. vivax into Africa (Hamblin et al., 2002; Seixas et al., 2002). Regarding P. schwetzi, some considered it related to Plasmodium ovale (Coatney et al., 1971), an assertion consistent with recent findings of ovale-like parasites in chimpanzees (Duval et al., 2009). P. schwetzi could simply be P. vivax found in chimpanzees (Krief et al., 2010), since these parasites are identical to Asian isolates suggesting a recent introduction from that part of the world rather than a new species. Thus, all available evidence so far supports, or is consistent with, an Asian origin for P. vivax. Nevertheless, the model of an Asian origin for P. vivax is still a work in progress. New data could still change our perspective on the origin of this parasite. Part of the problem is that phylogenetic studies are still missing data from most Southeast Asian apes (Pacheco et al., 2012). A possibility that should be systematically explored is whether ancestors of 206 Jane M. Carlton et al.

Figure 5.1 Phylogenetic tree of P. vivax and closely related taxa from Southeast Asia. Bayesian phylogenetic tree using complete mitochondrial genomes for parasites of Southeast Asian primates, using parasites found in African Cercopithecidae as out- groups (P. gonderi and Plasmodium sp). This phylogenetic tree was inferred under the general time reversible + gamma model (GTR + Γ) model with MrBayes (Ronquist and Huelsenbeck, 2003). The search was performed with 6,000,000 Markov chain Monte Carlo steps and discarded 50% as a burn-in. Sampling was performed every 100 generations. Values above branches are posterior probabilities. Branch length is representative of number of nucleotide substitutions per site, as indicated by the scale bar. Maximum likelihood analyses yield comparable results. (For colour version of this figure, the reader is referred to the online version of this book). Genomics, Population Genetics and Evolutionary History of Plasmodium vivax 207

Southeast Asian apes could have introduced nonhuman primate parasites into Asia. Although unlikely, such an event is still possible given that orangutans and gibbons are naturally infected by many species of malaria parasite [1] and the fact that there was a host switch from Asian apes into Asian Cer- copithecidae as evidenced by the relative position of Plasmodium inui in the phylogeny (Fig. 5.1). Thus, an ape-driven introduction of primate malarias into Asia cannot be completely ruled out without a comprehensive survey of ape malarias. The extraordinary biological diversity of these nonhuman primate malarias closely related to P. vivax offers an invaluable resource to understand the genetic basis of the adaptations that allowed this human parasite to successfully thrive in humans. It also provides a framework to better understand the extant population variation of P. vivax and to ascertain the genetic bases of important biologic characteristics of this very successful group.

3. THE P. VIVAX ENOMEG AND COMPARATIVE GENOMICS P. vivax ‘-omics’ has had a slow and chequered history, primarily due to problems with obtaining sufficient material for whole genome sequencing, but also due to a lack of resources exacerbated by the perceived lesser importance of vivax malaria (Carlton et al., 2011). However, there are now five completely assembled and annotated reference genomes (Table 5.1), more than is available for Plasmodium falciparum, providing a solid resource for the malaria community. In addition, the genomes of several closely related monkey malaria parasites are becoming available (Table 5.2), broadening the usefulness of the P. vivax genomes and enabling comparative genomics of the monkey malaria clade. Below we provide a historical overview of the P. vivax whole genome sequencing projects, describe main features of the P. vivax genome and some of the comparative genomics studies between different members of the monkey malaria clade, and finally provide a brief summary of the P. vivax transcriptome and proteome studies undertaken so far. Some of the difficulties of sequencing P. vivax genomes are exemplified by the first genome to be sequenced, that of the strain Salvador I isolated from a patient from El Salvador. Initial attempts to obtain sufficient genetic material from P. vivax-infected patients for sequencing were unsuccessful, so an alternative source had to be pursued: the Salvador I strain of the parasite that had been adapted to growth in New World monkeys. P. vivax is unable to grow in Old World monkeys such as macaques, and instead must be 208 Jane M. Carlton et al.

Table 5.1 Whole Genome Sequences of P. vivax, with their Country of Isolation, Source and Data Type Indicated P. vivax strain Country Source Data References Salvador I El Salvador Monkey- Reference Carlton et al. adapted lab assembly (2008) strain and annotation India VII, India Monkey- Reference Neafsey et al. North adapted lab assembly (2012) Korean, strains and Mauritania annotation I, Brazil I IQ07 Peru Patient isolate Unassembled Dharia et al. (2010) SA-94, SA-95, Peru Patient isolates Unassembled Bright et al. SA-96, (2012)) SA-97, SA-98 Belem, M08, Brazil, Monkey- Unassembled Chan et al. M09, C08, Madagascar, adapted lab (2012) C15, C127 Cambodia strain, and five patient isolates 26 isolates Thailand & six Patient isolates Unassembled S. Auburn travellers et al., unpublished cultivated in small, expensive Aotus and Saimiri monkeys found in Central and South America. This adaptation to growth in a nonhuman host can take many months, but fortunately a large number of such adapted strains are available as part of the research resources of the Centers for Disease Con- trol and Prevention (http://www.cdc.gov/malaria/tools_for_tomorrow/ research_resources.html), and now made available to researchers through the malaria repository MR4 (http://www.mr4.org/). However, no sooner were the problems with obtaining sufficient material overcome, then issues with funding the sequencing project arose. Contrary to popular belief, there was never a designated fund for sequencing the first P. vivax genome; instead, U.S. Department of Defence and National Insti- tutes of Health funds remaining from the first P. falciparum genome project published in 2002 (Gardner et al., 2002) were reassigned. However, these funds were depleted by 2003, and it was not until the Bur- roughs W ellcome Fund rescued the project by supporting the generation of Genomics, Population Genetics and Evolutionary History of Plasmodium vivax 209

Table 5.2 Whole Genome Sequences of Other Members of the Asian Old World Monkey Malaria Clade, with their Country of Isolation and Data Type Indicated Plasmodium Species and Strain Country Data Genome References Plasmodium Malaysia Reference assembly Pain et al. (2008) knowlesi and annotation H Plasmodium Malaysia Reference assembly Tachibana et al. cynomolgi and annotation (2012) Berok Plasmodium Malaysia Reference assembly Tachibana et al. cynomolgi and annotation (2012) B Plasmodium Cambodia Reference assembly Tachibana et al. cynomolgi and annotation (2012) Cambodian Plasmodium inui India Reference assembly J. Carlton, J. Barnwell, OS and annotation A. Escalante, unpublished Plasmodium gonderi Africa Reference assembly J. Carlton, J. Barnwell, and annotation A. Escalante, unpublished Plasmodium coatneyi Malaysia Reference assembly J. Carlton, J. Barnwell, and annotation A. Escalante, unpublished additional parasite material that the project was restarted in late 2004. The first P. vivax reference genome was finally published six years after the first P. falciparum genome. In 2012, four more P. vivax reference strains were sequenced (Table 5.1), once again using patient isolates adapted to growth in New World monkeys (Neafsey et al., 2012). These reference genomes from Brazil, Mauritania, India and North Korea were sequenced using next generation sequencing technology, and have been fully assembled and annotated. There are now five reference genomes available to the vivax research community, and biological material for each of these strains is available through MR4. An extraordinary finding from the analysis of these P. vivax genomes is that the species exhibits almost twice as much genetic diversity than P. falciparum (Neafsey et al., 2012). Analysis of two different types of mutation markers (SNPs and microsatellites) and in different sequence classes (intergenic, intronic, coding etc.) indicated a globally higher genetic diversity in P. vivax than P. falciparum, pointing to a distinct history of global colonization compared with P. falciparum. Additional analyses also suggested a capacity for 210 Jane M. Carlton et al. greater functional variation in the global population of P. vivax. Of course, the implications for this as regards control and eventual elimination of vivax malaria are enormous, and these studies although still preliminary serve as a warning that P. vivax is a very different beast from P. falciparum. The complete P. vivax genomes have begun to be supplemented by several unassembled and unannotated SNP-discovery sequences from Peru, Madagascar, Cambodia and Thailand that have started the trend of direct sequencing from patient infections (Table 5.1). These projects are pioneering the DNA extraction methods and bioinformatic analyses that are required to sequence parasites directly from human blood – tasks complicated by the small quantities of P. vivax genetic material, mixed genotype infections, and obtaining high quality sequence data from the highly [AT]- rich subtelomeric regions of P. vivax chromosomes. In addition, reference genomes of other members of the monkey malaria clade are becoming available. Three strains of the monkey malaria species P. cynomolgi, the closest living relative to P. vivax, were recently sequenced (Tachibana et al., 2012), joining a reference genome of the more distantly related zoonosis Plasmo- dium knowlesi sequenced several years ago (Pain et al., 2008), and reference genomes of P. inui, Plasmodium coatneyi and Plasmodium gonderi that are also underway (Table 5.2). What does the genome of P. vivax tell us about the biology of the parasite? Briefly, since this has been covered in several reviews (see for example (Carlton et al., in press)) as well as the original genome paper (Carlton et al., 2008), comparing the P. vivax ∼27 Mb genome with the genome of three other sequenced malaria parasites (P. falciparum, P. knowlesi and the rodent malaria species Plas- modium yoelii yoelii), the Plasmodium genomes were found to be similar in size (∼23–27 Mb), number of genes (∼5000), gene structure (>50% contain introns), but differ dramatically in [GC] content, repeat structure and DNA complexity. T he overall basic predicted metabolic pathways and transporter proteins in both P. vivax and P. falciparum were found to be very similar, including those metabolic pathways predicted in the apicoplast such as Type II fatty acid bio- synthesis, the isopentenyl diphosphate pathway, and a glyoxalase pathway, which can potentially be mined for drug targets. Interestingly, many of the same vaccine candidate genes were found in P. vivax as in P. falciparum, where there are ∼15–20 P. falciparum genes actively being studied as potential vaccine candidates versus only two in P. vivax). A major difference between Plasmodium genomes is the presence of species-specific gene families, and expansion of some gene families in certain lineages. In the P. vivax genome, the largest is the extremely divergent vir gene family. Members of the msp3 and msp7 are also massively Genomics, Population Genetics and Evolutionary History of Plasmodium vivax 211 amplified in P. vivax, although their functions are unclear. Many more genes coding for reticulocyte binding proteins (rbp) involved in invasion were found in P. vivax than had originally been thought to exist, suggesting that invasion of red blood cells by P. vivax may be more complex than previously envisioned. Several P. vivax-specific gene families were identified, including the pvtrag gene family that contains 36 members and localises to caveolae, part of the tubove- sicular structure found in infected red blood cells. Proteins of P. vivax orthologs implicated in drug resistance in P. falciparum were analysed using modelling and protein structure analysis, and using these data several predictions were made, namely that P. vivax should be more susceptible to artemisinin than P. falciparum because of certain predicted binding residues, and that should P. vivax develop resistance to atovaquone (used in combination therapy), the same mutations in the cytochrome B gene may be implicated as in P. falciparum. Finally, on a chromosome scale, kilobase stretches of the P. vivax chromosomes were found to be syntenic with other Plasmodium spp. chromosomes. From these synteny maps, it was possible to determine that the Plasmodium common ancestor probably had a P. vivax chromosome karyotype; and the evolutionary events that occurred between the time of this ancestral parasite form and the current chromosome forms of the existing species could be inferred. More recently, a comparison of the P. vivax Salvador I genome with three P. cynomolgi genomes and P. knowlesi has provided further insight into the monkey malaria clade (Tachibana et al., 2012). Interestingly, despite the close phylogenetic relationship between P. vivax and P. cynomolgi (Fig. 5.1), the latter was found to have some P. knowlesi-specific features, such as (1) intrachromosomal telomeric sequences (GGGTT[T/C]A) discovered in the P. knowlesi genome (Pain et al., 2008) but absent in P. vivax; (2) an example of a member of the P. cynomolgi cyir gene family containing a 56-amino acid region highly similar to the extracellular domain of a molecule involved in the regulation of T-cell function in primates (so-called ‘molecular mimicry’ as first identified in P. knowlesi); and (3) two genes similar to P. knowlesi SICAvar genes that are expressed on the surface of schizont-infected macaque erythrocytes and are involved in antigenic variation. The genomes of P. cynomolgi, P. vivax and P. knowlesi can almost be completely characterised by variations in the copy number of multigene families, with copy number of multigene families being generally greater in the P. cynomolgi/P. vivax lineage than in P. knowlesi, suggesting repeated gene duplication in the ancestral lineage of P. cynomolgi/P. vivax or potentially repeated gene deletion in the P. knowlesi lineage. Finally, an analysis of orthologs between P. vivax and P. cynomolgi revealed an extraordinary 83 genes under possible accelerated evolution but 212 Jane M. Carlton et al.

3739 genes under possible purifying selection, indicating that the genome of P. cynomolgi is highly conserved in single-copy genes when compared with P. vivax, and emphasising the value of P. cynomolgi as a biomedical and evolutionary model for studying P. vivax. One disappointment from these various genome sequencing projects of P. vivax and P. cynomolgi has been the lack of identification of a definitive hypnozoite developmental pathway or genetic determinant(s). Several candidate genes have been proposed, for example genes annotated as ‘dormancy-related’ or with the upstream ApiAP2 motifs necessary for sporozoite-specific transcription. However, the role of these genes in the relapse phenotype is speculative, and what is really required is hypnozoite biological material with which to do transcriptome and proteome analyses. While whole genome sequencing of P. vivax isolates is gradually gathering steam, functional genomic studies of P. vivax have been limited. The first microarray (a 60-mer long oligoarray with ∼5700 probes) developed from the Salvador I reference genome sequence was used to describe the 48-h intraerythrocytic cycle of three P. vivax isolates from Thailand (Bozdech et al., 2008). Strain-specific patterns of expression for genes predicted to encode proteins associated with virulence and host pathogen interactions were identified, and a comparison with P. falciparum intraerythrocytic expression revealed differences in the expression of genes involved in certain cellular functions. A second array developed using the Affymetrix platform and consisting of 4.2 million 25-bp probes covering 5419 P. vivax genes was used to generate transcriptome data from eight P. vivax patients from Peru (Westenberger et al., 2010), including a sample enriched for zygotes, ookinetes and gametes, and a sporozoite sample from mosquitoes fed on an infected chimpanzee. Interestingly, large differences in the expression profiles of asexual samples were found, with the most pronounced differences observed in genes involved in glycolysis. The orthologs of some genes shown to be upregulated in P. falciparum sporozoites (Le Roch et al., 2004) were found to be downregulated in P. vivax sporozoites, and vice versa. A number of uncharacterised genes showed substantial up-regulation of at least threefold in ookinetes. Interestingly, gene expression of the vir gene family, was lower than for other genes, perhaps as a consequence of genetic differences between the reference genome Salvador I on which the array was designed, and the Peruvian samples. Apart from a proteomics study that used P. vivax parasites from patients in India and identified ∼150 proteins expressed in the asexual blood stages Genomics, Population Genetics and Evolutionary History of Plasmodium vivax 213

(Acharya et al., 2011), no large-scale proteomics studies have been published in P. vivax. Obviously the lack of biological material is a major confounding factor, and it is to be hoped that with the awarding of several grants from the Bill and Melinda Gates Foundation to develop in vitro culture methods this situation will be rectified.

4. P. VIVAX ENETICGLOBAL G DIVERSITY AND POPULATION STRUCTURE Although Plasmodium genomics is advancing our knowledge of the organism’s complex biology, population-based investigations are still needed to explore the extent of the parasites’ genetic variation, how the observed variation is geographically distributed, and how such diversity affects the development/deployment of new interventions aimed to control and elimination (e.g. vaccines and antimalarial drugs). Unfortunately, several circumstances limit population genetic investigations of P. vivax. The lack of suitable in vitro culturing methods for P. vivax, as mentioned above, imposes the use of field specimens or adapted monkey strains on population studies. While population genomic investigations are desirable in many contexts (e.g. linkage analyses looking for gene associations to specific phenotypes), the high frequency of multiple infections in field isolates requires well- conceived studies in order to generate high-quality data usable by population genetic approaches that require defined lineages or haplotypes. In addition, population genomics demands equipment and expertise that make such approaches difficult to scale-up for supporting routine decision-making processes required by regional or national malaria elimination efforts. However, for many molecular epidemiologic applications, target gene approaches or more traditional multilocus analyses are not only sufficient but more appropriate; moreover, they are within reach of most malaria-endemic countries. Target gene approaches and/or multilocus investigations are still needed to deal with the prevalence of multiple infections in field specimens. In practical terms, direct sequencing of polymerase chain reaction products can generate false consensus that affects estimates of population genetic parameters. Multilocus approaches (e.g. microsatellites) have the same problem since lineages cannot be properly identified in parts of the processed samples. Those problems could make molecular population studies particularly expensive and laborious for endemic countries. Nevertheless, there have been several population-based studies using target genes including merozoite surface protein 3α (MSP-3α) (Mascorro 214 Jane M. Carlton et al. et al., 2005; Schousboe et al., 2011; Zhong et al., 2011); apical membrane antigen 1 (AMA1) (Putaporntip et al., 2009), circumsporozoite surface protein (CSP) (Chenet et al., 2012; Hernandez-Martinez et al., 2011; Imwong et al., 2005); MSP-1 (Imwong et al., 2005; Pacheco et al., 2007); Duffy receptor binding domain ( Ju et al., 2012; Premaratne et al., 2011); transmission blocking antigens Pvs25 and Pvs28 (Feng et al., 2011) and dihydrofolate reductase (DHFR) (Imwong et al., 2003). For most of those genes, genetic diversity was of interest because they are considered vaccine candidates or confer resistance to certain antimalarial drugs such as antifolates. MSP-3α, however, is used as a target for genotyping given its extraordinary diversity; unfortunately, high diversity does not necessarily make it a good tool. Restriction fragment length polymorphism-based essays are not easily interpretable in genetic terms beyond ‘this seems the same’ or ‘these two are different’; so given the plethora of other markers that are reproducible, it seems inappropriate to invest in this particular approach unless it involves sequencing. However, regardless of the fact that many of these studies have been geographically biased (with a few exceptions), all studies indicate a strong geographic structure. The same pattern emerges from microsatellite markers (Gunawardena et al., 2010; Imwong et al., 2007). Nowadays, the clearest picture of the global population genetic structure and the recent history of P. vivax populations come from the studies of mitochondrial genomes (Cornejo and Escalante, 2006; Jongwutiwes et al., 2005; Miao et al., 2012; Mu et al., 2005). The combined data set includes 390 sequences and 125 haplotypes from several populations across the P. vivax geographic distribution ( Jongwutiwes et al., 2005; Miao et al., 2012; Mu et al., 2005). The original published studies overlap in their estimates of TMRCA (the Time to the Most Recent Common Ancestor): Mu et al., 2005 concluded that P. vivax originated between 53,000 and 265,000 years ago, Jongwutiwes et al., 2005 estimated an origin between 217,000 and 304,000 years ago. However, there were differences in their sampling efforts and in the genetic patterns emerging from different geographic regions (Cornejo and Escalante, 2006) so the combined data sets gave older times, 346,000–464,000 Mya for the entire sample. Those estimates were supported using other methods and expanding the samples from other Asian regions, which yielded a TMRCA of 346,000–452,000 years ago (Miao et al., 2012). It is important to emphasise that the information available on the geographic origin of the isolates is still poor and the sampling has been ‘opportunistic’ rather than systematic. Thus, it is not possible to separate the effects of ‘hidden’ Genomics, Population Genetics and Evolutionary History of Plasmodium vivax 215 population substructure due to inappropriate sampling from the scenario of different local dynamics. Those problems could affect our TMRCA estimates. Nevertheless, even if we cut these estimates by half they are still consistent with an early origin of P. vivax and its introduction into Africa at a later time. Recent phylogeographic investigations that include human and chimpanzee P. vivax from Africa actually show that those lineages are identical to those found in Asia (Culleton et al., 2011; Krief et al., 2010). However, the sampling from Africa (including the Middle East) and India is still poor. Another important finding derived from sequencing mtDNA is the extensive number of unique haplotypes in several regions of the world, e.g. Melanesia (30 haplotypes out of 73 isolates) and Southeast Asia (24 haplotypes out of 56 samples). In fact, these two regions form separated blocks that are connected by haplotypes from the Americas (Cornejo and Escalante, 2006; Miao et al., 2012). This pattern is consistent with an ancient population expansion (Miao et al., 2012). Even further, the excess of rare mutations within some (but not all) of the subpopulations suggest that different demographic processes took place within each of the proposed subpopulations. However, an alternative explanation is that this excess of rare mutations is the result of poorly sampled subpopulations that were pooled together (Cornejo and Escalante, 2006). Information derived from the mtDNA studies will provide a framework to population genomics investigations in the context of defining areas that require special attention. Although population genomics actually could unveil complex demographic processes easier, given the richness of the data, it is not immune to sampling bias. Thus, there is much to learn from the mtDNA studies regarding the events that need to be taken into account by population genomic investigations. In addition, it is important to highlight that the integration of broad population genomic studies (e.g. global genetic diversity or linkage analyses looking for gene candidates) with local–regional gene target or multilocus investigations, will provide most needed information about how the parasite populations disperse and change at the different spatial–temporal scales required to coordinate elimination programs.

5. P. VIVAX POPULATION GENETICS IN INDIA The extensive polymorphism found in loci associated with P. vivax adaptive traits, such as those coding for surface antigens and drug resistance 216 Jane M. Carlton et al.

(Cui et al., 2003), reflects the combined effects of the parasite’s population history and selective constraints imposed by host immunity and drug usage (Escalante et al., 2004). Thus, these loci are less informative for determining P. vivax population structure, highlighting the need for neutral and nearly neutral markers which would allow for unbiased estimates of genetic variation, population structure and gene flow in natural parasite populations. Moreover, such kinds of inference can be drawn better from a country setting with variable P. vivax malaria epidemiology, such as exhibited in India. India is one of a handful of countries known to have widespread P. vivax infections. P. vivax used to be the principal cause of malaria in India, with few cases of P. falciparum infection. However, in recent years, P. falciparum has increased in India and the rates of infection for these two parasite species have equalised (Das et al., 2012; Singh et al., 2009). At present, although there are distinct geographic patches with high or low endemicity of both malaria species, P. vivax is widespread and present over almost the whole country ( Joshi et al., 2008). Such epidemiological features increase the complexity of P. vivax and have burdened the country with high morbidity and socioeconomic losses. A majority of P. vivax population genetic studies in India have employed antigenic genes, such as AMA1 (Rajesh et al., 2007; Thakur et al., 2008a), MSP1 (Kim et al., 2006; Thakur et al., 2008b), MSP3 (Kim et al., 2006; Prajapati et al., 2010), and CSP (Kim et al., 2006), and these studies generally reported high genetic polymorphism at these loci. However, since these markers are antigenic genes of P. vivax that are subjected to natural selection, population genetic structure (which is mainly influenced by demography) could not be well understood from these studies. In order to overcome the limitations imposed by these genetic markers, 12 multilocus, putatively neutral markers in noncoding regions of the nuclear genome were developed recently (Gupta et al., 2010). These were used to infer population genetic structure and demographic history of Indian P. vivax isolates using representative samples from low and high endemic regions of India – in total 10 different populations. Genetic diversity was shown to vary across all 12 neutral markers in Indian populations, and in addition, introns appeared to contain less diversity than intergenic regions, which might be an intrinsic property of P. vivax (Feng et al., 2003; Gupta et al., 2010). Furthermore, since the 12 markers came from a contiguous region of one P. vivax chromosome, it was possible to infer changes in genetic diversity across the region. A significant drop in nucleotide diversity was observed at two loci, which may represent Genomics, Population Genetics and Evolutionary History of Plasmodium vivax 217 interesting regions containing genes necessary for parasite survival and/or reproduction (Gupta et al., 2012). How does the population genetics of P. vivax in India fit into the global population structure of the species? A few samples of Indian P. vivax have been included in global studies using microsatellites as genetic markers (Imwong et al., 2006, 2007). In general, relatively less genetic differentiation between Indian P. vivax population samples was observed based on the presence of common alleles/genotypes ( Joshi et al., 1989, 2007). Using these observations, Indian P. vivax subpopulations were generally considered less or loosely structured ( Joshi et al., 2008). Using SNP markers, Gupta et al., 2012 found that Indian P. vivax exhibits higher overall nucleotide diversity than other global populations and the diversity was maintained within population samples but moderate amount of genetic differentiation could be seen between population samples (Gupta et al., 2012). In particular, Bayes- ian genetic cluster analysis (implemented in the program STRUCTURE (Pritchard et al., 2000)) grouped 10 population samples into three clusters based on the proportion of the genetic ancestries in each population. How- ever, the pattern of clustering does not correlate with sampling locations in India, confirming the fact that Indian P. vivax are loosely structured ( Joshi et al., 2008). Furthermore, analyses of past demographic events indicated reduction of population size in a majority of population samples, but when isolates from all the 10 regions were considered as a single population, the data fit a demographic equilibrium model (Gupta et al., 2012). The observed population genetic features of P. vivax point to a complex evolutionary history for this parasite in India; however, it seems likely that Indian P. vivax is part of the ancestral distribution range of this species. Interestingly, the reduction in population size of P. vivax in specific Indian areas has been found to be correlated with a higher prevalence of the FY*A allele of the human Duffy gene (known to confer some degree of resistance to vivax malaria (King et al., 2011)), and with a lower prevalence of P. vivax (specifically in the northeast, east and central Indian regions) than in other Indian regions (Chittoria et al., 2012). The distribution of the FY*A allele also correlated with population coordinates in India and was clinaly variable, indicating malaria-influenced evolution of protection in Indians through adaptive evolution of the Duffy gene by natural selection (Chittoria et al., 2012). Such genetic, environmental and epidemiological correlations underscore the need for P. vivax population genomics and associated human genetic studies if we are to understand evolution of host– parasite interactions in India. 218 Jane M. Carlton et al.

6. CONCLUSION P. vivax has a broad geographic distribution with an extraordinary phenotypic diversity exhibited in its life cycle (e.g. periodicity and morphology (Coatney et al., 1971)). Unfortunately, there are serious gaps in our basic biological knowledge concerning the parasite that will delay its elimination (Mueller et al., 2009). One such gap is our limited understanding of the genetic diversity observed from extant P. vivax populations. Despite stalwart efforts, we are just starting to understand the diversity and evolutionary history of P. vivax. For example, newly available whole genome data from four P. vivax genomes shows that it has high levels of genetic polymorphism, gene duplication events, and repetitive tandem repeats in its proteins (Neafsey et al., 2012). Whether such genetic polymorphism is maintained by positive selection or is the result of complex demographic processes is a matter that requires further investigation. Whole genome sequencing of isolates directly from patients paves the way for such deeper analysis of genetic variation worldwide. The fact that this human malaria parasite originated as part of a complex chain of evolutionary events involving host switches and a rapid species radiation with astonishing phenotypic differences, provides researchers with an extraordinary resource to better understand the origin of molecular adaptations in malaria parasites. This opens unique opportunities to both evolutionary biologists interested in understanding the dynamic of host– parasite interactions and biomedical researchers working on vivax malaria pathogenesis. Understanding the key molecular adaptations that allow this parasite to flourish in the human host will provide new targets for intervention. However, whereas population genomics will provide the framework to ascertain the extent of this parasite genetic diversity, well-defined field investigations are still urgently needed in under to understand the fine details of this parasite’s epidemiology and ecology. Such local and/or regional information is essential for deploying and evaluating intervention strategies.

ACKNOWLEDGEMENTS J.M.C. is supported by NIH/National Institute of Allergy and Infectious Diseases award U19AI089676, and by an NIH/Fogarty International Center Global Infectious Disease research training grant D43TW007884. A.D. is supported by the Indian Council of Medical Genomics, Population Genetics and Evolutionary History of Plasmodium vivax 219

Research and the Department of Biotechnology, Government of India, New Delhi. A.A.E. is supported by NIH/National Institute of General Medicine award R01GM080586. The content is solely the responsibility of the authors and does not necessarily represent the official views of the FIC or NIH. This paper bears the NIMR publication committee clearance number 27/2012. We thank Dr Steven Sullivan for critical reading and editing of this chapter.

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Malariotherapy – Insanity at the Service of Malariology

Georges Snounou*,†,1, Jean-Louis Pérignon*,†,‡ *Inserm, UMR-S 945, Paris, France †Faculté de Médecine Pitié-Salpêtrière, Université Pierre et Marie Curie-Paris 6, CHU Pitié-Salpêtrière, Paris, France ‡Faculté de Médecine Paris 5, Université René Descartes-Paris 5, CHU Necker-Enfants Malades, Paris, France 1Corresponding author: E-mail: [email protected]

Contents 1. Introduction 224 2. The Era of Malariotherapy 225 3. The Practice of Malariotherapy 228 4. Malariotherapy and Malariology 229 4.1. State of Malariology in the Early 1920s 229 4.2. Limitations to Malariological Observations 230 4.3. The Malariologists of Malariotherapy 231 5. Malariotherapy’s Major Contributions to Malariology 233 5.1. The Demise of the Unicity Theory 233 5.2. The Diversity of Malarial Parasites 234 5.3. The Characteristics of Naturally Acquired Immunity 234 5.4. The Natural Course of the Malaria Infection 236 5.5. The Hidden Cycle 237 5.6. Establishment of Laboratory-Bred Anopheline Colonies 239 5.7. The Dynamics of Transmission to Anophelines 239 6. Lessons from Malariotherapy: Caveats and Current Relevance 241 7. Conclusions 245 References 245

Abstract From the early 1920s until the advent of penicillin in the mid 1940s, a clinical course of malaria was the only effective treatment of general paresis, a common manifestation of tertiary syphilis that was nearly always fatal. For a number of reasons, Plasmodium vivax became the parasite species most often employed for what became known as malariotherapy. This provided an opportunity, probably unique in the annals of medicine, to observe and investigate the biology, immunology and clinical evolution of a dangerous human pathogen in its natural host. There is little doubt that the lessons learned from these studies influenced the malaria research and control agendas. It is equally true that over the last 40 years, the insights afforded by malariotherapy have remained

largely undisturbed on the dusty shelves of institutional libraries. In this chapter, we broadly review the published data derived from malariotherapy, and discuss its r oelevance t current challenges of P. vivax epidemiology, immunology and pathology.

1.NTRODUCTION I Towards the close of the nineteenth Century, malaria, tuberculosis and syphilis were the three infectious diseases most feared by humankind. At that time all three were globally distributed. Syphilis, a sexually transmitted disease whose dissemination was only checked by morality, was the least prevalent. The highly contagious tuberculosis thrived on poverty especially in densely populated areas. There was no effective cure for either disease. Malaria, though recorded in all climes, was primarily a disease of warm countries. Its prevalence was such that only few would remain free of infection while residing in the humid tropical and subtropical areas. Malaria could be cured by quinine, a remedy that emerged from Spanish-colonised South America in the middle of the seventeenth century. However, quinine supplies were limited and restricted to a minority who could afford it. The discovery of the causative agents of malaria (Plasmodium) in 1880 by Charles Louis Alphonse Laveran, that of tuberculosis (Mycobacterium tuberculosis) in 1882 by Robert Koch, and that of syphilis (Treponema pallidum) in 1905 by Fritz Richard Schaudinn and Erich Hoffmann, opened the way for scientific investigations aimed at finding a means to cure, prevent and eventually eliminate these diseases. Research was intense and it was conducted independently on each of these three biologically and clinically distinct pathogens. There was, however, one extraordinary period where the paths of malaria and syphilis scientists and clinicians converged, namely the 40 or so years during which induced malaria became the standard treatment of neurosyphilis. Malariotherapy, as it became known, is remarkable for many reasons. From a medical point of view, it is a unique example of one pathogen being used to reverse the pathology caused by another. Curiously, few if any of the syphilologists or the malariologists switched specialities. Whereas malariotherapy added little to the knowledge of the treponemal infection, it provided the bulk of our knowledge on the natural course of clinical malaria and substantial insights into the biology of the life cycle of the malaria parasites of humans. Given the unique value of the observations to malariology, it is regrettable that the publications on the subject are, to say the least, poorly known or explored. The bulk of the relevant observations were published between 1920 and 1950, often in journals that are now difficult to access, and most are Malariotherapy – Insanity at the Service of Malariology 225 not indexed in the usual databases. Relatively few books were devoted to malariotherapy and its techniques (Gerstmann, 1928; Kupper, 1939; Leroy and Médakovith, 1931; Rudolf, 1927; Shute and Maryon, 1966); and it is likely that others escaped our notice. Chapters dealing with this treatment appeared in many of the syphilology books of that time. To date we have gathered about 600 primary publications and we estimate that at least half as many remain to be uncovered. In this article, we wish to provide a historical perspective of the era of malariotherapy, and to present an overview of the rich advances it afforded malariology. We will finally discuss their relevance to current questions, in particular those related to P. vivax, the parasite that was most often used for malariotherapy.

2. THERAF E O MALARIOTHERAPY The syphilitic origin of the general paralysis of the insane (GPI), first described in 1798 by Haslam in an inmate of Bethlehem Hospital in Lon- don, was suggested in 1822 by Bayle in Paris (Anonymous, 1922). The 300 years gap between the introduction and spread of syphilis in Europe and the hypothesis of Bayle is not only due to the fact that GPI develops in some but not all syphilitics, but also that it does so only after many years of latency that follow the relatively closely spaced localised primary and systemic secondary episodes of the infection. Moreover, in tertiary syphilis, T. pallidum can affect different organs, though most often the ascending aorta or the central nervous system. In those that develop neurosyphilis, a third remains clinically silent while the remainder progress to primary optic atrophy, tabes dorsalis or GPI (also known as progressive paralysis or dementia paralytica). Patients in who GPI declares, deteriorate inexorably and few survive beyond 4 to 5 years after onset, though temporary remissions occur in some. The symptoms, which include partial paralysis, loss of memory and reason, delusions, hallucinations, and aphasia, remove the patients from normal society into the care of mental institutions. In the early decades of the twentieth century, patients with GPI accounted for 10–20% of the inmates in mental institutions. The fact that persons who had syphilis had to live for years under the palpable threat to develop GPI, from which they were then condemned to a slow descent into wretchedness before death, amply justified the view that GPI was the most terrible of all human diseases. By the turn of the twentieth century, GPI patients, their families and the clinicians who cared for them were prepared to endure heroic treatments, principally injections of toxic formulations of mercury, bismuth or arsenic, in the hope of bettering their fate. 226 Georges Snounou and Jean-Louis Pérignon

The discovery of malariotherapy was primarily due to the single- mindedness of one person, the Austrian psychiatrist Julius Wagner-Jauregg (1857–1940) (Fig. 6.1), and indirectly to the First World War’s campaign in the Balkans. The details are best related by Wagner-Jauregg (1946) in a monograph he composed in 1935 but that remained unpublished until it was translated by Walter Bruetsch in 1946. In the nineteenth century, many psychiatrists espoused the view that a bout of fever can improve mental disorders, a notion prevalent since Hippocratic times. In 1887, Wagner-Jauregg proposed to infect patients by erysipelas or by malaria to obtain such febrile episodes. After trying erysipelas unsuccessfully (no fever was obtained), he employed tuberculin but this was stopped in the face of objections from the medical community. Wagner-Jauregg was eventually presented with an opportunity to try malaria when a P. vivax-infected soldier returning from the Balkans in 1917 was admitted by mistake to Wagner-Jauregg’s psychiatric ward in Vienna; a gap of 30 years between conception and execu- tion. Nine paretics were infected in that summer (Fig. 6.1). They were then followed up for 1 year before the results were published. Of the nine, six had improved significantly, an outcome so encouraging that by 1922 more than 200 GPI cases had been treated (Wagner-Jauregg, 1922). Of these, an astounding 50 patients were effectively cured (including three of the nine originally inoculated in 1917), in that they resumed their erstwhile life and activities. Many more had partial remission or saw the progression of their symptoms halted. Unlike his earlier publications, Wagner-Jauregg published these results in an American journal in English rather than in German or Austrian journal in German. Thus, the world at large was finally made aware of the success of Wagner-Jauregg’s treatment. Given GPI’s dire prognosis, this was a momentous discovery. Malario- therapy was immediately tried elsewhere with a rate of success (Delgado, 1922; Grant, 1923; McAlister, 1923; Moll, 1924; O’Leary et al., 1926; Worster-Drought and Beccle, 1923) that ensured that it became the most favoured standard treatment of GPI. Many tens of thousands were treated with malaria (often supplemented with the traditional heavy metal-based therapies) and generally, nearly half could be expected to resume normal life or improve significantly, while only one in five failed to respond (Austin et al., 1992; Chernin, 1984). It was not surprising that Wagner-Jauregg was promptly rewarded with the Nobel Prize in 1927. The dominance of malariotherapy waned after the arrival of the antibiotic penicillin in the late 1940s, though its practice persisted in some institutions until the late 1970s (Maurois et al., 1979; Ungureanu et al., 1976). Figure 6.1 The proponent of malariotherapy, Wagner-Jauregg, kindly acceded to a request by Shute to re-enact the first inoculations ofP. vivax into patients suffering from neurosyphilis. The photograph, dated December 1937(4?), has been reproduced from the preface of Shute and Maryon’s book in which they distilled the wealth of practical knowledge gained over many decades of malaria investigations, principally at the Horton Mental Hospital (Shute and Maryon, 1966). Yorke (England) was one of the first malariologists outside Germany who exploited malariotherapy to study malaria parasites. James (England), Ciuca (Romania) and Boyd and Young (United States) were the first directors of the most important, productive and longest running malariology units associated with the practice of malariotherapy. 228 Georges Snounou and Jean-Louis Pérignon

3.HE T PRACTICE OF MALARIOTHERAPY For the syphilologists, malariotherapy was most effective when the patient experienced at least 8–15 malarial distinct paroxysms after which the malarial infection was terminated. Generally, improvements in the clinical signs related to GPI clinical were only observed weeks or months following the malarial episode. In patients where no such improvements could be noted, further courses of induced malaria are attempted. The likelihood of cure and the magnitude of any remission significantly increased when malariotherapy was administered early in the course of GPI. For the clinician, the Hippocratic primum non nocere was pre-eminent. First, in a nonimmune person, malarial paroxysms, irrespective of the infecting Plasmodium species, constitute a serious physical and physiological assault. Thus, only those paretics who were in relative good health and of sufficiently strong constitution were selected for malariotherapy. They were then monitored closely during their malarial infection and any sign of clinical severity, hyperpyrexia, or parasite burdens exceeding 100,000 parasites per microlitre of blood precipitated immediate administration of quinine, the only antimalarial available at that time. Depending on the condition of the patient, the dose of quinine was varied to effect rapid eradication of the parasites or to dampen the infection and alleviate symptoms temporarily. Of the three species known at that time P. vivax was the species most employed for malariotherapy, principally because it was relatively benign and was more sensitive to quinine than Plasmodium malariae. The use of Plasmodium falciparum was generally avoided because of the risk of sudden and unpredict- able shift to life-threatening clinical severity, though it was used to treat persons found to be insusceptible to infection with P. vivax. Second, some methods of inducing the malaria infection were not without danger. The inoculation of infected blood was the most practical; typically, a few millilitres of infected blood were injected subcutane- ously or intramuscularly. Some workers preferred intravenous inoculation because it reduced the prepatent period to a few days (as opposed to 1 to 2 weeks), but this carried the risk of introducing blood clots into the recipient’s circulation. The major disadvantage of using infected blood was the potential to transfer unwanted pathogens from donor to recipient. Given that the donors were predominantly GPI patients undergoing malariotherapy, there was justifiable unease that the recipient could become infected with other infectious agents or be re-infected with Malariotherapy – Insanity at the Service of Malariology 229

T. pallidum, the pathoge responsible for initiating malariotherapy in the first place. In some centres malariotherapy was initiated by the bite of infected mosquitoes, but for many the expenditure needed to maintain anopheline colonies were prohibitive. Finally, from a public health standpoint, it was important that the malaria infections induced in the inmates should not spread unintentionally to others in the wards, or to those outside the walls of the institution where malariotherapy was provided. This concern and those above were raised early in the era of malariotherapy, and in some countries official guidelines for the employment of malaria, such as those drafted in 1924 in the United Kingdom (Dickinson, 1924), were drawn.

4. MALARIOTHERAPY AND MALARIOLOGY 4.1. State of Malariology in the Early 1920s Intermittent fevers (the ague, or malaria) had long been recognised and equally feared by the common inhabitant and the military, and thwarted many an effort to expand into and colonise vast tracts of the world. The discovery of the causative agent in 1880 and then of the method of their transmission in 1898 (independently by Ronald Ross and Patrick Manson, and the Italian workers Giovanni Battista Grassi, Amico Bignami and Giuseppe Bastianelli) were followed by a plethora of important epidemiological, clinical and biological observations. The wealth of knowledge thereby acquired sustained the hope, for the first time in history, that the strategies to control and eventually eradicate malaria were at hand. By the 1920s it had become clear that the impeccable logic of an all-out attack on mosquitoes, or of quinine mass administration (championed by Ross and Koch, respectively), was all too frequently ineffective at implementation. It became increasingly clear that much needed to be learnt about the natural history of malaria if effective control measures were to be devised. The only experimental models known then were avian malarias, but their relevance to human malaria was dubious. On rare occasions, experimental infections of a small number of healthy volunteers were conducted to provide a definitive answer (for example Blacklock and Adler, 1922; Mitzmain, 1916c). For the most part malariologists relied on clinical and epidemiological observations. However, it was often difficult to draw any firm conclusions because the subjects (or mosquitoes) were selected opportunistically and mainly without knowledge of their prior malaria history. The practice of malariotherapy provided a unique 230 Georges Snounou and Jean-Louis Pérignon opportunity for malariologists to decipher behaviour of the parasite in its hosts under scientifically controlled conditions.

4.2. Limitations to Malariological Observations Whereas it is true that large numbers of paretics were infected with malaria parasites in a large number of widely situated institutions, the opportunities to derive new knowledge on the malaria parasites were limited by practical and ethical considerations. First, malariotherapy was a major and potentially dangerous clinical procedure that was primarily administered by syphilologists in specialised wards where the presence of a malariologist was considered surplus to requirement. It was not too difficult to procure parasites, generally from locally infected patients, from persons who acquired it in the tropics, or from other clinics. Usually, a relatively mild P. vivax highly susceptible to quinine was selected, and was then maintained from one patient to the other. Malario- therapy patients were closely monitored (Sweeney, 1929), but the detailed data collected were principally used for the day-to-day management of the malaria infection (history of the parasite strain, dose and mode of inoculation, prepatent period, parasitaemia, temperature charts, clinical signs, haematological changes, etc.). This information was often lost to malariologists. It appeared only infrequently and parsimoniously in the publications reporting treatment outcome. Thus, observations of interest and value to malariology were only made in very few of the institutions where malariotherapy was practiced. Second, the type of experiments that a malariologist might have wished to conduct was ethically restricted. The selection of the patient for malariotherapy and the decision to terminate treatment were the pre- serve of the clinicians and/or the syphilologists in charge. Malariologists selected post hoc those patients whose induced fever history best served to probe a particular aspect of malaria, or they waited until one suitable for this purpose became available. The ethics of malariotherapy and its exploitation by malariologists might be considered somewhat dubious when viewed through the prism of the ethical rules that regulate today’s experimentation on human subjects. This point was discussed cogently in an introduction to a series of articles that analysed retrospectively data acquired during the malariotherapy era (Collins and Jeffery, 1999a, 1999b, 1999c, 1999d). It was clearly concluded that there was no evidence that the patients were exploited or that the standards for informed consent of the day were violated (Weijer, 1999). Malariotherapy – Insanity at the Service of Malariology 231

4.3. The Malariologists of Malariotherapy The malariotherapy clinics to which malariologists were associated for sustained periods were few. Nonetheless, it was this tiny group of malariologists who provided the lion share of the new knowledge on malaria, which many considered to have vastly exceeded all that was learnt in the 40 years since Laveran’s discovery of Plasmodium. In England, malariotherapy was adopted very soon after the publication of Wagner-Jauregg (1922). Starting in June 1922, Warrington Yorke of the Liverpool School of Tropical Medicine and Hygiene (Fig. 6.1) was the first to derive malariological knowledge from observations on GPI patients inoculated with malaria at the County Mental Hospital of Whittingham. Although his work in this domain did not extend beyond a few years, it clearly demonstrated the value of malariotherapy to malariology ( Yorke, 1925, 1926; Y orke and Macfie, 1924a, 1924b; Yorke and Wright, 1926). The officials, who administer the Acts relating to the care of mental patients in England, took the view that there were serious ethical objections to inducing malarial attacks by the direct inoculation of blood from patient to patient. Malariotherapy for GPI patients and those suffering other mental diseases was to be initiated by the bites of infected mosquitoes. Sydney Price James (Fig. 6.1), an eminent malariologist at the Minis- try of Health, was charged with producing infected mosquitoes that could then be dispatched to mental hospitals elsewhere in the world. Thus, the Mott Clinic for Malaria Therapy was born at the Horton Mental Hospital. Directed first by James and then by John Alexander Sinton, Gordon Covell and finally Percy Cyril Claude Garnham, the Mott Clinic remained active for 40 years (1923–1973) and 17,000 patients underwent malariotherapy in its wards. The success and fame of this clinic was in no small part due to technical prowess and the biological insights of Percy George Shute (Fig. 6.1) and his companion Marjorie E. Maryon, both of who worked there for more than 40 years. The contribution of the Horton workers to malariology is inestimable as is evident by a selection of their published observations (Covell, 1956; Covell and Nicol, 1951; Covell et al., 1949a, 1949b; Garnham et al., 1975; James, 1926, 1931, 1934; James and Ciuca, 1938; James and Shute, 1926; James et al., 1927, 1932a, 1932b, 1936; Shute, 1940, 1946, 1951, 1958; Shortt and Garnham, 1948; Shute and Covell, 1967; Shute and Maryon, 1948, 1963, 1968; Shortt et al., 1948a, 1976; Sinton, 1938, 1939, 1940a, 1940b; Sinton et al., 1939a, 1939b; Ungureanu et al., 1976). 232 Georges Snounou and Jean-Louis Pérignon

In Romania, Mihael Ciuca (Fig. 6.1) initiated in 1926 an impressive series of investigations into malaria parasites in a number of malariotherapy clinics that continued for 50 years. The breadth and significance of these studies is illustrated by a selection of their published observations (Ballif et al., 1963; Ciuca et al., 1928, 1930, 1934, 1937a, 1937b, 1937c, 1943, 1947a, 1947b, 1962, 1964a, 1964b; Lupascu et al., 1967, 1968; Ungureanu et al., 1976). In the United States of America, two centres for malariotherapy boasted an associated malariology unit. The first, the National Institute of Health’s Williams Malaria Research Laboratory, was based at the South Carolina Mental Hospital at Colombia, South Carolina. The malariological research was initiated by Bruce Mayne, a malaria expert at the United States Public Health Service, and was considerably expanded by Martin Dunaway Young (Fig. 6.1), who joined this laboratory in the late 1930s as a junior zoologist, becoming its director from 1937 to 1961. The data generated from this unit are of the highest interest (Coatney and Young, 1942; Coatney et al., 1950a, 1950b; Cooper et al., 1950; Ehrman et al., 1945; Mayne, 1932a, 1932b, 1933, 1937; Mayne and Young, 1938; Y oung, 1944; Young and Burgess, 1947; Young and Eyles, 1949; Young et al., 1940a, 1940b, 1945, 1947). In the late fifties, Geoffrey M. Jeffery, who also worked at Milledgeville State Hospital, Georgia, and William E. Collins joined the team, and expanded the malariological observations (Eyles and Jeffery, 1949; Jeffery, 1952, 1954b, 1956, 1958, 1960; Jeffery et al., 1950, 1952, 1954a, 1956, 1963; Jeffery and Eyles, 1955; Young et al., 1955). These two outstanding scientists are likely to be among the last, if not the only, malariologists still alive who had participated in administering malariotherapy to neurosyphilitics. Moreover, they still regularly publish remarkably informative retrospective analyses of the data gathered from the records that were kept from the patients that were treated (Collins and Jeffery, 1999a, 1999b, 1999c, 1999d, 2002, 2003, 2005, 2007; Collins et al., 2003, 2004a, 2004b, 2009, 2012; Diebner et al., 2000; Jeffery, 1966; McKenzie et al., 2001, 2002a, 2002b, 2007; Molineaux et al., 2001, 2002; Simpson et al., 2002). The second, the Station for Malaria Research, funded by the Rock- efeller Foundation, was based in Tallahassee, Florida in association with the Florida State Hospital. The considerable scope and the excellence of the research carried out by Mark Frederick Boyd (Fig. 6.1) and his colleagues, most notably Stuart F. Kitchen, can be appreciated in their prolific publications (Boyd, 1932, 1935, 1938, 1939, 1940a, 1940b, 1940c, 1942a, 1942b, 1944, 1946, 1947, 1949a, 1949b; Boyd and Coggeshall, 1938; Boyd and Kitchen, 1936a, 1936b, 1936c, 1937a, 1937b, 1937c, 1937d, 1938a, 1938b, Malariotherapy – Insanity at the Service of Malariology 233

1938c, 1939, 1943, 1944; Boyd and Matthews, 1939; Boyd and Stratman- Thomas, 1932, 1933a, 1933b, 1933c, 1933d, 1934a, 1934b, 1934c; Boyd et al., 1934, 1936a, 1936b, 1936c, 1938a, 1938b, 1939, 1941; Kitchen, 1938, 1949c; Putnam et al., 1947).

5. MALARIOTHERAPY’S MAJOR CONTRIBUTIONS TO MALARIOLOGY As we delved into the malariotherapy literature, it became increasingly evident that a review embracing all the myriad observations, the novel insights to which they led, and their impact on the field of malariology, would in equal measure overwhelm both authors and readers. We have opted to highlight the more important malariological advances consequent to the practice malariotherapy, many of which could not have been made otherwise. We limited ourselves to the biological aspects of the infection, leaving discussion of equally important observations on various aspects of chemotherapy including drug discovery to another occasion. We sincerely hope that this will encourage the reader to peruse the original articles, many of which are now available electronically, in order to appreciate the full extent of these fascinating and unique observations.

5.1. The Demise of the Unicity Theory It is an oft forgotten fact that malariologists in the early 1920s were still debating whether malaria was due to one or more species of Plasmodium. Laveran, the discoverer of Plasmodium, proposed that there was only one species of malaria parasite, but that it was capable of changing its morphology and clinical course depending on factors related to the state of the person it infects (Laveran, 1890). This notion, termed the unicity theory, was contradicted by the careful observations of Italian researchers in the late 1880s of parasites in untreated malaria, which led them to conclude that there were three distinct species (P. falciparum, P. vivax, P. malariae). Laveran steadfastly maintained his view to his last days in 1922; demonstrating that a professorship at the Institut Pasteur, a fellowship of the Royal Society, and a Nobel Prize can confer plausibility but not validity to a theory. The observations from malariotherapy, where each of the three Plasmodium species invariably maintained their distinctive biological and morphological features in both vertebrate and insect hosts, laid the unicity theory to rest. This became the standard test to validate a species, for example Plasmodium ovale ( James et al., 1932a) a parasite first described in 1922 that many argued was merely a P. vivax variant, or to 234 Georges Snounou and Jean-Louis Pérignon disprove its existence as was the case for P. falciparum quotidianum, an aestivo- autumnal parasite with a 24-h periodicity ( James et al., 1932b).

5.2. The Diversity of Malarial Parasites The idea that within a given species there exists races or varieties that breed true and that are distinguishable from each other had long been accepted. This was after all the bedrock of the Darwinian theory of evolution. Whereas it was a relatively simple matter to investigate diversity in multicellular free-living organisms where individuals or groups can be observed in isolation, it was particularly challenging to observe unicellular parasitic pathogens in their hosts. Nonethe- less, by the early 1920s it was clear to all observers (who did not subscribe to the unicity theory) that the parasites within each of the Plasmodium species were not homogeneous. The differences most frequently noted were based on the morphology of the blood stages, and observations of peculiar forms prompted some to propose new species, such as Plasmodium tenue and Plasmodium perniciosum, two parasites that resemble P. falciparum (Russell, 1928). Otherwise, clinical observations indicated differential susceptibility to quinine for some strains and suggested that others caused specific pathologies (such as blackwater fever or hyperpyrexia). However, in the absence of experimental data, the nature of the diversity in malaria parasites remained a matter of debate, often restricted to opinion and speculation. Malariotherapy provided the means to gather a wealth of experimental data, which by the mid 1930s irrevocably established that within each species of malaria parasite there are races or strains distinguishable by their natural parasitological and clinical course, infectivity, antigenic content, and susceptibility to drug treatment. The data clearly did not encompass the rich diversity of the malaria infection in the varied endemic zones, in that observations were predominantly limited to infections of a specific subset of mostly malaria-naïve subjects with a rather small and select number of parasites strains. Nonetheless, the data were sufficient to elaborate a conceptual framework of the malaria infection incorpo- rating the diversity within each parasite species. In the next few sections, we will briefly describe the salient discoveries stemming from malariotherapy that made this possible. We strongly recommend, and gratefully acknowledge to have been inspired by, an excellent recently published essay on the evolution of the notion of strain for malaria parasites (McKenzie et al., 2008).

5.3. The Characteristics of Naturally Acquired Immunity The fact that a form of immunity to malaria could be acquired had been suspected long before the discovery of Plasmodium; but it was only with Malariotherapy – Insanity at the Service of Malariology 235 the advent of accurate microscopic diagnosis of malaria (and measurement of spleen rates) that it became possible to investigate how this immunity is acquired. In the absence of animal models, the investigations were limited to epidemiological observations. Building on the pioneering observations of Koch (1901) and Schüffner (1938), Christophers conducted a seminal study of hyperendemic malaria in Singhbhum District (Bihar State, India) and one of his principal conclusions sums up the state of knowledge at that time: ‘These results show that there is a definite acquired immunity in malaria, evidenced by a restricted numerical prevalence of parasites in the blood and the ability of the individual to live infected, but free from sickness, under conditions that to the new comer (or newly born) means intense, almost continuous heavy infection.’ (Christophers, 1924). The efficacy of malariotherapy relied on producing paroxysms, and it was natural to attempt inducing a second malaria infection should a first malaria episode fail to improve the mental state of the patient. It quickly became apparent that this was easier said than done. The authors of one of the first reports of reinoculation (conducted with the same strain 6–20 months after the first infection) were surprised that this failed to produce any clinical signs in 14 patients, though all developed a patent asexual parasitaemia (Nicole and Steele, 1926). At the same time, the observations of Ciuca in Romania confirmed and added to those of Yorke, when he demonstrated that the immunity acquired to infection by one parasite species does not protect against infection or disease by another (Ciuca et al., 1928). Back in England, James added to these observations by showing that the solid immunity acquired against a homologous strain is only partially efficient at protecting against challenge with a heterologous strain ( James, 1931). Across the Atlantic, Boyd (1947) and others significantly added to the knowledge of acquired immunity against malaria in humans. Thus a new dimension, the immunological, was added to the concept of strains, races or varieties in malaria parasites. We will burden the reader but little more on this topic, as immunity is discussed in another article of this series. We strongly recommend a recent review where the contributions of induced malaria in humans to the acquisition of immunity are discussed with insight and elegance (McKenzie et al., 2008). Briefly, the principal conclusions reached from years of induced infections in neurosyphilitics were that the immunity acquired against one particular strain can eventually protect against challenge with another strain, but the magnitude of this protection (clinical and parasitological) is inversely propor- tional to the antigenic ‘distance’ between these strains. On the other hand, 236 Georges Snounou and Jean-Louis Pérignon even the most robust immunity induced against parasites of a given Plasmo- dium species offers little protection against challenge by parasites from another species. Furthermore, acquired immunity is stage-specific and it wanes relatively quickly (a few years or less) in the absence of further exposure to the parasite. Given these characteristics of acquired immunity in malaria, and the recognition that each species comprises a large number of antigenically distinct strains, most of the malariologists at that time were united in their opinion that ‘…it is more than apparent that there is little reason to hope for an effective vaccine for malaria’ (Yount and Coggeshall, 1949).

5.4. The Natural Course of the Malaria Infection It could be argued that the core of our knowledge of the course of naturally acquired malarial infection in humans and of the factors that govern the complex interactions between the parasite and its vertebrate host could not have been acquired without the observations made principally in mental patients undergoing malariotherapy, and thereafter in healthy volunteers. Indeed, as the number of neurosyphilitics dwindled after the introduction of penicillin, observations on the course of induced malaria continued unabated in army and prisoner volunteers throughout World War II. The fact that experimental infections of humans (often untreated but always very closely monitored) were allowed until the late 1970s at the Atlanta Fed- eral Penitentiary, United States of America, under the aegis of the National Institute of Health (Neva, 1974), is in no small part due to a recognition of the major contributions of malariotherapy. The insights into the biology of Plasmodium parasites profoundly altered perceptions of malaria epidemiology and significantly improved the efficacy of control measures worldwide. The practice of malariotherapy provided the malariologist, for the first time, with a steady supply of human subjects who could be inoculated by the bite of mosquitoes infected with one or other established strains of the four species of malaria parasites. The sole aim was to obtain a clinical attack that should be allowed to evolve, preferably unhindered, so as to procure the patient with 10–15 paroxysms, before curative administration of quinine. In a few centres, some patients were left untreated after spontaneous resolution of the infection and monitored over the following months for renewed malarial activity. In this manner, myriad data were collected on the prepatent period (the time to the first observation of parasites in the blood), the incubation period (the time to the first clinical symptoms), the evolution of the asexual and sexual parasitaemia, the magnitude and periodicity of paroxysms, the Malariotherapy – Insanity at the Service of Malariology 237 fever curves, the clinical manifestations, changes in parasite morphology, the timing and frequency of recrudescences and relapses, and the duration of the infection. Side-by-side comparisons of infections induced by different parasite strains made it possible to define and investigate their biological and clinical characteristics, thus contributing to define the concept of strain in malaria (Boyd, 1940b; Ciuca et al., 1937b; James and Ciuca, 1938; Y oung et al., 1947). Excellent summaries have been prepared for each of the four species ( James et al., 1949; Kitchen, 1949a, 1949b, 1949c). The value of this type of data has recently come to the fore in the elaboration of mathematical models of the malaria infection (Diebner et al., 2000; McKenzie et al., 2001, 2002a, 2002b, 2007, 2008; Molineaux et al., 2001, 2002; Simpson et al., 2002), and the malaria community is grateful to Collins and Jeffery for collating hitherto unpublished data gathered from neurosyphilitics subjected to malariotherapy at the Carolina State Hospital between 1940 and 1963 (Collins and Jeffery, 1999a, 1999b, 1999c, 1999d, 2002; McKenzie et al., 2001). One of the more baffling observations made early after the introduction of malariotherapy in the United States was the refractoriness of many persons of African origin to infection with P. vivax, as opposed to universal susceptibility of persons of other origins (Boyd and Stratman-Thomas, 1933d, 1934b). This was baffling to malariologists because at that time many still considered P. ovale as a variant of P. vivax, and thus considered that the Africans in their native continent were susceptible to P. vivax in general. The basis for this absolute resistance was only uncovered in the mid 1970s (Miller et al., 1976), when it was demonstrated that the Duffy blood group was necessary for red blood cell invasion by P. vivax, whereas nearly all West Africans and a majority of the population in subtropical Africa are Duffy negative.

5.5. The Hidden Cycle The relapses of P. vivax are now considered as the major obstacle to an eventual extirpation of this parasite. A relapse is the appearance, in a person bitten by an infected mosquito on a single occasion, of the blood stage parasites with or without clinical symptoms many weeks, months or years after parasites from the primary episodes have been fully eliminated from the blood. Such episodes, which are quite distinct from recurrences due to sub- curative treatment, were noted early last century (Thayer, 1901), and posed a major public health problem when it was feared that World War I and World War II soldiers who fought in malarious areas would reintroduce P. vivax in 238 Georges Snounou and Jean-Louis Pérignon countries from which it has disappeared. Relapse aetiology was elucidated only in 1980 (Bray et al., 1981; Krotoski et al., 1980): quiescent liver stage parasites, hypnozoites that mature to yield hepatic merozoites long after the initial infective bite. This topic is the subject of another article of this series, and the same author recently extensively and superbly reviewed it (White, 2011). The hypnozoite remains the more elusive and least investigated stage of the Plasmodium parasites. Its discovery would have no doubt been indefi- nitely delayed had the hepatic stage of the malaria infection not been discovered 32 years previously. We will limit ourselves to highlighting the role that malariotherapy played in leading to the discovery of the generally very short and discreet hepatic phase of the malaria life cycle. Having spent nearly two decades observing malaria parasites exclusively in blood, the minds of the malariologists at the turn of the nineteenth century readily accepted the notion that the newly discovered sporozoite, the form injected by the mosquito, would naturally invade a red blood cell to initiate the infection. Indeed, Schaudinn’s one-off observation of such an event in a solution where P. vivax sporozoites were mixed with red blood cells (Schaudinn, 1903) was taken as sufficient confirmatory proof. It was only 20 years later that this view was put in doubt by Yorke and Macfie. In the course of early observations on malariotherapy patients, they demonstrated that quinine failed to prevent infection when it was administered as a short course around the time of mosquito inoculation, but not when the treatment is initiated a week or more later the mosquito bite (Yorke and Macfie, 1924b). Yorke then showed that patients inoculated with infected blood whose primary infection is curatively treated tend not to relapse, whereas they do so frequently if they were initially inoculated by mosquito bite (Yorke, 1925). James independently obtained similar results ( James, 1926, 1931), and despite numerous attempts neither Yorke nor others could observe a sporozoite invading and developing in a red blood cell. The hunt was on to discover what happened to the sporozoite between its deposition by the mosquito into the skin and the appearance of blood stage parasites. Access to malariotherapy patients and infected mosquitoes was the only means to investigate the biology of the sporozoite and the initial short and clinically silent period of the infection. Remarkable experiments, in particular those based on blood transfusions from a malariotherapy patient bitten by an infected mosquito into other malaria naïve patients (classic subinoculation experiments), were conducted by Boyd (Boyd, 1940a; Boyd and Stratman-Thomas, 1934a, 1934c; Boyd and Kitchen, 1937a, 1939), Ciuca et al. (1937a), and in volunteers by Fairley (1947). These provided crucial Malariotherapy – Insanity at the Service of Malariology 239 insights into the behaviour of the sporozoite pre-and postinoculation, and allowed to determine the minimal duration of the tissue phase of the Plas- modium parasites in humans with some accuracy. It was Shortt and Garnham who first identified the hepatocyte as the site where pre-erythrocytic development occurs. This demonstration was first reported using Plasmodium cynomolgi (Shortt et al., 1948b) a parasite of macaques. Soon thereafter the work was repeated using P. vivax (Shortt et al., 1948a) in a malariotherapy patient who consented to a liver biopsy 7 days after inoculation with a large number of sporozoites. To our knowledge, the last observations on hepatic stages to be made in malariotherapy patients were those of Shute and his Romanian colleagues (Shute et al., 1976; Ungureanu et al., 1976) who wished to investigate the difference between sporozoites (and relapses) from two P. vivax strains distinguished by the duration of their prepatent periods and relapse patterns.

5.6. Establishment of Laboratory-Bred Anopheline Colonies The requirement for mosquito-induced infections for malariotherapy was initially met with locally caught anophelines ( James and Shute, 1926; Y orke and Macfie, 1924b). However, as the number of malariotherapy patients grew, a year-round supply of susceptible uninfected mosquitoes became indispensable. Thus were established the first insectaries for mass rearing of anophelines: Anopheles maculipennis (Shute, 1936), Anopheles quadrimaculatus (Boyd et al., 1935), and thereafter other species. The availability of mass- reared anophelines, to quote Boyd et al. (1935), ‘afford opportunities for research on entomological, parasitological, and epidemiological problems of malaria that have heretofore either been approached with difficulty or not at all’. Thus, a wealth of data accrued on the factors concerned with the transmission of malaria from humans to mosquitoes and from mosquitoes to humans (see below), and on anopheline biology, bionomics, and ecology. This illuminated the nature of malaria endemicity, and significantly ben- efited the design and efficiency of malaria control measures.

5.7. The Dynamics of Transmission to Anophelines ‘In order that anopheline mosquitoes may be infected from malarial blood it is necessary that the sexual forms of the parasite be present in sufficient numbers, of proper maturity and suitable proportion of sexes’. This sentence in Deaderick’s (1909) book on malaria sums up the views prevalent prior to malariotherapy on malaria transmission to mosquitoes. Indeed, there were only scanty studies in which gametocyte dynamics (numbers as well 240 Georges Snounou and Jean-Louis Pérignon as proportions of micro- and macro-gametocytes) were examined (Ross and Thomson, 1910; Thomson, 1911). In only few studies were mosquitoes (usually field-caught) applied to the patient with a view to investigate factors influencing infectivity to the insect (Mitzmain, 1916a, 1916b, 1916d). However, the history of the infection was poorly known, if at all, the infecting parasites varied between patients some of who harboured mixed species infections, and finally many had been or were under quinine treatment. Thus, interpretation of the observations was fraught, and knowledge of the dynamics of the transmission of malaria to Anopheles was to say the least fragmentary. It was important for the practice of malariotherapy to have access to a constant supply of infected mosquitoes. Thus, the factors that impinged on the efficiency of transmitting the parasite to anophelines were investigated with great care. James, who was tasked with providing infected mosquitoes to malariotherapy centres in Great Britain, was one of the first to tackle this problem. His early observations on the transmission by wild caught A. maculipennis of P. vivax ( James, 1926), and of P. falciparum and P. malariae ( James, 1931; James et al., 1936), were rich in novel observations and unexpected conclusions, of which a selection is presented. Despite the fact that many patients were infected with the same strain of P. vivax only a few proved to be consistently good infectors to mosquitoes. Equally surprising was the fact that it was not the number of gametocytes present in the blood that correlated with the proportion of infected mosquitos (i.e. where sporozoites appeared in the salivary glands), but the presence and quality of the microgametocyte (principally their ability and the time they take to exflagellate). In the patients, gametocytes were rarely observed less than 2 weeks after mosquito inoculation, and were generally not infective until the 10th day after the first rise in fever. By contrast, gametocytes appeared and were infective on the first day on which a relapse occurred. Boyd reported broadly similar results for the transmission of the local (Florida) P. vivax strains by wild-caught and then laboratory-bred A. quadrimaculatus (Boyd, 1942b; Boyd and Stratman-Thomas, 1932; Boyd and Kitchen, 1937b, 1938b; Boyd et al., 1936c). The mosquitoes themselves were also found to differ in their ability to transmit the infection. Observations of consistently refractory individuals from the same batch of wild-caught mosquitoes woke entomologists to the existence of cryptic species and contributed to explain the phenomenon of anophelism without malaria, which perplexed malariologists early last century. It was further demonstrated that mosquito species could differ not Malariotherapy – Insanity at the Service of Malariology 241 only in their susceptibility to different parasite species, but also to different parasite strains of the same species. The classic example was the refractoriness of the European A. maculipennis to African strains of P. falciparum but not to the strains indigenous to Italy ( James et al., 1932b; Shute, 1940). Boyd noted a similar phenomenon for nearctic and neotropical mosquito species with respect to P. vivax and P. falciparum (Boyd et al., 1938a). These and a wealth of other observations relevant to the transmission of malaria parasites between its hosts have helped understand how subtle variations in environmental conditions, mosquito bionomics and human behaviour could lead to highly contrasting epidemiological pictures (Boyd, 1949a, 1949b). This in turn substantially influenced the manner in which control measures were selected and deployed to the best effect.

6. LESSONSROM F MALARIOTHERAPY: CAVEATS ANDURRENT C RELEVANCE Given the foregoing list of the more remarkable discoveries that the practice of malariotherapy brought within the grasp of malariologists, the reader might have gained the impression that an in depth analysis of all the related material and publications would leave little to be learnt about malaria infections and their parasites. Such is not the case. There are some caveats that must be taken into consideration if one wishes to apply and/or extrapolate the lessons from the malariotherapy era to some questions r elevant to today’s efforts to control malaria. First, the data on the malaria infection were nearly exclusively collected in neurosyphilitic adults generally of sound constitution and under constant care, who for the most part had not been previously exposed to malaria. This question, elegantly phrased in the title of an article 70 years ago, ‘Are the data of therapeutic malaria applicable to conditions obtaining in nature?’ ( Winckel, 1941), remains one that some could formulate today. There are indeed differences between the acquisition and eventual resolution of the malaria infection in malariotherapy patients and in endemic residents. However, we do not feel that these are sufficient to justify the view that the malariological observations made in the course of malariotherapy merely represent special cases with little connection or relevance to what happens in nature. In areas of high endemicity, it is the children who bear the brunt of symptomatic malaria. Moreover, as endemicity increases fewer and fewer persons can be considered truly malaria naïve, as many would have been exposed to 242 Georges Snounou and Jean-Louis Pérignon the parasite or its antigens in utero, and to infection from birth onwards. For some unknown reason, cases of congenital and/or juvenile tertiary syphilis were poorly responsive to malariotherapy (Anonymous, 1936; O’Leary and Welsh, 1933; Wile and Hand, 1935), consequently reports of malariotherapy in children are rare. On the other hand, there is little to indicate that the natural course of the malaria in children is fundamentally different from that observed in adults. As for concurrent neurosyphilis, it could be argued that it might have modified the course of the induced malaria infections in a special way. However, this is unlikely to be the case because the biological and immunological observations made in the course of malariotherapy were in all respects similar to those that were made later in healthy volunteers (also exclusively adults in good health). Furthermore, few if any of the conclusions derived from the malariotherapy data are inconsistent with field-based observations. Second, the substantial malariological observations made in the various malariotherapy centres were derived from infections by a few selected strains of parasites. The use of P. malariae was somewhat restricted because it was relatively difficult to obtain and quite difficult to transmit by mosquito, but it was the parasite species favoured to infect African American patients who were refractory to P. vivax. Induced P. falciparum infections were restricted to the few centres where expert malariologists were in atten- dance, and even there it was used sparingly (Ciuca et al., 1943; James et al., 1932b). Throughout the malariotherapy era, P. vivax was the parasite species of choice. Nonetheless, the P. vivax strains that were used for malariotherapy had been selected to fulfil the requirements for malariotherapy: primarily to be of relative low virulence yet leading to the reliable production of a sufficient number of paroxysms, to be highly susceptible to quinine, and for some centres, to produce an adequate number of infective gametocytes. Thus, the vast majority of observations made by the leading malariologists were based on infection with only a few strains. Yorke based his observation from a single strain isolated from a patient who acquired the infection in India (Yorke and Macfie, 1924b). At Horton, it was the Madagascar strain that was predominantly used for more than 40 years (Covell, 1956; James, 1931; James et al., 1936; Shute, 1958). Although its origin is presumed to be Madagascar, it was actually derived from an Indian seaman who developed malaria on arrival to London from India, and whose last port of call was Madagascar. The parasites recovered from his blood were equally likely to be from a chronic infection or a relapse from an infection acquired in India. The Madagascar strain was distributed very widely in Europe. In Romania Malariotherapy – Insanity at the Service of Malariology 243

Ciuca relied on two local strains, TBR102 and TBAp (Ciuca et al., 1937b, 1943) that he isolated prior to World War II. In the United States of Amer- ica, Young mainly employed a local strain, St. Elizabeth (Coatney and Young, 1942; Young, 1944) and subsequently the Chesson strain that was isolated from a soldier who became infected in Papua New Guinea (Ehrman et al., 1945). Although Boyd derived a number of distinct strains from patients who acquired P. vivax in Florida, the McCoy strain that had been isolated in 1931 was the most frequently inoculated. It is unlikely that this eclectic collection of strains were representative of the gamut of biological, immunological and clinical diversity that the global P. vivax population encompassed then, or for that matter today. Indeed, it is probable that many of the parasite strains prevalent in the temperate northern climes, such as the long incubation period P. vivax strains used by the Dutch (Swellengrebel et al., 1929) and the Russian malariologists (Tiburs- kaja et al., 1968) became extinct as a result of the WHO’s Global Eradica- tion Campaign of the 1950s to 1960s. This is possibly also the case for the Italian strains of P. falciparum that were shown to be particularly virulent and tolerant to quinine in head-to-head comparison with P. falciparum strains originating from India ( James et al., 1932b). Finally, although the strains that were used tended to remain phenotypically constant, they were not necessarily genetically homogeneous, and it is possible that their genetic heterogeneity might have varied over the numerous passages from human to human or between humans and mosquitoes. Third, much of the published data obtained through malariotherapy would be considered to be inadequate as judged by the standards of clinical trial designs and statistical analyses that would be deemed acceptable today. This is by no means a criticism of the clinicians and malariologists who, it must be stressed, generally carried out their work investigations to the highest scientific standards of their time. Moreover, it is not obvious that it would have been practical or ethically acceptable to conduct the type of placebo-controlled, double-blind or other types of clinical trials that have now become standard. It is with these main caveats in mind that one should interpret, or extrapolate from, the malariological data obtained through the malariotherapy era, and the subsequent series of experimental infections in human volunteers. The data should be approached with an open frame of mind. Most if not all of the biological, clinical and immunological phenomena that were revealed during the course of malariotherapy are obviously valid because they were directly observed. However, the frequency of these phenomena under 244 Georges Snounou and Jean-Louis Pérignon natural conditions and the manner and extent to which each contributes to shape malaria epidemiology in a particular setting remain to be determined. For example, it was conclusively demonstrated that in two lots of 20 or so untreated or subcuratively African American patients who were inoculated with either one or the other of two P. falciparum strains (the local Santee- Cooper, and the Panamanian El Limon), the blood infection persisted for a median of 222 or 279 days, respectively, with a maximum observed duration of 480 and 503 days in a few individuals (Eyles and Young, 1951; Jeffery and Eyles, 1954). Clearly P. falciparum infections can last for nearly 2 years. Nonetheless, many important questions need to be answered if these data are to be useful to the epidemiologist or the malaria control person. Do all P. falciparum strains have the potential to persist for many years? Does infection longevity vary with the ethnic/genetic background or the immune or health status of the human host? Do infectious gametocytes circulate during the chronic phase, in which case for how long? The validity of many an elegant mathematical model of malaria depends on the answers to such questions. Finally, there are aspects of malaria that remained outside the practical or ethical scope of the observations or the investigations made in the context of malariotherapy. This is the case for issues of clinical severity. With the recent reinstatement of P. vivax to its rightful place as a harmful parasite that deserves the attention of the malaria community (Baird, 2007; Price et al., 2007), the dogma of its relative clinical mildness has been challenged (Anstey et al., 2009; Baird, 2009). Unfortunately, it is quite difficult to derive data from the malariotherapy literature that are robust enough to favour one or other side of the argument. The mortality rates observed in malariotherapy patients varied between centres and with time. Average values around 10% were often reported in the early years of malariotherapy (Anonymous, 1929; Graham, 1925). By the mid 1930s, though some individual centres still reported double-figure rates, it was less than 1% in others; the average recorded mortality rates to 2.4–5.4% (Fong, 1937; Krauss, 1932; O’Leary and Welsh, 1933). This might reflect the accumulated expertise of medical and nursing staff in caring for and monitoring patients subjected to malariotherapy, or a switch to parasite strains with reduced virulence. Further- more, in those centres where only those patients deemed to be physically capable of withstanding a clinical malaria infection were selected, the mortality rate was up to nine times lower that in those where no such selection was made (Krauss, 1932). It is unfortunate that Krauss did not provide any details as to the nature and origin of the parasite species and strains that were used in the selected and unselected patients. The infecting dose and route of Malariotherapy – Insanity at the Service of Malariology 245 administration (intravenous, intramuscular, or by mosquito bite) are potential confounding factor when interpreting mortality rates (Hoch and Kusch, 1940; Kusch et al., 1936); however, the infecting dose is rarely provided. Moreover, there does not seem to be a standard set of criteria for attributing mortality directly to the P. vivax infection, except maybe the presence of high numbers of parasites at the time of death. For these and other reasons, it is rather complicated to derive an accurate estimate of the malaria infection’s contribution to mortality from the malariotherapy publications. Nonethe- less, it is undisputed that deaths consequent to infection with moderately virulent P. vivax strains occurred despite close medical supervision in physically fit patients. A conservative case fatality rate of 0.1%, low as it might appear, is sufficient to sustain the notion that P. vivax infections are a threat to life.

7. CONCLUSIONS ‘Prior to the introduction of malaria therapy, the description of a typical malarial attack given in most text-books on tropical medicine was based on a study of the disease in persons resident in, or recently returned from a malarious country. Observations of induced malaria attacks in subjects who have never before been exposed to infection have shown that in many respects these descriptions were incorrect.’ (Covell, 1956). These are humbling words from an eminent malariologist of 30 years experience, many of which were spent investigating the epidemiology and treatment of malaria in India. The observations of induced malaria revolu- tionised the way in which Covell (1887–1975) and the malariologists of his generation perceived malaria. We feel confident in thinking that few of today’s generation malariologists are aware of the existence of these observations. Our hope in writing this review (with its unapologetically abridged list of relevant references) is that it will encourage our colleagues to become acquainted with this unique record of the natural history of malaria and its parasites and inspire them to devise investigations to fill the many gaps that are still there. We should warn potential readers that this literature could be addictive.

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Note: Page numbers with “f” denote figures; “t” tables.

A Apicoplast, 210–211 A-variant Artificially induced infections, naturally G6PD deficiency, 147, 168 acquired immunity features based in sub-Saharan Africa, 170, 172 on, 88t variant dependency, 180 Asia Pacific Malaria Elimination Network primaquine sensitivity, 180–181 (APMEN), 154 Abs. See Antibodies Atabrine, 137–138 Acute haemolytic anaemia (AHA), 137, 149 B Altered redox equilibrium, 177–178 Bayesian geostatistical model, 156–157 AMA1. See Apical membrane antigen 1 Benign tertian malaria, 31–32 8-aminoquinoline clinical development BinaxNOW® G6PD assay, 154–155 programme, 140–141 Biological discovery era 8-aminoquinoline pamaquine, 137, 137f cell biology and germ theory, 29–30 Anaemia, 151–152 neurosyphilis observation, 30–31 AHA. See Acute haemolytic anaemia treatments, 31–32 (AHA) human variation and blood groups CNSHA. See Chronic nonspherocytic blood group systems, 36t–37t haemolytic anaemia (CNSHA) cell biology and infectious disease, sickle cell anaemia, 61–62 34 Anopheles farauti, 33–34 heritability and human population Anopheles mosquito, 3 biology, 34–35 Anophelines malariotherapy and African-based malariotherapy practice, 240 resistance, 32–33 malarial infection, 106 Anopheles farauti, 33–34 mosquito species, 10–11 natural exposure, 33 mosquito-induced infections, 239 treatments, 32 observations, 240–241 Biomarkers, 81 sexual stage of, 5 Blood group systems, 34–35, 36t–37t transmission dynamics to, 239–240 human variation and, 34–35 zygote of, 111 Blood-stage cycle, 3 Antibodies (Abs), 80 Blood-stage immunity effector mechanisms, cross-reacting antibodies, 34–35 93–94 specific for PvDBP, 46–47, 60 merozoite, 94–95 Antigen-specific T-cell responses, 106 P. knowlesi, 93–94 Antimalarial drugs, 213–214 RBC and Pv membrane, 93–95 Aotus (owl or night monkeys), 14–15, 97–98 Blood-stage immunity targets Apical membrane antigen 1 (AMA1), immunological memory, 105 94–95, 213–214 P. vivax merozoite proteins, 96–97 Apical membrane antigen-1 of P. vivax Plasmodium merozoites, 95, 95f (PvAMA1), 100 protective immune responses, 96

257 258 Index

Blood-stage immunity targets (Continued ) CVCs. See Caveola-vesicle complexes PvAMA1, 100 Cytoadherence ligands, 9 PvDBP, 102 Cytokines, 103–104 PvDBPII-based vaccine, 102–103 Cytotoxic lymphocyte (CTL), 107–109 vaccine-induced anti-DBP Abs, 102–103 D PvMSP1, 97–98 DBLEBP. See Duffy-binding-like erythro- PvMSP3, 98–99 cyte binding proteins PvMSP9, 99–100 DBP. See Duffy binding protein PvRBPs, 101 Deletion of GYPC exon 3 (GYPCDex3), P. vivax genome, 102 58–59 PvDBP-II, 101–102 Differential acquisition of immunity pvrbp2 gene, 101 to P. falciparum, 81 T-cell immune responses, 103–104 of clinical immunity, 83 Blood-stage infection, force of, 89 endemic areas, 83–84 epidemiological observations, 83 C modulator, 84 Carboxy-primaquine, 175 prevalence, 83 Care-Start™ G6PD screening test, 154–155 to P. vivax, 81 Caveola-vesicle complexes (CVCs), 6, 91 of clinical immunity, 83 CD4+ memory T cell, 103–104 endemic areas, 83–84 CD8+T cells, 109 epidemiological observations, 83 Chemokine receptor, 91–93 evidence for rapid acquisition of Chesson strain, 138–139 immunity, 82f Chimpanzee infections, 13–14 modulator, 84 Chronic nonspherocytic haemolytic prevalence, 83 anaemia (CNSHA), 149, 150t treatment Circumsporozoite precipitation reaction development, 85–86 (CSP reaction), 107 experimental infections, 87 Circumsporozoite protein (CS protein), NAI features, 87–88, 88t 106, 213–214 P. vivax, 84–86 CNSHA. See Chronic nonspherocytic P. vivax parasitaemias, 84–85 haemolytic anaemia strain-specific immunity, 86–87 Comparative genomics, and P. vivax Dose dependency, haemolytic effect of genome, 207–213 primaquine, 179–180 Bill and Melinda Gates Foundation, Duffy antigen grants, 212–213 for blood group, 35–38 difficulties of, 207–208 primary structure, 42f–43f whole genome sequences Duffy binding protein (DBP), 35–38, 45–46 of Asian Old World Monkey malaria antigen interaction, 46 clade, 209t genes, 15–16 of P. vivax, 208t in P. cynomolgi, 15–16 CS protein. See Circumsporozoite protein parasite ligand–host receptor relationship, CS-specific cytolytic CD4+T cells, 110 46–47 CSP reaction. See Circumsporozoite of parasites, 8 precipitation reaction PvDBP, 45–46 CTL. See Cytotoxic lymphocyte Duffy blood group nomenclature, 45t Index 259

Duffy polymorphism enzyme activity defect, 148 FY*AES allele in Papua New Guinea, NADPH, 147–148 47–48 residual enzyme activity, 146 Fybweak antigen, 48–49 G6PD genetics and inheritance, 143 global distribution of, 54–57 13 exons, 143 P. knowlesi Duffy binding protein, 49 gene’s X-linked inheritance, 145 semi-quantitative PCR assay, 48 mutations, 143–145, 144f Duffy protein, 40–41 G6PD Mediterranean, 173 Duffy-binding-like erythrocyte binding G6PD gene, diversity of mutations in, 144f proteins (DBLEBP), 45–46 Gametocyte, 9–10 Duffy-independent blood-stage infection, P. falciparum, gametocyte development, 50–52 9–10 Duffy-independent red cell invasion, 52 P. vivax- gametocyte-infected erythrocyte, 9–10 E P. vivax gametocytes, 6 Erythrocyte membrane protein band 3. See General paralysis of the insane (GPI), Solute carrier family 4, anion 29–30, 225 exchanger, member 1 (SLC4A1) Genetic resistance factor Erythroid silent (ES), 41 blood-stage infection, 39 Extensive polymorphism, 100, 215–216 distribution, 38–39 Duffy blood group negativity, 39–40 F Germ theory, 29–30 Falciparum malaria, 31–32, 48 neurosyphilis observation, 30–31 α-thalassaemia, 58 treatments, 31–32 immune regulation, 93 Global genetic diversity and SAO and Gerbich negativity, 59–60 mitochondrial genomes, 214–215 Favism, 136–137, 149 MSP-3α, 213–214 FDA. See US Food and Drug Association mtDNA, 215 Fluorescent spot test (FST), 150–151 multilocus investigations, 213 FY alleles, global frequencies of, 55f population genetic investigations, 213 Fy a+b-phenotype, 102 population genomic investigations, 213 Fy blood group antigen, 102 target gene approaches, 213 FY*A allele, 102 Glucose-6-phosphate dehydrogenase FY*B allele, 102 deficiency (G6PD deficiency), 54, Fybweak polymorphisms, 44 57–58, 135 Fyx polymorphism, 43–44 in Africa+, 169–170 in America, 168–169 G antimalarial primaquine, 135 G6PD deficiency. See Glucose-6-phosphate in Asia Pacific, 164f–165f, 166–168 dehydrogenase deficiency clinical manifestations, 148, 150t G6PD enzyme, 143 AHA, 149 cell age, 146 CNSHA, 149 dimer bind, 146 G6PD alleles, 149–150 haemolytic risk, 147 NNJ, 148–149 Mediterranean variant, 147 P. vivax therapy, 150 mutations, 146 symptoms, 148 PPP as anti-oxidative defence in countries East of India, 167 260 Index

Glucose-6-phosphate dehydrogenase pamaquine, 137–139 deficiency (G6PD deficiency) predisposing factor, 139 (Continued ) primaquine tolerability and safety diagnosis methods, 150 daily dosage, 142 evidence of selective advantage 14-day regimen, 140–141 case-control in vivo evidence, 172–173 growing acknowledgement, 142–143 epidemiological evidence, 171 haemolysis, 141–142 in vitro evidence, 171–172 primaquine sensitivity, 141 evolutionary drivers of distribution, toxicity, 140–141 170–171 in Saudi Arabia, 169 favism, 136–137 in sub-Saharan Africa, 170 in Indian subcontinent, 167 in Yemen, 169 molecular diagnostic tests, 152 Glutathione dimers (GSSG), 147–148 enzyme activity, 152–153 Glutathione monomers (GSH), 147–148 high-end laboratory requirements, Glycophorin A (GYPA), 58–59 152–153 Glycophorin C (GYPC), 58–59 mutation mapping, 163 Glycosylphosphatidylinisotol anchor, national allele frequency, 162f 97–98 neglecting P. vivax role, 174 GPI. See General paralysis of the insane G6PD Mediterranean, 173 GSH. See Glutathione monomers protective role, 173 GSSG. See Glutathione dimers with P. vivax endemicity, 164–170 GYPA. See Glycophorin A P. vivax transmission, 164–166, 166f GYPC. See Glycophorin C phenotypic diagnostic tests, 150–151 GYPCDex3. See Deletion of anaemia, 151 GYPC exon 3 heterozygotes, 151–152 in large-scale population, 151 H molecular methods, 152 Haemoglobin, 141–142 screening methods, 150–151 Haemoglobinopathies, 58 sensitivity, 152 Haemolysis, 141–142 in Philippines, 168 drug-induced, 188–190 point-of-care G6PD test, unavailability, primaquine-induced, 175–179 153 Haemolytic risk haemolysis, 154 assessment RDT, 154–155 drug-induced haemolysis, 188–190 total RBC count, 154 higher dosage schedule, 190 population, estimates of, 161–163 at individual level, 187 prevalence, 136 national risk index, 189f prevalence mapping, 155–156 WHO treatment guidelines, 188 evidence-base, 156 factors affecting, 179 mapping model, 156–157 dose dependency, 179–180 overview, 157–159 red blood cell age dependency, 183–184 primaquine, path to sex dependency, 184 8-aminoquinoline pamaquine, 137 variant dependency, 180–183 atabrine, 137–138 prediction, 184 drug–drug interaction, 138–139 Hepatocyte, 239 efficacy, 140 HepG2-A2 cells, 14 Index 261

Heterozygotes, 145 Infected RBC (iRBC), 4f, 9 Hexose monophosphate shunt. See Pentose and blood-stage immunity targets, phosphate pathway (PPP) 95–105 Hibernans types. See Temperate strains P. vivax- infected, 11 Hidden cycle PvHIST/CVC-8195 release in, 91 hepatocyte, 239 Infected red blood cell membrane, 89–91 patients and infected mosquitoes, Interferon-γ (IFN-γ), 99–100, 108f 238–239 Interleukin (IL) relapses of P. vivax, 237–238 human interleukin 8 receptor, 40–41 Schaudinn’s one-off observation, 238 IL-4 responses, against PvMSP9 peptides, HLA. See Human leukocyte antigen 99–100 5H6MQ. See 5-hydroxy-6-methoxy-8- Intermittent fevers, 30–31, 229–230 aminoquinoline Interquartile range (IQR), 159–161 Human leukocyte antigen (HLA), 98–99 in uncertainty quantification, Human malaria species, 10–11 159f–160f Human relapsing parasite, 10–11 IQR. See Interquartile range 5-hydroxy-6-methoxy-8-aminoquinoline iRBC. See Infected RBC (5H6MQ), 175 Hypnozoites, 6–8, 88–89, 106–107 K Knickerbocker Blood Bank of New York I City, 35–38 IE. See Infected erythrocyte Korean War (1950–1953), primaquine use, IFN-γ. See Interferon-γ 139 IL. See Interleukin Küpffer cells (KC), 3, 107–109 Immune responses, against malaria Ab- and T-cell-specific responses, 111 L Anopheles mosquito, 106 Light microscopy (LM), 47–48, 83 CD8+T cells, 109 Long-lasting insecticide-treated nets cell-mediated mechanisms, 107–109 (LLINs), 80–81 CS-specific cytolytic CD4+T cells, 110 Lyonization, 145, 151–153 genomic and proteomic technologies, 110–111 M hepatocyte invasion, 106 mAb. See Monoclonal Ab hypnozoites, 106–107 Macaca mulatta red blood cells, P. knowlesi lymphocyte, 107–109 invasion of, 28, 29f at pre-erythrocytic stages, 107, 108f Macrogamete, 5 sporozoites movement, 106 Mahidol variant, 181–182 Immunological memory, 105 Malaria, 204, 224 In vitro models, 11 causative agents, 224 liver-stage investigation, 13 immune response P. vivax-infected RBC, 11 Ab- and T-cell-specific responses, 111 in rhesus monkey RBC, 11–12 Anopheles mosquito, 106 In vitro evidence, 171–172 CD8+T cells, 109 In vivo evidence, case-control, 172–173 cell-mediated mechanisms, 107–109 Infected erythrocyte (IE), 81 CS-specific cytolytic CD4+T cells, 110 hemozoin pigment granules in, 9–10 genomic and proteomic technologies, Schüffner’s stippling, 6 110–111 262 Index

Malaria (Continued ) Caucasian patients vs. African-American hepatocyte invasion, 106 patients, 32–33 hypnozoites, 106–107 caveats and relevance lymphocyte, 107–109 acquisition and eventual resolution, 241 at pre-erythrocytic stages, 107, 108f clinical severity, 244–245 sporozoite movement, 106 eclectic collection of strains, 243 infection grasp of malariologists, 241 malariotherapy, practice, 236 high endemicity, 241–242 myriad data, 236–237 through malariotherapy, 243 natural course, 236 malariological observations, 242–243 with P. vivax, 237 contributions to malariology, 233 malariotherapy, 224 hidden cycle, 237–239 Malaria Atlas Project (MAP), 156 laboratory-bred anopheline colonies, G6PD mapping model, 156–157 239 generation, 156 malaria infection natural course, map’s prediction confidence, 159–161 236–237 prevalence map, 157–159, 157f–158f malarial parasites diversity, 234 regional prevalence, 161 naturally acquired immunity uncertainty, 159–161, 159f–160f characteristics, 234–236 Malaria Hypothesis, 34 transmission to anophelines dynamics, Malaria red cell invasion, 35 239–241 Duffy binding protein unicity theory, demise, 233–234 DBP–Duffy antigen interaction, 46 in England, 231 parasite ligand–host receptor relation- GPI, 225 ship, 46–47 dire prognosis, 226 PvDBP, 45–46 and malariology genetic resistance factor observations, limitations to, 230 blood-stage infection, 39 malariologists, 231–233 Duffy blood group negativity, 39–40 malariology state, 229–230 impressive distribution, 38–39 natural exposure, 33 molecular and cellular basis in paretic patients, 31–32 comparative sequence analyses, 44 practice, 228 Duffy antigen, 42f–43f hippocratic primum non nocere, 228 Duffy serology, 43–44 using infected blood, 228–229 Fybweak polymorphisms, 44 public health standpoint, 229 methodical progress, 40–41 proponent of, 227f resident PNG, 41–43 single-mindedness, 226 SNP, 41 Station for Malaria Research, 232–233 working guidelines, 45t in United States of America, 232 serological recognition, 35–38 MAP. See Malaria Atlas Project Malaria tropica, 31–32 MBC. See Memory B cell See also Falciparum malaria Mediterranean variant, 147, 183 Malarial parasites, diversity of, 234 Memory B cell (MBC), 105 Malariotherapy, 32–33, 224 Mendelian X-linked gene, 143 and African-based resistance to P. vivax, Merozoite apical organelles, 8 32–34 Merozoite surface protein 3α (MSP-3α), and treatment of neurosyphilis, 32 213–214 Anopheles farauti, 33–34 Mepacrine. See Atabrine Index 263

Methaemoglobin (metHb), 150–151, 177 Naturally acquired immunity (NAI), 78 Mitochondrial genomes, 214–215 characteristics, 234–236 Molecular diagnostic methods to malaria, 78–79 Duffy polymorphism and population biomarkers, 81 distribution blood-stage infections, 79–80 Duffy-negative protection, 56f premunition, 80 frequency distribution, 53f transmission in sexual stages, 80 FY alleles, 55f to P. vivax, 80–81 G6PD deficiency, 54 Neonatal jaundice (NNJ), 148–149 PvPAR, 54–57 Neurosyphilis, 225 Duffy polymorphism on resistance primary cure, 84–85 FY*AES allele in PNG, 47–48 New World monkey species, 12, 14–15 Fybweak antigen, 48–49 NHP experimentation. See Non-human P. knowlesi DBP, 49 primate experimentation semi-quantitative PCR assay, 48 NK cells. See Natural killer cells Duffy-independent red cell invasion, 52 NNJ. See Neonatal jaundice blood-stage infection, 50–52 Non-Duffy gene polymorphisms, 57 Duffy-negative P. vivax resistance G6PD deficiency, 57–58 factor, 49–50 haemoglobinopathies, 58 Duffy-positive or Duffy-negative Southeast Asian ovalocytosis, 58–60 people, 52 Non-human primate experimentation Giemsa-stained thin smear prepara- (NHP experimentation), 2 tions, 51f New World monkey species, 14–15 malaria-transmission regions, 50 molecular and cell biology technologies, O 47 Omics technology, 17–18, 207 non-Duffy gene polymorphisms, 57 On the Origin of Species (Darwin), 34 G6PD deficiency, 57–58 Open reading frame (ORF), 40–41 haemoglobinopathies, 58 Oxidative stress, 176–177 Southeast Asian ovalocytosis, 58–60 Molecular force-of-infection (molFOI), 83 P Molecular mimicry, 211–212 Pamaquine, 137–138 molFOI. See Molecular force-of-infection chemical structure of, 137f Monkey malaria clade Parasitaemia, 48 comparative genomics, 207 blood-stage, 32–33, 38–39 P. knowlesi, 211–212 and fever, 84–85 reference genomes, 210 and NAI, 79–80 whole genome sequences, 209t PCR. See Polymerase chain reaction Monoclonal Ab (mAb), 99–100 Pentose phosphate pathway (PPP), Mosquito-induced infections, 239 147–148 MSP-3α. See Merozoite surface protein Heinz bodies, 148 3α PfEMP1. See PfVSA erythrocyte mtDNA sequencing, 215 membrane protein 1 PfMSP3, 98–99 N PfVSA erythrocyte membrane protein 1 NAI. See Naturally acquired immunity (PfEMP1), 89–90 Natural killer cells (NK cells), 103–104 PHIST protein. See Plasmodium helical Natural regulatory T cells, 103–104 interspersed subtelomeric protein 264 Index

Plasmodium cynomolgi (P. cynomolgi), 5, 15–16, gametocytes, 9–10 205 genome and comparative genomics Plasmodium diversity apicoplast, 210–211 global genetic diversity, 213–215 biological material, 212–213 importance of, 204 genome sequences, 208t–209t, 212 Plasmodium falciparum (P. falciparum), 5, 81, molecular mimicry, 211–212 207, 228 Plasmodium coatneyi, 210 Plasmodium fieldi (P. fieldi), 5 P. falciparum genome, 207–209 Plasmodium helical interspersed subtelo- P. gonderi, 210 meric protein (PHIST protein), 91 using Sanger sequencing technology, Plasmodium ovale (P. ovale), 5–6, 233–234 207–208 Plasmodium perniciosum, 234 SNPs and microsatellites, 209–210 Plasmodium semiovale (P. semiovale), 5, 17 sporozoite-specific transcription, 212 Plasmodium vivax (P. vivax), 2, 28, 81, 204. See from genomics to systems biology, 17–18 also Molecular diagnostic methods functional interaction networks, 18–19 biological characteristics, 88. See also machine learning techniques, 18–19 Naturally acquired immunity (NAI) metabolomics high-throughput force of blood-stage infection, 89 technologies, 17–18 immunosuppressive networks, 93 metabolomics sensitivity, 19 infected RBC membrane, 89–90 systems biology approaches, 19 predicted VSA role, 90 global genetic diversity Pv infections, 91 mitochondrial genomes, 214–215 PvHIST/CVC-8195, 91, 92t MSP-3α, 213–214 red cell invasion ligands, 91–93 mtDNA, 215 relapses, 88–89 multilocus investigations, 213 variation of surface antigens, 89–90 population genetic investigations, 213 biological discovery era population genomic investigations, cell biology and germ theory, 29–32 213 human variation and blood groups, target gene approaches, 213 34–35 life cycle, 3, 4f malariotherapy and African-based blood-stage cycle, 3 resistance, 32–34 growing stage, 3–5 endemicity, 166f infection stage, 6 evolutionary history for liver-stage development, 6–7 Asian and African origins, 204 P. ovale, 5–6 extraordinary biological diversity, 207 penetrating stage, 5 molecular phylogenies, 205 in Southeast Asian macaque monkeys, 5 P. inui, 205–207 sporozoite infection, 7–8 P. schwetzi, 205 malaria red cell invasion, 35 phylogenetic tree, 206f DBP, 45–47 ex vivo models genetic resistance factor, 38–40 aseptic P. vivax sporozoites production, molecular and cellular basis, 40–44 14 serological recognition, 35–38 chimpanzee infections, 13–14 merozoite interaction, 63f from neotropical New World monkey neotropical NHP models, 14–15 species, 12 phylogenetic tree of, 206f in P. knowlesi blood-stage gene Plasmodium knowlesi invasion, 29f expression, 12–13 population genetics Index 265

adaptive traits, 215–216 Population genetics antigenic genes, 216–217 adaptive traits, 215–216 human Duffy gene, 217 antigenic genes, 216–217 in India, 216 human Duffy gene, 217 using microsatellites and SNP markers, in India, 216 217 using microsatellites and SNP markers, RBC, 28–29 217 relapse treatment, haemolytic risk Population genomic investigations, 213 assessment, 185 PPP. See Pentose phosphate pathway drug-induced haemolysis, 188–190 Pre-erythrocytic stages, immune responses, haemolytic risk prediction, 186–187 205, 224 higher dosage schedule, 190 Ab-specific responses, 111 at individual level, 187 Anopheles mosquito, 106 national level, 185 CD8+T cells, 109 national risk index, , 189f cell-mediated mechanisms, 107–109 proposed framework, 185–186 CS-specific cytolytic CD4+T cells, 110 WHO treatment guidelines, 188 genomic technologies, 110–111 resistance to, 61f hepatocyte invasion, 106 reticulocyte host cells, 8 hypnozoites, 106–107 CVCs, 8–9 lymphocyte, 107–109 iRBCs, 9 at pre-erythrocytic stages, 107, 108f parasite-encoded proteins, 8–9 proteomic technologies, 110–111 RBCs, 9 sporozoite movement, 106 reticulocyte-binding proteins, 8 T-cell-specific responses, 111 sexual life strategies Premunition, 80 Anopheline mosquito species, 10–11 Primaquine, 137, 174. See also gametocyte, 9–10 Glucose-6-phosphate dehydroge- sexual maturity, 9–10 nase deficiency (G6PD deficiency) Southern Asian macaque monkeys, 15 chemical structure of, 137f NHP-relapsing malaria species, 16 primaquine-induced haemolysis, 175 P. cynomolgi, 15–16 altered redox equilibrium, 177–178 P. simiovale, 17 methaemoglobin, 177 P. vivax characteristics, 15–16 oxidative stress, 176–177 specific antibodies, 105 primaquine and metabolites, 175–176 in vitro models, 11 Primaquine (Continued ) liver-stage investigation, 13 significance of, 178–179 P. vivax-infected RBC, 11 sensitivity, 180 in rhesus monkey RBC, 11–12 therapy, 185 whole-genome sequences of, 208t–209t haemolytic risk prediction, 186–187 Plasmodium vivax DBP (PvDBP), 45–46, ranking national-level risk, 185–186 101–102 Pv antigen-specific effector, 103–104 Point-of-care G6PD test, requirements, 153 PvAMA1. See Apical membrane antigen-1 haemolysis severity, 154 of P. vivax RDT, 154–155 PvDBP. See Plasmodium vivax DBP total RBC count, 154 PvHIST/CVC-8195, 91 Polymerase chain reaction (PCR), 83 PvMSP1, 97–98 semi-quantitative, 47–48 PvMSP3, 98–99 for target gene approaches, 213 PvMSP9, 99–100 266 Index

PvPAR, 54–57 SLC4A1. See Solute carrier family 4, anion pvrbp2 gene, 101 exchanger, member 1 PvRBPs, 91–93, 101 SNP. See Single nucleotide polymorphism P. vivax genome, 102 Soluble immune mediators, 109 PvDBP-II, 101–102 Solute carrier family 4, anion exchanger, pvrbp2 gene, 101 member 1 (SLC4A1), 58–59 Southeast Asian ovalocytoses (SAOs), Q 58–60 Quinacrine. See Atabrine Southern Asian macaque monkeys, 15 NHP-relapsing malaria species, 16 R P. cynomolgi, 15–16 Rapid diagnostic test (RDT), 154–155 P. simiovale, 17 Rapid acquisition of immunity, evidence P. vivax characteristics, 15–16 for, 82f Spatial distribution mapping. See also RBC. See Red blood cell Glucose-6-phosphate dehydroge- RBL proteins. See Reticulocyte binding- nase deficiency (G6PD deficiency) like’ proteins population estimation, 161 RDT. See Rapid diagnostic test IQR, 161 Red blood cell (RBC), 3, 28–29, 35, 135 malaria endemic regions, 163, age dependency, 183–184 164f–165f Red cell invasion mutation mapping, 163 Duffy independent, 49–52 national allele frequency, 161, 162f ligands, 91–93 prevalence map, 155–156 Red fluorescent protein gene, 16–17 MAP, 156 Relapse aetiology, 237–238 national-level map, 155–156 Reponema pallidum, 29–30 regional prevalence, 161 Reticulocyte, 8–9 uncertainty in, 159–161, 159f–160f ‘Reticulocyte binding-like’ proteins (RBL Sporozoite infection, 7–8 proteins), 101 Sporozoite surface protein (SSP), 106 Sporozoite-specific transcription, 213 S SSP. See Sporozoite surface protein Salvador I reference genome sequence, 213 SSP2/TRAP proteins, 106 Saimiri (squirrel monkeys), 14–15 Station for Malaria Research, 232–233 SAO. See Southeast Asian ovalocytosis Syphilis, 224 Schüffner’s stippling, 6 Sex dependency, 184 T Sexual stage parasites, 111 T helper type (Th1-type), 103–104 in malaria endemic areas, 111 Tafenoquine, 137 P. falciparum TBI, 112 chemical structure of, 137f P. vivax TBI, 112 TBI. See Transmission blocking Anopheles albimanus mosquitoes, 113 immunity gametocyte infectivity, 112 Temperate strains, 7–8 patients’ sera, 112–113 Th1-type. See T helper type TBI, 111 Transmission blocking immunity (TBI), target molecules against, 113 111–112 Single nucleotide polymorphism (SNP), 41, Tropical strains, 7–8 173 Trickle effect, 87 Index 267

U Whole genome sequences Unicity theory, demise of, 233–234 of Asian Old World monkey malaria US Food and Drug Association (FDA), 141 clade of P. vivax, 208t, 212 V Wild-type erythrocytes, 183–184 Variant dependency, 180–183 Variant surface antigen (VSA), 81 X Vivax malaria. See Benign tertian malaria X-linked inheritance of G6PD gene, 145 W mechanism of, 156 West Africans and P. vivax absence of, 38–39 Y FY*BES allele observed in, 50–52 Young children, P. vivax in resistance to, 29–30 less than 2 years, 81 WHO school-aged children, 60 Global Eradication Campaign of the Young RBCs, 8 1950s to 1960s restricting infection to, 9 recommendations, 180–181 for Mediterranean variants, 183 Z ‘mild’ and ‘severe’ deficiency, 188 Zygote formation, 5, 111 on primaquine, 181–182 studies, using Affymetrix platform, 212 re-assigning of, 188 and TBI action, 111