Detection and Isolation of Plasmodium Liver Stages and Analysis of Circumsporozoite Protein Antigen Processing

Home , CD81, Dumoulin, Meuleman, Plasmodium relictum

STUDIES ON EXOERYTHROCYTIC DEVELOPMENT OF PLASMODIUM FALCIPARUM IN VITRO: DETECTION AND ISOLATION OF PLASMODIUM LIVER STAGES AND ANALYSIS OF CIRCUMSPOROZOITE PROTEIN ANTIGEN PROCESSING

by Peter Dumoulin

A dissertation submitted to Johns Hopkins University in conformity with the

requirements for the degree of Doctor of Philosophy

Baltimore, Maryland

May, 2015

Abstract

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden despite efforts to eradicate the parasite. Infection is initiated when a mosquito deposits sporozoites into the dermis that then actively invade hepatocytes. Sporozoites and the resulting liver stage of Plasmodium falciparum infection are leading targets for generation of a protective vaccine. The circumsporozoite protein is the major surface protein on sporozoites and is the target of vaccines aimed at preventing blood stage infection. During the process of invasion, sporozoites cleave the N-terminus of CSP and consequently two forms of CSP are potentially exposed to the immune system. We utilized ectopic expression of two natural form of CSP to quantify their abilities to serve as targets for MHC class I-restricted CD8+ T cells. We determined that presentation of both forms of

CSP on MHC class I depends on proteasomal activity, however, presentation of full length

CSP is more efficient and is conferred by the presence of N-terminal lysine residues absent in the cleaved form of CSP. To evaluate the presence of these two forms during in vitro infection, we developed methods to allow for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. We showed that exoerythrocytic forms can be detected and isolated from in vitro infected cultures of the

HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Furthermore, we present an experimental evidence, that our

ii methods can be applicable for the analysis of human host factors limiting development of

P. falciparum EEFs.

iii

Main goals

1. Assess the ability of two forms of Plasmodium falciparum CSP, to serve as targets for MHC class I-restricted CD8+ T cell responses

Specific Aims:

A. To develop an in vitro system for monitoring the MHC class I processing and presentation of peptide epitopes derived from the P. falciparum CSP antigen

B. To assess the ability of targets expressing one of the CSP forms to induce specific CD8+ T cell activation in a MHC class I-restricted and non-restricted manner

C. To examine the role of major cellular proteolytic machineries in the processing of two forms of CSP

D. To investigate the role of the ubiquitin system in the MHC class I-restricted processing of peptide epitopes from two forms of the CSP antigen

2. To develop an experimental system for detection and isolation of Plasmodium falciparum EEFs in vitro

Specific Aims:

A. To develop an in vitro hepatocellular model permissive for propagation of P. falciparum EEFs

B. To develop flow cytometry-based detection and specific isolation of P. falciparum EEFs

C. To compare dynamics of P. falciparum EEF development in vitro using different experimental hepatocellular systems

D. To validate the applicability of our experimental model for the analysis of human host factors limiting development of P. falciparum EEFs.

Table of Contents

Abstract ...... ii Main goals ...... iv Table of Figures...... viii Chapter 1 : General Introduction ...... 1 History of elucidating the Plasmodium life cycle ...... 5 From skin to merosome ...... 9 Targeting exoerythrocytic forms: whole organism vaccines ...... 13 Mechanisms of immune protection ...... 17 Biology of CSP ...... 23 Techniques to study the Plasmodium liver stage ...... 26 Concluding remarks ...... 28 Chapter 2 : Processing and presentation of two forms of CSP on MHC class I ...... 29 Introduction ...... 30 Presentation of peptides on MHC class I ...... 30 Targeting Plasmodium: role of the MHC class I antigen processing and presentation pathway ...... 34 Two forms of CSP as targets of the CD8+ T cell immune response ...... 38 Results ...... 40 Ectopic expression of CSP in hepatocytes ...... 40 Identification of CSP in traversed and infected hepatocytes during in vitro infection .. 41 Two forms of CSP differentially activate CD8+ T cells...... 43 Targets expressing one of two forms of CSP are differentially lysed by CD8+ T cells... 49 Two forms of CSP do not alter immune phenotypes of targets or overall susceptibility to death stimuli ...... 51 Half-life of two forms of CSP and dependence of proteasomal degradation on MHC class I presentation ...... 53 CSP-Full forms polyubiquitin chains more efficiently than CSP-Short ...... 54 Individual lysine residues in the N-terminus of CSP are required for efficient MHC class I-restricted antigen presentation ...... 56 Discussion ...... 59 Chapter 3 : Specific detection and isolation of P. falciparum EEFs in vitro ...... 67

Introduction ...... 68 Results ...... 72 Immortalized human hepatocyte cell lines are permissive for traversal by P. falciparum sporozoites ...... 72 Human hepatocyte cell lines exhibit differential ability to support development of P. falciparum EEFs ...... 76 P. falciparum-infected HC-04 hepatocytes can be specifically isolated ...... 81 Primary human hepatocytes support P. falciparum 3D7HT-GFP EEFs in vitro ...... 84 Discussion ...... 93 Chapter 4 : Flow cytometry based assays of P. falciparum liver stages in vitro: from sporozoite infectivity to EEF development ...... 100 Introduction ...... 101 Results ...... 104 CD81 is required for P. falciparum infection of primary human hepatocytes, but is not essential for the infection of HC-04 ...... 104 Humanized CSP-specific antibody 2A10 inhibits traversal and reduces the number of P. falciparum EEFs in human hepatocytes in vitro ...... 107 Discussion ...... 109 Chapter 5 : Future Directions ...... 113 Chapter 6 : Materials and Methods ...... 119 Human hepatocyte culture ...... 120 Collagen coating and hepatocyte plating densities ...... 121 Infection with P. falciparum sporozoites ...... 121 Detection of hepatocyte traversal ...... 123 Detection of EEFs by flow cytometry ...... 123 Quantification of parasite development by RT- PCR ...... 124 Immunofluorescence of EEFs in HC-04 and primary human hepatocytes ...... 124 CD81 detection and surface neutralization ...... 125 Evaluation of the efficacy of mouse serum containing humanized anti-CSP mAb 2A10 on parasite motility ...... 126 Generation and maintenance of CTLs ...... 126 Construction of CSP containing mammalian expression plasmids ...... 127 Transfection of hepatocytes and purification by MACS ...... 128 Detection of CSP by western blot ...... 128 Detection of ectopically expressed CSP ...... 129

T-cell activation assay: IFNγ release ...... 129 Cytotoxicity lactate dehydrogenase assay ...... 129 Chemical inhibitor treatments ...... 130 Immunoprecipitation of HA-ubiquitin complexes ...... 130 Peptide synthesis and target pulsing ...... 131 Statistical analysis ...... 131 References ...... 132 Curriculum Vitae ...... 157

vii

Table of Figures

Figure 1.1 The lifecycle of the human Plasmodium parasites...... 2

Figure 1.2 First microscopic identification of the pre-erythrocytic stage of P. falciparum. .... 8

Figure 1.3 Cartoon representation of the sporozoite interacting with the liver sinusoid...... 11

Figure 1.4 Cartoon representation of merosomes budding and release into the blood stream.

...... 13

Figure 1.5 Visualization of the CSP reaction...... 15

Figure 1.6 Schematic of CSP and the RTS,S vaccine...... 20

Figure 1.7 Estimated relative population sizes (biomass) between stages of the P. falciparum lifecycle...... 23

Figure 1.8 Schematic of the structure of CSP from P. knowlesi based on nucleotide sequence...... 24

Figure 1.9 Regions of sequence identity in CSP between P. knowlesi and P. falciparum sequences...... 25

Figure 2.1 Schematic representation basic antigen presentation on MHC class I...... 31

Figure 2.2 Schematic of the structure of HLA-A2...... 33

Figure 2.3 Schematic representation of CSP during infection...... 39

Figure 2.4 Ectopic and natural expression of the two forms of P. falciparum CSP...... 42

Figure 2.5 Identification, isolation and expansion of HLA-A2 restricted viral peptide specific CD8+ T cells...... 44

Figure 2.6 Verification of CTL specificity...... 46

Figure 2.7 Identification and purification of transfected HC-04...... 47

Figure 2.8 MHC Class I restricted recognition of cells expressing CSP chimeras by peptide specific CD8+ T-cells...... 48

viii

Figure 2.9 Specific lysis of targets expressing CSP-Full or CSP-Short by CD8+ T-cells...... 50

Figure 2.10 Susceptibility of targets expressing two forms of CSP to T cell effector functions...... 52

Figure 2.11 Degradation of ectopically expressed CSP in HC-04 cells...... 54

Figure 2.12 Identification of ubiquitination patterns of two forms of CSP...... 56

Figure 2.13 Role of the N-terminus on presentation of CSP and the contributions of individual lysines...... 58

Figure 3.1 Specific detection of traversal by P. falciparum sporozoites in vitro...... 74

Figure 3.2 Human hepatocyte cell lines can be traversed by P. falciparum sporozoites...... 76

Figure 3.3 Identification of P. falciparum parasites by flow cytometry...... 77

Figure 3.4 Specific detection of infection by P. falciparum sporozoites in HC-04 cultures in vitro (48 hours postinfection)...... 78

Figure 3.5 Quantification of parasite-encoded 18S rRNA in cultures of human hepatocytes infected with P. falciparum...... 81

Figure 3.6 Visualization of parasites in HC-04 cells infected with P. falciparum...... 83

Figure 3.7 Primary hepatocytes have a high degree of autofluorescence and require adjustment to voltages...... 85

Figure 3.8 Comparison of detection of P. falciparum infection between primary human hepatocytes and HC-04...... 87

Figure 3.9 Specific detection of infection by P. falciparum sporozoites in primary hepatocytes in vitro (96 hours postinfection)...... 88

Figure 3.10 Parasite morphology and size in cryopreserved human hepatocytes from three donors...... 90

Figure 3.11 Parasite detection and persistence over time...... 92

Figure 4.1 Expression of surface CD81 by human hepatocyte cell lines and primary hepatocyte donors...... 105

Figure 4.2 Influence of CD81 blocking by mAb 1D6 on P. falciparum hepatocyte infection.

...... 106

Figure 4.3 Influence of a humanized anti-CSP mAb 2A10 on P. falciparum traversal and invasion...... 108

Chapter 1 : General Introduction

Malaria is a mosquito-borne infectious disease caused by protozoan parasites of the genus Plasmodium. The parasite is initially deposited by an infected mosquito into the dermis of the intermediate host [1,2] (Figure 1.1A) where it actively enters the blood stream and develops as a clinically silent stage in the liver [3,4] (Figure 1.1B). Mature liver stage parasites eventually seed the blood and initiate the erythrocytic cycles associated with clinical malaria [5,6] (Figure 1.1C,D). A portion of asexually reproducing parasites in the blood will lead to male and female gametocyte production, which can initiate infection of a new mosquito if taken up in a blood meal (Figure 1.1E).

C A

Figure 1.1 The lifecycle of the human Plasmodium parasites. (A) Infection of the human host begins when sporozoites are deposited in the dermis. (B) Sporozoites actively reach hepatocytes and invade. (C) Once infectious merozoites are formed the host cell is ruptured and merozoites are released. (D) Parasites invade red blood

2 cells. (E) A subset of parasites will commit to formation of gametocytes capable of infecting a mosquito if ingested with a blood meal. Adapted from [7]

Five species of Plasmodium are capable of causing disease in humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and

Plasmodium knowlesi [8,9]. Of these species, infection with P. falciparum results in the most complications and deaths, primarily in sub-Saharan Africa [10]. An estimated 207 million new cases of malaria occurred in 2012, resulting in an estimated 627,000 deaths

[10]. The current morbidity and mortality of malaria are concentrated in impoverished countries [11], particularly in tropical and subtropical regions. Examples of GDP growth rate increases after local eradication suggest that malaria is a cause of poverty and not just a confounding characteristic of malaria endemic countries [11]. Undoubtedly, resource- poor countries also have difficulty in sustaining control measures, resulting in a “poverty trap” [12]. Successful elimination has historically occurred in relatively temperate geographic regions with less intense transmission and a higher degree of seasonality [13].

Consequently, current efforts of eradication or elimination are now focused on the most intransigent bastions of malaria. A vaccine capable of slowing transmission has the potential to aid in these efforts.

Pathogenesis of P. falciparum can manifest as metabolic acidosis, severe anemia, respiratory distress, or coma and with complications during pregnancy [14–17]. Persistent infection and disease progression are often the result of strategies employed by P. falciparum to avoid clearance by the immune system [18] or mechanical destruction by the spleen [19]. A disproportionate number of polymorphisms in parasite invasion genes [20–

23] allow the parasite to persist at a population level. In the case of individual subunit

3 vaccination against AMA1, a significant allele-specific efficacy has been observed [24] but was ineffective at targeting most circulating parasite strains. Considerable effort has been aimed at replicating the naturally acquired immunity to blood stage parasites and at generating a vaccine capable of halting blood stage growth completely. However, these approaches are largely confounded by the same evolutionary escape mechanisms that persist at the population level of natural parasite transmission.

In addition to allelic variation between parasites, individual parasites have the ability to change expression of important genes within a single host. Cytoadherence is a tactic employed by the parasite to sequester in the blood vessels and tissues to escape clearance. This process is achieved through expression of PfEMP1 by the parasite at the

RBC surface to mediate adherence [25]. The parasite encodes a family of these genes that are antigenically distinct, and which are expressed one at a time [26]. The ability of the parasite to sequester has been implicated in serious clinical outcomes such a cerebral malaria and complications in pregnant women [27,28]. As the host generates an adaptive immune response to one variant of PfEMP1, the fitness of parasites expressing that gene decreases and a minor subset of parasites expressing an alternative allele gains the selective advantage within the host [29]. In this case, many of the same principles of selection and diversity that apply at the population level are present in an individual host. This antagonistic relationship is not surprising considering the extensive period of co-evolution that has existed between Plasmodium and its intermediate hosts.

Based on comparisons of Plasmodium gene sequences collected from wild apes in

Africa, P. falciparum originally entered the human population as a gorilla parasite [30].

The timing and number of cross-species transmission events remain controversial [31,32]

4 and largely based on assumptions of mutation rate in P. falciparum. Even without knowing an exact timeframe, it is apparent that Plasmodium parasites have generated considerable selective pressure on the human genome [33]. These protective variants are often loss of function mutations that affect the red blood cell and include hemoglobinopathies, G6PD deficiency and ovalocytosis.

History of elucidating the Plasmodium life cycle

Prior to the dissection of the etiological details of malaria, many theories existed to explain the presence of the cyclical fevers originally described by Hippocrates [34]. Even before usage of the term malaria, the general disease theory of antiquity focused on the concept of “bad-air” (later miasma) as contagion combined with the notion that individual susceptibility is mediated by a balancing of the four humors, as first proposed by Galen

(reviewed in [35]). The characteristic fevers observed in malaria patients were first attributed to an imbalance in one of the humors, either yellow or black bile. These theories relied on the concept of spontaneous generation of toxic air or organisms from non-related matter. The underpinning of this theory, in which living things are generated from the non- living, were left unchallenged for centuries. In 1668 Francesco Redi experimentally tested and refuted the theory of spontaneous generation by determining that the formation of maggots on meat required contact from flies [36]. Soon after, Antonie van Leeuwenhoek directly observed single celled organisms by using his newly developed single lens microscope [37]. Louis Pasteur was instrumental in providing formal experiments describing microbial causes of disease and suggesting that common diseases have the potential to be explained by germ theory [38].

These conceptual and technological advancements in microbiology set the stage as scientists began to search for a microbial determinant of malaria. The first observations of a parasite associated with malaria pigment in a patient’s blood were made by the French military doctor Alphonse Laveran [39]. His samples were unstained, which allowed him to observe motility or what we now know is the process of exflagellation of male gametocytes. In 1897 William George MacCallum described an additional non-motile parasitic form in avian blood and postulated that this was due to sexual dimorphism and observed temperature dependent ookinete formation [40]. However, this observation still did not explain the mechanism of parasite transmission or the reason behind geographic risk factors. Considerable attention was paid to environmental sampling as others suggested the possibility that mosquitoes vectored the parasite. Ronald Ross worked under the premise that mosquitoes could cultivate the malaria parasite, while noting that:

“The study is a difficult one, as there is no a priori indication of what the derived

parasite will be like precisely nor in what particular species of insect the experiment

will be successful…”[41].

After two years of attempts, he was successful at identifying pigmented parasites larger than typical mosquito cells only in mosquitoes fed on patients whose blood contained

“crescents” [41]. It was also noted that these cells were larger when observed later after blood meal, suggesting parasite development within the mosquito. However, before he was able to establish transmission back to humans he was posted to Calcutta, forcing him to utilize an avian model, Plasmodium relictum, due to the paucity of human malaria in the region [42]. Using this model he was able to observe sporozoites in the mosquito salivary gland and transmission to a new host [43]. Ross correctly assumed that a similar process

6 of mosquito incubation and transmission occurred in human malaria parasite transmission but was unable to conclusively demonstrate the life cycle of such parasites. This formal proof was provided in 1899 by Bignami and Grassi in Italy, which conclusively demonstrated transmission from the mosquito to human [44]. This new understanding of the lifecycle, from human to mosquito back to human, provided an important framework to begin public health measures aimed at vector control.

However, a “cryptic” or “negative” phase was repeatedly observed, in which parasites were not observable until about 10 days after mosquito bite. It was noted during this phase that blood was not infectious and that quinine was ineffective [45]. Grassi himself suggested that the morphological difference between the sporozoite nucleus and early stage blood trophozoites might be indicative of a developmental stage prior to blood stage parasitemia [46]. Yet the spurious observation that Plasmodium vivax sporozoites were able to penetrate and invade red blood cells persisted as evidence that sporozoites could progress directly to erythrocytic development [47,48]. The theory of direct progression to blood stage failed to explain the details of the observed cryptic phase and after several decades the theory was challenged by experiments with avian parasites showing development of sporozoites outside of erythrocytes [49,50]. None of these studies, however, directly implicated the site of development of primate trophic Plasmodium species. The study of Hepatocystis kochi in Kenya and Uganda by P.C.C. Garnham led to the discovery of developmental stages in the liver parenchyma [51]. Since H. kochi lacks an erythrocytic cycle and only invades red blood cells for gametocyte formation, Garnham rightly postulated that the liver may also serve as a developmental site for Plasmodium.

Soon after, using Plasmodium cynomolgi infected Anopheles maculipennis fed on rhesus

7 macaques, Garnham observed “plasmodial masses undergoing schizogony” in the liver

[52]. Similar forms were found in liver biopsies from human subjects experimentally exposed to “enormous” doses (770 mosquito bites) of P. falciparum and P. vivax infected mosquitoes [45,53] (Figure 1.2). These works represent the completion of the basic understanding of the Plasmodium life cycle begun by Laveran almost 70 years prior.

Figure 1.2 First microscopic identification of the pre-erythrocytic stage of P. falciparum. Biopsy of a liver from a volunteer after 770 mosquito bites from P. falciparum infected mosquitoes. The above image (x 700) is attributed to parasites that have been developing in the liver for 116 hours. Since multiple mosquito feeds were done on different days and only one biopsy was performed the age of the parasite in the liver can only be assumed by size. Adapted from [45] Fig. 2

From skin to merosome

Understanding the life cycle of Plasmodium was the first step in designing interventions aimed at interrupting parasite transmission. The concept that malaria transmission was caused by mosquitoes was quickly followed by the proof that individuals could be protected from malaria by protecting them from mosquito bites [42,46]. In a similar fashion, the discovery of the liver stage as an obligate replicative cycle prior to symptomatic blood stage infection offered the possibility of generating immunity that could eliminate the parasite prior to clinical episodes. Since the definitive demonstration in the 1940s that the cryptic phase is indeed an obligate liver stage of development, a considerable amount of work has elucidated the processes that allow for sporozoite targeting to the liver and progression to the blood stages. P. falciparum has a limited number of hosts, including humans and some primates [54–56], and consequently study of the liver stage has been undertaken largely with the use of rodent trophic parasites originally isolated from Thamnomys and Grammomys rodents in Africa [57,58].

The journey to the liver begins when sporozoites are injected into the host dermis

[1,2] and gradually move from the bite site through a process of non-productive cell invasion termed traversal [59,60], a process first observed as sporozoite penetration of macrophages [61]. Once sporozoites enter the circulation they attach through CSP region

II to the heparin sulfate proteoglycans (HSPGs) present in the extracellular matrix of the liver [62,63] (Figure 1.3A). Sporozoites detect these sulfation levels and convert from a migratory mode to an active mode for invasion [64]. The presence of HSPGs are not obligatory for invasion as shown during in vitro infections [65] but rather are important under flow conditions [66] similar to what the parasite experiences in vivo. This suggests

9 that HSPGs serve as a ligand for sporozoites to concentrate near hepatocytes after reaching the circulation in vivo and become activated for productive invasion.

Identification of the parasite genetic determinants of traversal [67,68] has confirmed the necessity of traversal in reaching the liver in vivo where direct access is blocked by the sinusoid endothelium and Kupffer cells [69]. Depletion of sentinel Kupffer cells allows entry of traversal-deficient sporozoites to the liver when injected intravenously

[67,70]. Traversal competent parasites have multiple routes available to traverse that can include but are not restricted to direct movement through Kupffer cells [71,72] (Figure

1.3B,C). Once sporozoites reach the liver parenchyma they continue to traverse hepatocytes [71] but induce a switch in motility to allow for productive invasion allowing for proper parasitophorous vacuole (PV) formation in hepatocytes [59,60,73] (Figure

1.3D). The particular hepatocyte ligands that mediate productive parasite invasion are not completely characterized.

B C

Figure 1.3 Cartoon representation of the sporozoite interacting with the liver sinusoid. (A) Sporozoites recognized sulfation levels in the liver sinusoid and become activated for infection. (B,C) Sporozoites cross the sinusoid layer through cell traversal activity that does not require Kupffer cell invasion (D) Sporozoites actively invade hepatocytes. Adapted from [5] Fig 2.

One of the host surface molecules that has been shown to be important for P. falciparum sporozoite invasion into hepatocytes is CD81 [74,75]. Even though CD81 is present at the hepatocyte surface, it is not liver specific and is commonly found on the

11 surface of lymphocytes [76]. Prior to the discovery of the importance of CD81 for hepatocyte invasion by P. falciparum, the extracellular loop had been shown to be important for the binding of hepatitis C virus E2 and hepatocyte infection [77]. Initial experiments found that CD81-deficient mice are resistant to P. yoelii exoerythrocytic form

(EEF) formation [74]. In the same report, blocking of CD81 was shown to inhibit P. falciparum EEF formation in a dose-dependent manner. Subsequent work utilizing ectopically-expressed CD81 in the CD81 negative cell line HepG2 established that in the case of P. yoelii EEF formation, CD81 was sufficient to drive infection [75]. However, expression of CD81 was necessary but not sufficient to allow for P. falciparum EEF formation. These data suggest that additional receptors and/or mechanisms exist that are necessary for P. falciparum sporozoites to initiate productive infection in hepatocytes.

Interestingly, the type I scavenger receptor (SR-BI) has been shown to be important for both invasion and development of Plasmodium sporozoites [78] in vitro but was found to be unnecessary in vivo [79].

After sporozoites productively invade a host hepatocyte and establish a parasitophorous vacuole (PV), they take up residence adjacent to the host nucleus [80] and recruit host organelles to the PV. The developmental time required for Plasmodium EEFs to reach maturity varies widely between species. For instance, the rodent parasites P. berghei and P. yoelii complete full development in less than three days post-infection

[81,82]. In contrast, P. falciparum requires a minimum of six to seven days to reach maturity [83] and at maturity the parasites are considerably larger [84,85]. From the mature

EEF of the parasite, bags of hepatic merozoites termed “merosomes” bud into the liver sinusoid and avoid phagocytosis by inhibiting the presentation of phosphatidylserine

12 residues at the cell surface [6] (Figure 1.4). In the case of P. yoelii, the merosomes reach the lung where individual merozoites are released and begin the erythrocytic cycle [86].

Figure 1.4 Cartoon representation of merosomes budding and release into the blood stream. Merosomes bud to gain access to red blood cells to initiate an erythrocytic infection.

Targeting exoerythrocytic forms: whole organism vaccines

Vaccines protecting against infectious diseases have been one of the most potent and cost effective public health measures. Smallpox is the only human infectious disease to be successfully eradicated, and is also the first disease to be safely vaccinated against

[87]. The notion that smallpox-infected or cowpox-exposed individuals remained resistant to smallpox infection was well established before the 1750s [88] but it was not until 1796 that Edward Jenner performed the first definitive vaccination. Arguably more importantly, he also communicated his findings to the scientific community at large [89]. Eradication efforts begun in the 1960s by the World Health Organization (WHO) were aided by the absence of an animal reservoir, a stable vaccine, and a protective immune response that lasted for five years after a single immunization [90]. The characteristics of both the pathogen and vaccine were optimal in the case of smallpox eradication. When D.A.

Henderson, the head of the successful smallpox eradication campaign, was asked about prospects for additional human pathogen eradication campaigns he said:

“No. Not at this time given presently available relevant technologies, our

understanding of the epidemiology of the infectious diseases, and pilot programmes

of national disease elimination programmes.” [91]

This sentiment highlights the need for continued research and development of novel vaccine strategies and public health solutions for diseases such as malaria.

The generation of a vaccine capable of blocking either Plasmodium sporozoite invasion of hepatocytes or development of EEFs has been a focus of research for decades.

A successful formulation targeting the liver stage has the potential to protect the individual from symptomatic blood stage infection while simultaneously eliminating transmission.

The first evidence that a sterilizing immune response was capable of halting the liver stage was generated with whole parasite vaccination, using first irradiated P. berghei sporozoites

[92] and subsequently using the bites of infected, irradiated whole mosquitoes [93]. This concept of attenuated whole-organism vaccination was shown to be effective in protecting humans using hundreds of irradiated, infected mosquitoes [94]. This study established the possibility of generating a sterilizing immune response and demonstrated that immune sera reacted to sporozoites (circumsporozoite precipitation test) (Figure 1.5). This anti- circumsporozoite reaction was correlated with protection from infection, suggesting an important role for antibodies in whole-organism vaccination with sporozoites [95].

Figure 1.5 Visualization of the CSP reaction. Sporozoites treated with normal serum (top) and immune serum (bottom). Adapted from [96] Fig 2.

The stage specificity of this response was established by demonstrating that individuals protected from the bite of non-irradiated, infected mosquitoes were still completely susceptible when directly transfused with blood stage parasites [94]. Strain transcendent immunity was evident following vaccination with attenuated P. falciparum sporozoites (Burma) and subsequent protection following challenge with strains from

Panama, Malaya and the Philippines [97]. However, this same cross-strain immunity did not protect against the Chesson strain of P. vivax, suggesting a species-specific immunity

[97]. These seminal studies established the possibility of sterilizing immunity, even when natural exposure is unable to generate this response [98]. The protective immunity in these studies persisted for up to six months. Ideally, protective immunity would persist through entire transmission and dry seasons and considerable work has been done to extend the immune response following whole-organism vaccinations [99]. Since the administration of

15 heat-killed sporozoites did not elicit a protective response, further efforts have been made using metabolically active attenuated parasites [100].

A major consideration since the demonstration of sterilizing protection using attenuated sporozoites for vaccination has been the production hurdles and economic feasibility of large scale formulations [101]. However, various whole-organism vaccines have been successfully developed for veterinary purposes [102]. Development of these veterinary vaccines have benefited from the ability to test formulations directly in their cognate host and in some cases due to a need for only short lived immunity [102].

An alternative to irradiation, attenuation through elimination of liver stage specific genes resulting in immature parasite arrest in the liver, is also being developed [103]. This method has several theoretical advantages compared to irradiation which include exposure of the host to late stage parasite antigens and generation of a homogenously attenuated parasite. Parasites that progress further into the liver stage before arresting have been shown to produce a greater immune response [104]. This observation has directed the discovery of parasite genes important for late stage development in the liver [105].

However, the generation of an immune response against late liver stage antigens is not boosted by additional attenuated sporozoite vaccinations. The inability to further boost immunity against these antigens is due to a dominant immune response against early stage parasites inhibiting EEF development or invasion (as measured by parasite 18S rRNA), including a strong response to CSP [106].

It is critical to note for whole-organism vaccine development that genetic attenuation must be complete because breakthrough parasites have the potential to cause disease [107]. Candidate parasite genes are typically identified in rodent species and often

16 do not translate into completely attenuated human parasites resulting in the necessity of multiple gene deletions and substantial safety testing prior to human vaccine administration

[105,108].

Mechanisms of immune protection

The protective mechanisms of the immune response to irradiated sporozoites have been studied to identify correlates of protection. This information has been used to inform subunit vaccine design with the goal of generating a sterilizing immune response comparable to the “gold standard” of irradiated sporozoites. Initial observations on the ability of sera from mice immunized with irradiated sporozoites to specifically react with the surface of sporozoites [109] and the observation of the “CSP reaction” correlation with protection in humans [95] suggested that antibodies are important for a sterilizing immune response. Additional studies found that passive transfer of antibodies could protect against

P. berghei infection and that monoclonal antibodies against a 44,000 (Pb44) molecular weight protein could abolish infectivity of P. berghei sporozoites in vitro [110,111]. The protein identified in these studies (Pb44), now referred to as the circumsporozoite protein

(CSP), is the major surface protein of the sporozoite [112,113] and is shed during sporozoite motility. Inhibition of P. falciparum sporozoite invasion into hepatocytes by anti-CSP antibodies was confirmed in vitro [114] suggesting that the observations originally made with rodent Plasmodium species may be applicable to human trophic parasites. However, antibody responses to CSP occur in areas of natural high transmission and do not correlate with protection from development of parasitemia, both symptomatic and asymptomatic [115]. This observation supports the notion that the immune response generated in endemic areas to the pre-erythrocytic stages does not protect against clinical

17 infection (blood stage parasitemia) [98]. Several scenarios are possible to explain the presence of serum anti-CSP antibodies without measurable protection: 1) the antibodies generated are of a different specificity or avidity (possibly enhancing or neutral for infection) than those generated with irradiated sporozoites, or 2) the antibodies are maintained in the serum at levels below what is required for protection.

Even though the ability of anti-sporozoite antibodies to react and inhibit development of EEFs is well characterized, it was first shown in 1977 that mice deficient in B-cell generation could still be protected by irradiated P. berghei vaccination without detectable anti-CSP serum reactions [116]. This observation became the start of defining the cell-mediated immune response to pre-erythrocytic stages of Plasmodium. It was then shown, in a similar manner to transfer of protective serum, that cell-mediated protection can also be adoptively transferred [117]. A recombinant Pb CSP vaccine that generated antibody titers higher than those achieved by irradiated sporozoite vaccination, had lower efficacy than whole-organism vaccination [118]. By comparison, the irradiated sporozoite vaccination induced a stronger cell-mediated immune response than the recombinant vaccine. This work highlighted the ability of irradiated sporozoite vaccination to generate both humoral and cell-mediated immune responses, but also demonstrates the importance of the cellular immune response with regards to sterilizing protection. Selective neutralization or depletion of components of the cellular immune response aimed to determine the mechanism of this protection. Studies found that in vivo depletion of CD8+

T cells but not CD4+ T cells ablated the protection from irradiated sporozoites [119,120].

Combinations of T cells and sera in adoptive transfer acted synergistically, supporting the

18 notion that irradiated sporozoites induce a complex immune response with a strong cellular response important for sterilizing immunity [119].

Experiments aimed at defining the specificity of the cytotoxic immune response determined that an epitope from P. berghei CSP (249-260) could provide protection through passive transfer of T cells in the context of MHC class I molecule H-2Kd [121].

Yet, generation of an anti-CSP cytotoxic immune response that provides sterilizing protection requires a higher number of memory CD8+ T cells than other pathogens [122].

The genetic background in murine models is important when determining the details of protection [123]. For instance, to generate a sterilizing immune response to P. yoelii,

BALB/c mice require CD8+ T cells, IFNγ, IL-12 and iNOS whereas C57BL/6 additionally required CD4+ T cells [124]. Host genetics both inside and outside the MHC class I locus determine the ability to elicit sterilizing immunity from irradiated sporozoite vaccination

[125].

As noted above, CSP is a major target of the immune response to pre-erythrocytic stages of Plasmodium and has subsequently been of considerable focus for human vaccine design (Figure 1.6).

Figure 1.6 Schematic of CSP and the RTS,S vaccine. (A) Representation of the CSP molecule with an N-terminal signal sequence, conserved regions RI and TSR along with a central repeat region. (B) Portion of P. falciparum CSP fused to the Hepatitis B surface antigen with immunogenic potions indicated. Adapted from [126] Fig 2.

Transgenic mice that express P. yoelii CSP in the germ line to force tolerance demonstrated

that CSP is the immunodominant antigen in irradiated sporozoite vaccination [127].

However, tolerized mice can be protected when hyper-immunized, showing that even

though an anti-CSP response is sufficient to drive sterilizing immunity, it is not necessarily

required, or that repeated immunization overcame CSP tolerance [128]. This mouse model

is important in establishing the presence of non-CSP CD8+ dependent antigens and

suggests that antigens processed and presented by Kupffer cells or traversed cells could

control infection through bystander activation [127]. This mechanism would be in contrast

to a direct (or proximal) recognition and killing of P. berghei EEFs by CD8+ T cells

[129,130], a possible explanation for the discrepancies in species-specific protection of

CSP-tolerized mice.

The presence of persisting parasites in the liver post-irradiated sporozoite vaccination has been proposed to be a potential mechanism driving sterilizing protection.

This process is absent in subunit vaccination and may help explain differences observed in clinical trials [131,132]. This phenomenon was initially identified in rats immunized with

P. berghei sporozoites that had detectable parasite DNA up to six months post-vaccination

[133]. Administration of primaquine was able to clear persisting parasites and subsequently abrogated the time of protection post-immunization, suggesting that persisting parasites maintain or enhance the immune response [133]. Later work provided evidence that persisting parasites are capable of being presented to the immune system and enhance a prolonged CD8+ T cell response important for protection [134]. In addition, primaquine treatment of mice immunized with genetically attenuated parasites (Pb uis3(-)) lost CD8+ mediated sterilizing immunity, implying that irradiated sporozoite vaccination and genetically attenuated parasite vaccination both rely on parasite persistence [135].

The choice of inbred mice as a model for Plasmodium infection has been, and will continue to be, paramount in understanding basic parasite biology and the critical components of the host immune response. However, the natural reservoir for P. berghei is the tree rat Grammomys surdaster, which requires a lower infectious dose of sporozoites for progression to a blood stage infection and which is considerably more difficult to protect with irradiated sporozoite vaccination than mice [136]. This is expected when considered in the context of extensive host-parasite co-evolution and mirrors the difficulty of whole-organism vaccination in humans [137]. These differences could be attributable to

21 factors such as parasites driving hepatocyte resistance to apoptosis [138] or antigenic mimicry resulting in T cell tolerance and immune evasion [139]. Consequently, mice represent an invaluable resource to study Plasmodium, but their use with a non-native parasite may not always provide conclusions relevant to all Plasmodium species or to relevant hosts. In this sense, the difficulty in protecting cognate parasite-host combinations would speak to the difficulty in generating a broadly sterilizing vaccine.

Yet, the liver stage of Plasmodium has remained an attractive target for vaccination because parasite biomass is not equal throughout the parasite lifecycle and has a considerable bottleneck during establishment of a liver stage infection. These points in the lifecycle have been proposed to be ideal targets of intervention [140] when considering strategies of elimination and eradication (Figure 1.7). However, the converse argument would suggest that any intervention against these stages requires some degree of perfection since one surviving parasite can continue transmission. At a population scale, even if sterilizing protection is only achieved in a subset of individuals it has the potential to alter transmission.

Figure 1.7 Estimated relative population sizes (biomass) between stages of the P. falciparum lifecycle. (A) Low numbers of Oocysts in the mosquito and (B) injection of few sporozoites represent bottlenecks in the lifecycle. Adapted from [140] Fig 3.

Biology of CSP

Since the identification of CSP as the major target of the humoral response to irradiated sporozoite vaccination in 1980 [110,111], considerable work has aimed to define its structure, conservation, and importance to parasite biology. Initially the amount of cDNA obtainable was a limiting factor in characterizing the sequence of CSP. In 1983, using P. knowlesi sporozoites, a cDNA library was constructed containing CSP [141], and the first CSP sequence was reported [142]. This report identified the central tandem repeat region (12 amino acids, 12 times) and demonstrated competitive binding of synthetic repeat peptides with native CSP [142] (Figure 1.8). This central repeat region has since been

23 shown to be characteristic of CSP from all Plasmodium species, but varies in the composition and total number of repeats [143].

Figure 1.8 Schematic of the structure of CSP from P. knowlesi based on nucleotide sequence. Areas of charge and tandem repeats were discerned from the cDNA sequence of P. knowlesi CSP. Adapted from [96] Fig 3.

A protocol to digest intragenic regions of P. falciparum genomic DNA facilitated the generation of libraries to screen for P. falciparum CSP using asexual cultures not expressing CSP mRNA [144]. In 1984 [145], the CSP sequence (412aa) was obtained from

P. falciparum and several architectural observations were made. This original sequence had 41 tandem repeats, Asn-Ala-Asn-Pro (37 times) and Asn-Val-Asp-Pro (4 times) composing the central repeats region. At the N-terminus the first 16 amino acids were predicted to be a parasite signal sequence and the remaining amino acids up to the repeat region carried an overall positive charge. The C-terminal region to the repeats was predicted to be hydrophobic, and it was postulated that it may constitute an anchor region

[146]. Since the P. knowlesi CSP sequence had previously been published, several observations about conserved regions were made. Two regions of homology were noted, region I directly N-terminal to the repeats and region II C-terminal to the repeats (Figure

1.9). The high degree of conservation of region II was suggested to be due to a role in liver invasion by binding to a putative receptor; this region also bears resemblance to the

24 adhesive protein thrombospondin and is identified as a type I thrombospondin repeat (TSR)

[139]. Experimentally, the TSR has been shown to be important for HSPG binding [147] and mediates initial parasite attachment in vivo [66]. Region I has been demonstrated to be a cleavage site [148] for a parasite protease that removes the N-terminus [149]. This cleavage process is triggered by high levels of HSPGs [64] and unmasks the C-terminal

TSR [148]. The N-terminal cleavage event is not required for parasite traversal but is important for productive invasion of hepatocytes [148].

Figure 1.9 Regions of sequence identity in CSP between P. knowlesi and P. falciparum sequences. Region I and Region II (TSR) were identified as being conserved between P. knowlesi and P. falciparum. This observation implied a potential functional role of these regions. Adapted from [145] Fig 6.

Paradoxically the repeat regions of CSP appear immunodominant (Pf (NANP3)) in the humoral immune response [150] but the repeat sequences are conserved within each species of Plasmodium [143]. Conservation of the repeats could be due to a functional constraint on parasite biology, but may also suggest that immune pressure is not exerted on this portion of CSP. Antibodies are protective in vitro [150] and in vivo [110,111] but the inability of anti-CSP antibodies to protect in settings of natural transmission [98,115]

25 suggests that naturally acquired immunity is insufficient to drive selective pressure of the repeats.

In contrast, the T cell responses from persons living in malaria-endemic areas were greater against known polymorphic regions of CSP [151,152], implying that certain T cell epitopes may be under immune pressure. However, these polymorphisms in the C terminus appear stable over time in a given region and do not vary between age groups or correlate with hazard of infection [23].

The role of CSP in attachment and invasion in the liver [62,63,65,66,147–149] has been well characterized. In addition, several reports have suggested that CSP is involved in modulation of the host immune response through inhibition of host cell protein synthesis

[153], modulation of Kupffer cell activation [154,155], or inhibition of NFκB nuclear import [156].

Techniques to study the Plasmodium liver stage

Since direct access to Plasmodium-infected patient material (especially hepatocytes) is difficult and often cost prohibitive, the development of both in vitro and in vivo models of liver stage infection are paramount in basic research. Study of the erythrocytic stage of P. falciparum has benefited from two achievements. First, the demonstration that asexual cultures could be grown ex vivo using human red blood cells, serum, and a low oxygen environment [157] allowed for generation of considerable parasite material without the need for an infected primate host, such as Aotus. Second, a technique utilizing electroporation was shown to be capable of introducing foreign DNA into the parasite [158]. These methods provided the ability to grow continuous cultures of asexual parasites and also to manipulate their expression.

Study of the pre-erythrocytic stages of Plasmodium have depended heavily on the establishment of parasite lines capable of infecting laboratory rodents [57] and the development of in vitro methods for cultivating EEFs [159]. Rodent parasites can also be transfected in a similar manner to P. falciparum [160,161]. Using this technique fluorescent parasites have been generated to constitutively express GFP [162]. These lines allow for live parasite identification and the utilization of FACS to purify and profile EEFs [163,164] and their host cells. Plasmodium yoelii parasites expressing luciferase have provided a noninvasive measure of parasite liver development in vivo [165].

The use of these rodent parasites in vitro has benefited from a broad host cell line tropism that allows for complete EEF development in vitro [159]. P. falciparum EEFs can be obtained in primary human hepatocytes [55] but have a limited ability to infect many cells lines that are susceptible to rodent parasites [159]. Primary hepatocytes have long been the only method in which to routinely generate P. falciparum EEFs [55,166] and techniques have been developed to optimize this procedure [167]. Identification of P. falciparum EEFs in vitro has relied on simple staining techniques. Considerable effort has been made to identify cell lines that could replace the need for primary human hepatocytes for in vitro infections and thereby reduce costs and to better standardize experiments across labs. To date only two cell lines have been reported to allow for complete development of

P. falciparum EEFs in vitro: the cell line HHS-102 [168] reported extremely low levels of infection and the cell line HC-04 [169] reported more substantial levels of infection of both

P. vivax and P. falciparum. Recent development of a P. falciparum strain constitutively expressing GFP [170] and the availability of susceptible cell lines suggest that techniques

27 pioneered using rodent parasite lines may also be capable of routinely isolating and identifying P. falciparum EEFs in vitro.

An alternative approach to the growth of P. falciparum EEFs in vitro has been the development of mice with a liver partially reconstituted with human hepatocytes [85].

Generation of these liver chimeric mice is prohibitively expensive for routine use and the technique used to reconstitute the liver completely ablates the immune system, making study of an in vivo immune response impossible [171]. Similar to rodent lines expressing luciferase, a P. falciparum line expressing a GFP-luciferase transgene has been developed

[172] but only luciferase is detectable in hepatocytes. Therefore, the development of new techniques that can be applied to both in vitro and in vivo models of P. falciparum EEF development will aid in answering basic questions about host-pathogen interaction and will potentially serve as methods to validate interventional approaches such as vaccine development.

Concluding remarks

The transmission of Plasmodium parasites has continued throughout written human history. Only relatively recently has a microbial cause of malaria been determined. Yet, considerable strides have been made to parse the determinants of Plasmodium persistence and the correlates of the host response. This understanding takes place in the context of millennia of co-evolution, resulting in an evasive parasite that has left a distinct imprint on the human genome. The scientific community continues to investigate unknown facets of the host-pathogen interaction and to design potentially protective measures. An expanding understanding of parasite biology will continue to rely on the available techniques and the relevance of our models.

Chapter 2 : Processing and presentation of two forms of CSP

on MHC class I

Introduction

Presentation of peptides on MHC class I

The importance of the thymus in the immune response was initially characterized by thymectomy. These experiments showed profound, systemic reductions in lymphocytes and impaired cell-mediated immunity [173]. The difference in this phenotype compared to removal of other lymphoid organs designated the thymus as a “central” lymphoid organ.

Subsequent work focused on defining the cell populations present in the thymus and their roles in the immune response. Using allogeneic stimulation, it was shown that a subset of the thymus-derived cells had specific lytic activity [174] and that other cells could provide

“helper” activity to the immune response [175]. The commitment step of T cells to follow these different functional subsets was shown to occur before stimulation and could be delineated by surface markers from unlinked genetic loci [176]. These experiments using allogeneic stimulation determined the ability of T cells to specifically lyse or to provide help to non-thymus immune cells during the immune response. However, classification of effector function subsets did not determine the stimuli responsible for an immune response in a syngeneic system.

In the context of lymphocytic choriomeningitis (LCM) infection it was found that adoptively transferred T cells could home to lymphoid tissues in both syngeneic or allogeneic hosts, but could only continue to proliferate in a syngeneic host (specifically the

H-2 gene complex) [177,178]. Yet, the restriction of lysis to genetically matched targets did not explain the mechanism of recognition of viral antigen and could not be explained by activation from free virus. Rather, the viral infection resulted in what was originally

30 termed “altered self” and suggested the importance of viral “modifications” to a component of the H-2 complex [178]. These findings set the original framework wherein specific lysis by T cells requires recognition of pathogens (peptides) in the context of self (MHC class

I). Target cells are recognized during the effector phase of a CD8+ T cell immune response through TCR recognition of peptides presented in the context of MHC class I. For antigen presentation to occur, proteins are degraded and the resulting peptides are loaded onto

MHC class I, which is trafficked to the cell surface [179] (Figure 2.1).

Figure 2.1 Schematic representation basic antigen presentation on MHC class I.

Degradation of antigens and translocation of peptides for presentation of peptides in the context of MHC class I at the cell surface. Adapted from [179] Fig. 1.

CD8+ T cell recognition of peptide complexed with MHC class I at the cell surface triggers the activation of CD8+ T cells with cytolytic potential [179]. Direct cytotoxic T lymphocyte (CTL)-mediated lysis can be achieved through two main mechanisms. First, mice deficient in the pore-forming protein perforin were shown to be deficient in lysis of both virally infected and allogeneic targets [180]. Alternatively, death receptor mediated cytotoxicity, including members of the tumor necrosis factor (TNF) family such as

Fas/FasL [181,182] and TRAIL [183,184] can induce apoptosis of a target cell through a perforin-independent process. In addition to direct cytotoxicity of target cells, CTLs have the potential to control infection through non-cytolytic mechanisms [185] that include activation-dependent cytokine and chemokine production [186].

The MHC molecule is a cell surface glycoprotein with two structural motifs. The distal portion to the cell membrane is composed of eight antiparallel β-strands with two α- helix domains above [187,188]. The pocket between helices forms a groove that is filled by 8-10 amino acid peptides (Figure 2.2).

Figure 2.2 Schematic of the structure of HLA-A2. Top down view of the binding pocket for peptides in the groove of MHC class I. β-strands form the bottom of the groove with two α-helix domains shown. Adapted from [187] Fig. 2.

The binding of peptides in this groove provides the necessary stability for the MHC class

I heterodimer to be trafficked from the endoplasmic reticulum (ER) to the cell surface and prevents the translocation of empty MHC molecules to the cell surface [189,190]. Unlike the presentation of peptide on MHC class II, the presentation of peptides on MHC class I is generally unaltered by the inhibition of endosomes or lysosomes [191]. This finding implicated cytoplasmic or nuclear generation of peptides for subsequent presentation.

Using inhibitors of the proteasome it was shown that a majority of peptides loaded onto

MHC class I molecules were generated through proteasomal-dependent degradation [192] and ubiquitination of target proteins [193]. However, alternative pathways for the generation of peptides for presentation on MHC class I have been suggested, including a role for autophagy in antigen processing [194]. These peptides have been shown to be transported from the cytoplasm to the ER for loading onto MHC class I molecules by the transporter of antigen presentation (TAP) protein [195]. Each MHC class I allele has the potential to bind a myriad of peptides, but the exact repertoire is dependent on the individual characteristics of the binding pocket that accommodates peptide loading [195].

Consequently, on a population scale, the MHC locus represents the most polymorphic portion of the human genome and also has some of the highest recombination potential [196]. In fact, HLA-B is the most polymorphic gene identified in the human genome with a calculated 86 single nucleotide polymorphisms (SNPs) per kilobase and a recorded 1,605 alleles with 1,268 unique peptide sequences [197,198]. This diversity is attributed to the “Red Queen” hypothesis in which organisms must continually evolve to avoid extinction [199]. However, many pathogens, most notably viruses, have evolved capabilities to impair antigen presentation on MHC class I, presumably to prevent host cell lysis by CTLs. These methods include inhibition of antigen degradation, transportation, and MHC class I complex trafficking [200,201].

Targeting Plasmodium: role of the MHC class I antigen processing and presentation pathway

Several facets of vaccine design have focused on blocking sporozoite entry into the liver and/or eliminating EEFs before they reach maturity to prevent progression to blood

34 stage. The circumsporozoite protein (CSP) is the major protein on the surface of

Plasmodium sporozoites and contains both B and T cell epitopes [132]. Rodent models of malaria, particularly in the BALB/c host, have demonstrated the importance of a CD8+ T cell mediated, CSP-specific response in protective immunity [121,202]. In rodent models

CSP is the major antigen involved in protection from whole organism-based vaccines and is a leading candidate for generation of a protective vaccine for use in humans [127,203].

The only stage of the Plasmodium life cycle that involves the infection of MHC class I expressing cells is the liver stage. In mouse models of vaccination with irradiated sporozoites, the gold standard of inducing a sterilizing immune response to the Plasmodium liver stage, is dependent on the generation of CD8+ T cells [119,120]. CSP is the immunodominant antigen in this model [127]. CD8+ T cell clones specific to C-terminal peptides of CSP are sufficient to provide liver stage specific immunity and protection in both P. berghei [121] and P. yoelii [204]. These protective CD8+ T cells are initially primed in the draining lymph node [205] and are maintained as a resident memory population in the liver [206]. In mouse models utilizing irradiated sporozoite immunization, sterilizing protection that is dependent on CD8+ T cells was not mediated by perforin/granzyme or

Fas mechanisms of killing [124]. Rather, in these mouse models IFN-γ produced by activated CD8+ T cells was a correlate of protection, suggesting the potential for control of

EEFs without direct recognition. However, in a P. yoelii model, IFN-γ deficient CS specific

CD8+ T cells were able to eliminate liver stage parasites similarly to wild-type [207]. This study was in contrast to data obtained using IFN-γ deficient mice and IFN-γ neutralization experiments, suggesting a potential non-CD8+ source of IFN-γ or potential systemic defects in IFN-γ deficient mice.

During natural transmission, CD8+ T cells are primed against liver stage antigens

[152,208] and antibodies are generated that target the sporozoite [209]. Yet, a sterilizing effector immune response is not achieved in naturally exposed individuals but rather they gain clinical protection from disease caused by the erythrocytic stage [98]. Possible reasons for the absence of a naturally generated protective liver stage immune response include parasite allelic diversity [23,210], parasite resistance to immune effector mechanisms [138] or failure to maintain a protective memory T cell population [211].

However, the HLA-B53 allele has been implicated in protection from severe malaria by recognition of liver stage antigen-1 (LSA-1) [212–214]. This HLA-B allele is enriched in West African populations, possibly due to selection for a particularly protective immune response. Immune protection of naturally exposed individuals through a specific

MHC class I allele directed against a liver stage antigen [212] may seem paradoxical since sterilizing liver stage immunity fails to develop [98]. Measurement of protection of HLA types used severe malaria as a metric of protection [213] as assessed by passive surveillance and not protection from development of blood stage parasitemia. Yet, a lack of natural sterilizing immunity was found using a prospective study [98] that did not rely on clinical metrics and measured time to blood stage infection in each individual.

Importantly, this study did not utilize a drug treatment baseline to avoid recrudescent infections [215] nor skewing of the immune response [216,217]. Therefore, a natural immune response targeting the liver stage may not provide sterilizing protection, but could, through an undefined mechanism, influence the severity of a resulting blood stage infection and influence clinical disease. This could help explain the protective nature of a MHC class

I allele in the absence of an observed sterilizing immunity.

The inability of immunity to protect against Plasmodium infection in naturally exposed individuals [98] even in the presence of CD8+ T cells specific to CSP [152] suggests that the parasite may have evolved to inhibit presentation of CSP on MHC class

I. In fact, CSP has been shown to be capable of altering host cell protein synthesis [153] and promoting development through interference of NFκB signaling [156]. The generation of a protective CD8+ T cell mediated response to CSP requires a high level of memory

CD8+ T cells [122] in animal models. This requirement may reflect a parasite driven resistance to apoptosis [138] or an ability of the parasite to limit the amount and/or scope of peptides capable of being presented on MHC class I. Alternatively, the observation that immunodominant T cell epitopes of CSP are also regions of a relatively higher number of polymorphisms [151] suggest that the parasites may be capable of escaping natural T cell immune pressure. In contrast to blood stage immunity [210], clear, CSP allele-specific protection has not been established in humans [23]. This may be due to the differences in

CSP derived peptides presented by various HLA types [152], extensive HLA diversity in

Africa [218] or inability of naturally exposed individuals to be protected by low percentage anti-CSP CD8+ T cell response. In this case, the interaction between parasite antigen alleles and individual HLA would ultimately determine the ability of CSP to be processed and presented in a single individual.

Design of a vaccine capable of completely halting the liver stage must overcome these hurdles inherent in the natural immune response. Various adjuvant formulations are capable of enhancing the magnitude of the immune response but do not alter the breadth of the immune response that may be importance in a setting of heterologous challenge.

Additionally, identify mechanisms the parasite may employ to misdirect or avoid a

37 protective immune response is essential in informing an intelligent vaccine formulation.

The RTS,S vaccine is designed to target the sporozoite and liver stage of P. falciparum and is currently seeking licensure. The formulation is composed of a portion of

CSP fused to the hepatitis B surface antigen (HBsAg) in combination with an adjuvant formulation (Figure 1.6). Active detection has observed a reduction in time to measurable parasitemia [131,219] and clinical data show a reduction in severe disease [131,219–221].

However, protection is not sterilizing and differs between age groups. The roles of antibodies and CD4+ T cell in protection have been modeled [222] and suggest that infections may be the result of individual sporozoites productively infecting the liver.

Antibody titers against CSP correlate with protection in non-endemic human volunteers

[223,224]. In these studies a level of 20µg/ml anti-CSP antibody was considered a general threshold necessary for protection.

Re-formulations and adjuvant designs are aimed at boosting the immune response to RTS,S with the goal of inducing sterilizing immunity. Additionally, the current CSP-

HBsAg fusion protein does not include the N-terminus of CSP and it is unclear if inclusion of a full length CSP could influence a protective immune response or could enhance the limited cytolytic response observed in prior studies [225,226].

Two forms of CSP as targets of the CD8+ T cell immune response

Prior to invasion, a parasite protease cleaves the N-terminus of CSP at the conserved region I, adjacent to the repeat region [149]. Consequently, CSP may exist in two forms in the host (Figure 2.3). The majority of work involving cleavage of CSP has

38 focused on the necessity of cleavage during invasion and subsequent importance to parasite biology. It is unclear what forms of CSP are present in host hepatocytes. Nothing is known about the individual contributions of these forms to antigen presentation and target cytotoxicity has not been addressed. Understanding both the nature of CSP found in host cells and the ability of these forms to be processed and presented is important for understanding any parasite mechanisms to avoid the immune response.

Figure 2.3 Schematic representation of CSP during infection. Proposed conformational changes to CSP that occur from the mosquito to the eventual infection of hepatocytes (N-terminus in green). Adapted from [148] Fig 8.

Here we describe differences in T cell activation, target cytotoxicity, and ubiquitination between two forms of CSP using an ectopic expression system. We identify which form of CSP predominates in cells traversed and infected by the Plasmodium parasite. These data suggest that inclusion of N-terminal lysine residues of CSP are important in antigen presentation and their removal may represent a parasite mechanism to lessen antigen processing and presentation.

Results

Ectopic expression of CSP in hepatocytes

We constructed mammalian expression plasmids capable of expressing either the full-length circumsporozoite protein (CSP-Full) or the cleaved N-terminal form (CSP-

Short). Due to the difficulty in obtaining and identifying lymphocytes specific to CSP from naturally exposed individuals we designed a chimeric CSP sequence that contained well characterized HLA-A2 epitopes. To evaluate presentation of CSP via MHC class I and the resulting CD8+ T cell activation we inserted known MHC class I viral epitopes from either influenza A matrix protein 1 or Epstein-Barr virus protein BMLF1 in the C-terminus

(Figure 2.4A). The amino acid substitutions used to alter the putative epitopes in CSP were not predicted to be appreciably different than the native epitope [227]. A HLA-A2 restricted response to the native epitope, GLIMVLSFL is primed and recall responses can be identified [152].

We utilized a model hepatocyte cell line, HC-04 that is reported to have the ability to support P. falciparum EEF development in vitro [169]. Using our CSP expression constructs we determined that CSP can be expressed in HC-04 following transfection, and that CSP-Full and CSP-Short can be differentiated by their electrophoretic mobility (Figure

2.4B). Both CSP-Full and CSP-Short contain the central repeat region (NANP) that is recognized by the monoclonal antibody 2A10 [150]. To determine if the N-terminus influenced the cellular localization of CSP in hepatocytes we transfected cells with either

CSP-Full or CSP-Short and stained the central repeat region (IFA). We observed that both

CSP-Full and CSP-Short are predominantly cytoplasmic and have similar localization

40 patterns in hepatocytes, when expressed ectopically (Figure 2.4C). In contrast to previous reports we do not observe localization of CSP to the host nucleus [156]. However, in vivo

CSP can be delivered or expressed in hepatocytes in a variety of ways, resulting in differential localizations over time including a diffuse cytoplasmic pattern (data not shown). Therefore, the localization of ectopically expressed CSP is representative of at least a portion of CSP found in vivo and has the potential to be classically processed for presentation on MHC class I.

Identification of CSP in traversed and infected hepatocytes during in vitro infection

Plasmodium sporozoites have been observed to interact with hepatocytes prior to productive invasion and parasitophorous vacuole formation [72]. This process of traversal, which also occurs in the skin, involves breaching of the host cell plasma membrane, intracellular movement, and egress [228]. Since CSP is shed during parasite motility [229] we aimed to identify which form of CSP is present in traversed cells and has the potential to activate CD8+ T cells. We sorted traversed hepatocytes from P. falciparum-infected cultures and identified the presence of both forms of CSP (Figure 2.4D). We primarily detect the presence of the cleaved form of CSP in traversed cells as compared to our ectopically expressed variants of CSP.

To identify the form of CSP present in infected cells, we sorted cultures infected with a GFP-expressing P. falciparum parasite 72 hours postinfection. Infected hepatocytes were found to contain CSP-Short (Figure 2.4E). Consequently, the short form of CSP is present and predominates in both traversed and infected cells. Even though CSP transcript

41 has been identified in EEFs [230], it is unclear if CSP is translated in the EEF and cleaved or the presence of cleaved CSP in EEFs originated in the sporozoite. Next, we asked if the two forms of CSP have a differential ability to be processed and presented on MHC class

I and presumably have different potential contributions to the effector phase of a CD8+ T cell immune response.

Figure 2.4 Ectopic and natural expression of the two forms of P. falciparum CSP. (A) Schematic representation of the mammalian expression construct pVITRO2-neo-mcs encoding CSP and a marker of transfection, the murine CD8 alpha chain. C-terminal chimeric sequences are indicated. (B) Western blot detection of CSP-Full (F) and CSP-

Short (S) at 12, 24 and 48 hours post-transfection. (C) Immunofluorescence detection of CSP in HC-04 cells ectopically expressing either CSP-Full or CSP-Short. (D) Western blot detection of CSP in HC-04 cells traversed with P. falciparum sporozoites compared to the electrophoretic mobility of ectopically expressed CSP-Full and CSP-Short. Traversed (dextran+) and non-traversed (dextran-) cells were separated by flow cytometry based cell sorting. (E) Western blot detection of CSP in HC-04 cells infected with P. falciparum 3D7- HTGFP compared to the electrophoretic mobility of ectopically expressed CSP-Full and CSP-Short. Infected (GFP+) HC-04 were isolated 72 hours postinfection by flow cytometry based cell sorting.

Two forms of CSP differentially activate CD8+ T cells

The conservative substitution of the native C-terminal sequence of CSP

(GLIMVLSFL) with two well characterized HLA-A2 restricted viral peptides allows the use of CD8+ T cells that are readily identifiable with existing reagents. The wild-type CSP epitopes and viral epitopes have similar predicted binding strengths to HLA-A2 [227]. To generate CD8+ T cell effectors and establish lines of monospecific, polyclonal CD8+ T cells we first identified HLA-A2 positive donor PBMCs. These donor lymphocytes positive for

HLA-A2 were stained with pentamer specific to either GLCTLVAML or GILGFVFTL in the context of HLA-A2 (Figure 2.5). These cultures were co-cultured with irradiated target hepatocytes pulsed with synthetic peptide (GLCTLVAML or GILGFVFTL) and expanded on allogenic feeders. After expansion, cultures were separated based on CD8 positivity and pentamer positivity by fluorescence-activated cell sorting (FACS) (Figure 2.5).

Figure 2.5 Identification, isolation and expansion of HLA-A2 restricted viral peptide specific CD8+ T cells. Lymphocytes of HLA A2-positive blood donors were screened for the presence of CD8+ HLA A2-restricted CTLs specific to GILGFVFTL (peptide sequence from influenza A,

44 matrix protein 1) and GLCTLVAML (peptide sequence from Epstein-Barr virus, BMLF1) peptide epitopes. Three lymphocyte cultures contained either GLC and/or GIL-specific CD8+ CTLs identified by pentamer staining (panel “screening”). Lymphocyte cultures were stimulated with relevant synthetic peptide and expanded on allogenic feeders (panel “expansion”). Peptide-specific CD8+ CTLs were sorted using specific pentamers and further expanded on allogenic feeders for expansion (panel “post-sorting and expansion”).

Specific activation of sorted and expanded CD8+ T cell cultures was demonstrated by using targets pulsed with either GLCTLVAML or GILGFVFTL synthetic peptides and subsequent measurement of intracellular IFNγ production by flow cytometry (Figure 2.6).

Figure 2.6 Verification of CTL specificity. HC-04 cells pulsed with either synthetic GLCTLVAML (EBV, BMLF1) or GILGFVFTL (Inf A, M1) peptide were used as stimulators for activation of sorted and expanded lymphocyte cultures. Intracellular IFNγ was measured after 8 hours of co-incubation using an anti-IFNγ antibody followed by flow cytometry.

Our CSP expression constructs also include an independent transcript encoding the murine CD8 alpha chain (mCD8a) (Figure 2.4A). This molecule is trafficked to the cell surface and can be used to identify transfected hepatocytes (Figure 2.7A). Live, transfected cells can be purified using magnetic-activated cell sorting (MACS) based on mCD8a surface expression (Figure 2.7B).

Figure 2.7 Identification and purification of transfected HC-04. (A) Staining of transfected HC-04 with anti-mCD8a or isotype control antibody 24 hours after transfection. (B) Purification of transfected cells 24 hours post-transfection by anti- mCD8a beads. MACS column flow-through (solid line) and purified fraction (dashed line) are shown.

Using purified effectors (CD8+ T cells) and targets (mCD8a+ HC-04 cells) we determined that targets expressing CSP-Short were less efficient at activating CD8+ T cells than targets expressing CSP-Full in a HLA-A2 restricted peptide-specific manner (Figure

2.8). CD8+ T cells were only activated by their cognate chimeric CSP and the observation of differential activation was seen in three of the four separate T-cell cultures.

Figure 2.8 MHC Class I restricted recognition of cells expressing CSP chimeras by peptide specific CD8+ T-cells.

HC-04 cells transfected with DNA constructs coding for different CSP chimeras were sorted by MACS and used as stimulators for viral peptide-specific CTLs at 1:1 ratio. Flow- cytometry-based detection of intracellular IFNγ was measured after 8 hours of co- incubation.

Even though the T-cell culture GIL-111006 displayed antigen specificity (Figure 2.6), it responded minimally to transfected targets.

Targets expressing one of two forms of CSP are differentially lysed by CD8+ T cells

After determining that hepatocytes expressing CSP-Full and CSP-Short differentially activate CD8+ T cells, we tested the ability of these targets to be lysed by

CD8+ T cells. We detected cytotoxicity by measuring the presence of an enzymatically active cytosolic protein, lactate dehydrogenase (LDH) using a commercially available kit, following addition of effector T cells. Similarly to experiments measuring T cell activation, targets expressing CSP-Short were less susceptible to lysis by antigen specific CD8+ T cells

(Figure 2.9A). We hypothesized that the observed difference in cytotoxicity was due to different molar amounts of processed and presented peptide at the hepatocyte surface.

Therefore, we attempted to indirectly measure the amount of peptide presented on the hepatocyte surface and quantify the difference we see between CSP-Full and CSP-Short

We generated a cytotoxicity curve by titrating synthetic peptide on hepatocytes and fit a line using a one site specific binding model (y=Bmax*x/(Kd+x)) (Figure 2.9B).

Simultaneously we repeated the cytotoxicity assay using MACS-purified chimeric CSP expressing targets. Using the cytotoxicity measurements of CSP expressing targets and the titration curve, we determined indirect estimations of presented peptide on the target

49 surface (Figure 2.9B). The relative molar values suggest that 2.5 fold more peptide is presented on the cell surface in CSP-Full than CSP-Short expressing hepatocytes.

However, these measurements assume that the inherent susceptibility of hepatocytes to cytotoxicity is the same between CSP-Full and CSP-Short.

Figure 2.9 Specific lysis of targets expressing CSP-Full or CSP-Short by CD8+ T- cells. (A) Release of LDH from MACS sorted HC-04 targets expressing CSP-Full-GLC or CSP- Short-GLC chimeras upon co-incubation with GLC-111006 CD8+ T cells used as effectors. Mean±SD of triplicates are shown. Significance was determined by two-way ANOVA, post-hoc comparisons shown, **p<0.01. (B) Titration of GLCTLVAML synthetic peptide on HC-04 targets and resulting cytotoxicity by GLC-111006 CD8+ T-cells. A best fit line was determined using a one site specific binding model. Concurrent cytotoxicity of transfected HC-04 is shown with indirect quantification of presented peptide.

Two forms of CSP do not alter immune phenotypes of targets or overall susceptibility to death stimuli

In addition to peptides presented on MHC class I, resistance or susceptibility to cytotoxicity can be influenced by the target cell surface phenotype or by an intrinsic resistance to apoptosis or apoptotic stimuli. To determine if the differential susceptibility to cytotoxicity is in fact due to differences in presented peptide, we surface stained for the presence of CD54, an adhesion molecule important for cell-cell contact, and total MHC class I (Figure 2.10A) on cells expressing the two forms of CSP. We did not observe a significant difference in the intensity of staining for the adhesion molecule CD54 or MHC class I complexes at the surface of hepatocytes expressing CSP-Full or CSP-Short.

Different responses to engagement of the death receptors Fas or TRAIL Receptors have the potential to influence target cytotoxicity. We engaged these death receptors with recombinant TRAIL or anti-Fas antibodies to evaluate their individual contributions to cytotoxicity and found no difference in cytotoxicity between CSP-Full and CSP-Short

(Figure 2.10B). Our initial measurement of cytotoxicity were made using CD8+ T cells.

Consequently, we evaluated if the presence of CSP alters overall target susceptibility to

CD8+ mediated cytotoxicity by using constructs that express wild-type CSP and therefore do not contain the chimeric viral epitope recognized by our CD8+ T cells. By pulsing these wild-type CSP expressing targets with synthetic peptide we activated T cells independently of peptides derived from CSP. Using this methodology we determined that targets expressing CSP-Full and CSP-Short are lysed with equal efficiencies when loaded with exogenous peptide (Figure 2.10C). These data support the conclusion that targets expressing either form of CSP maintain their susceptibility to apoptosis but differentially

51 process and present epitopes on MHC class I at the plasma membrane. Therefore, the difference in killing between targets expressing CSP-Full or CSP-Short may be due to different abilities of the two forms of CSP to generate peptides for presentation on MHC class I.

Figure 2.10 Susceptibility of targets expressing two forms of CSP to T cell effector functions. (A) Expression of CD54 and MHC Class I on the surface of HC-04 cells transfected with plasmids encoding CSP-Full or CSP-Short. (B) Susceptibility of transfected HC-04 cells to cytotoxicity mediated by SuperKiller TRAIL or CH11. (C) Cytotoxicity by GLC- 111006 CD8+ T cells of HC-04 cells expressing CSP-Full-WT or CSP-Short-WT pulsed with or without synthetic GLCTLVAML peptide. Mean±SD of triplicates are shown. Significance was determined by two-way ANOVA, post-hoc comparisons shown, ***p<0.001.

Half-life of two forms of CSP and dependence of proteasomal degradation on MHC class I presentation

Prior to presentation of peptides on MHC class I, proteins are degraded by proteolytic machinery into typically 8-10 amino acid long peptides [231]. We blocked global translation with cyclohexamide and monitored persistence of CSP-Full and CSP-

Short over time. We observed no difference in the rate of protein degradation over time between the two forms of CSP (Figure 2.11A) suggesting that they have a similar stability in hepatocytes. Next we sought to determine which pathways are responsible for CSP degradation and necessary for antigen presentation. One of the major pathways leading to generation of peptides on MHC class I is through the proteasome. Blocking proteasomal degradation of proteins using the inhibitor lactacystin significantly reduced the ability of hepatocytes to present both forms of chimeric CSP and to be lysed by CD8+ T cells (Figure

2.11B). Since proteasomal degradation of ectopically expressed CSP is required for presentation on MHC class I we speculated that access to the proteasome through formation of ubiquitin chains may influence the amount of peptides generated for presentation.

Figure 2.11 Degradation of ectopically expressed CSP in HC-04 cells. (A) Western blot detection and quantification of CSP band intensity following cyclohexamide treatment beginning at 24 hours post-transfection. (B) CTL-mediated lysis of HC-04 targets expressing CSP-Full-GLC or CSP-Short-GLC chimeras with or without prior exposure to lactacystin. Mean±SD of triplicates is shown. Significance was determined by two-way ANOVA, post-hoc comparisons shown, ***p<0.001.

CSP-Full forms polyubiquitin chains more efficiently than

CSP-Short

The formation and composition of ubiquitin chains on a substrate has the potential target the substrate to one of many independent proteolytic machineries in the cell [232].

Since presentation of CSP on MHC class I is proteasome dependent, degradation of CSP-

Ub complexes by alternative proteolytic pathways may alter the amount of peptide available for the proteasome and subsequent presentation. We used available plasmids

[233] that express ubiquitin (Ub) tagged with hemagglutinin (HA) to identify and compare ubiquitination of both forms of CSP. Hepatocytes were transfected with CSP expression

54 constructs and Ub-HA constructs at the same time. Lysates were immunoprecipitated for

HA and blotted for CSP. We first treated these transfected cultures with inhibitors of the proteasome (lactacystin), lysosome (chloroquine), and autophagy (spautin) to determine the major proteolytic systems that contribute to the degradation of ubiquitinated CSP.

Accumulation of CSP-ubiquitin linkages was most prominent during inhibition of the proteasome, suggesting that ubiquitinated CSP-Full and CSP-Short proteins are predominantly degraded by the proteasome (Figure 2.12A). However, we observed substantially more ubiquitin chains formed on CSP-Full than CSP-Short. The greater amount of ubiquitin chains formed on CSP-Full correlates with the observed greater susceptibility to cytotoxicity and dependence on proteasomal degradation for presentation.

Since ubiquitin has the potential to form chains using seven lysines we considered the possibility that the types of chains formed on CSP-Full and CSP-Short may also differ.

Classically, lysine-48 linked chains are targeted for proteasomal degradation and lysine-63 linkages traffic to the lysosome [234,235]. Using ubiquitin expression plasmids that restrict linkages to only lysine-48 (K48), lysine-63 (K63), or a plasmid that is devoid of lysine (K-

KO) we evaluated the contribution of individual chain linkages to Ub-CSP complexes. The two forms of CSP differed in their ubiquitination by wild-type ubiquitin but no appreciable difference was seen in their linkages restricted to K48 or K63 (Figure 2.12B). However, we noted that CSP-Full was able to form linkages independent of ubiquitin lysines (Figure

2.12B). The ability of ubiquitin to form lysine independent, “linear” chains has been reported but the contribution of these linkages to degradation and antigen presentation have not been well established [236].

Figure 2.12 Identification of ubiquitination patterns of two forms of CSP. (A) Transfection of HC-04 with plasmids containing HA-tagged ubiquitin and either CSP- Full or CSP-Short. Cells were lysed and supernatant was immunoprecipitated for HA. (B) Transfection of HC-04 with wild type HA-tagged ubiquitin or mutated ubiquitin to restrict lysine chain formation along with either CSP-Full or CSP-Short. Cells were lysed and supernatant was immunoprecipitated for HA.

Individual lysine residues in the N-terminus of CSP are required for efficient MHC class I-restricted antigen presentation

We constructed two additional N-terminal truncation mutants of CSP to evaluate the specific contribution of N-terminal amino acids to presentation on MHC class I and cytotoxicity. N-terminal truncations beginning at amino acids 59 and 68 of CSP resulted in a significant reduction in cytotoxicity when compared to CSP-Full (Figure 2.13A). The ability of the truncation mutants to serve as targets supports the notion that N-terminal

56 residues present in CSP-Full allow for efficient processing and presentation of C-terminal epitopes on MHC class I. This attribute of CSP would be in contrast to a system in which the N-terminal amino acids specifically found only in CSP-Full are the direct determinants of protein degradation [237]. However, it is uncertain if presentation is altered by the overall N-terminal structure or is dependent on specific N-terminal lysines for ubiquitin chain formation. Three lysine mutants were generated from CSP-Full, by substituting lysines to alanines. Mutation of lysine 58 to alanine significantly reduced cytotoxicity

(Figure 2.13B) and in parallel reduced ubiquitination (Figure 2.13C). Mutations at positions 66 and 67 of lysine to alanine did not significantly alter CTL mediated cytotoxicity (Figure 2.13B). However, substitution of both lysine 66 and 67 simultaneously reduced CTL mediated cytotoxicity and the concurrent amount of ubiquitinated CSP

(Figure 2.13B,C). These data support the hypothesis that individual lysines present in the

N-terminus of CSP contribute to antigen presentation of C-terminal epitopes and that cleavage of the N-terminus of CSP by sporozoites may alter the immunogenicity of CSP.

In our system, presentation of ectopically expressed CSP is dependent on peptide generation by the proteasome and can be regulated by altering the ability of ubiquitin chain formation, specifically at the N-terminus of CSP.

Figure 2.13 Role of the N-terminus on presentation of CSP and the contributions of individual lysines. (A) Cytotoxicity by GLC-111006 CD8+ T cells of HC-04 targets expressing N-terminal deletions of CSP. (B) Cytotoxicity by GLC-111006 CD8+ T cells of HC-04 targets expressing CSP with individual lysine to alanine mutations. Significance was determined by one-way ANOVA, post-hoc Tukey’s HSD comparisons shown, **p<0.01 ***p<0.001. (C) Transfection of HC-04 with HA-tagged ubiquitin and CSP with individual lysine to alanine mutations. Cells were lysed and supernatant was immunoprecipitated for HA.

Discussion

In rodent models of malaria, the circumsporozoite protein is the major target of the immune response in whole organism vaccination and can be protective in single antigen vaccination [238]. Currently, the only human vaccine against malaria in phase III trials targets CSP [221]. However, current forms of the vaccine do not provide complete, sterilizing protection [239]. Sterilizing protection is more difficult to achieve when native rodent hosts are infected with their natural Plasmodium pathogen [136]. This supports the idea that host-pathogen co-evolution alters the efficacy of the host immune response to favor transmission of the parasite.

Prior to productive invasion of the host hepatocyte, CSP must be cleaved by a parasite protease [148,149]. This cleavage event produces two forms of CSP that have potentially different abilities for presentation, independent of their functional roles in sporozoite invasion. Differences in presentation may be due to alternations in the N- terminal amino acids, overall protein structure or changes in the ability of the protein to be degraded [237]. As the sporozoite moves, CSP is shed from the surface and therefore has the potential to be processed and presented in cells traversed but not infected by the parasite. Additionally, CSP has the potential to be cross-presented by dendritic cells [240].

It is conceivable that shed CSP can activate CD8+ T cells and aid in elimination of EEFs in a bystander fashion.

To identify which form of CSP predominates during the process of sporozoite traversal prior to EEF formation, we sorted hepatocytes based on dextran retention six hours after infection. Both dextran positive and dextran negative populations from the same infected culture contained CSP but the amount of CSP in traversed cells was considerably

59 greater, suggesting that the act of traversal leads to deposition of CSP in hepatocytes. The presence of CSP in dextran negative cells is possibly due to extracellular, attached sporozoites, the presence of a proportion of traversed cells that did not take up dextran or from infected cells. In traversed cells we detect both forms of CSP, however, the cleaved form predominates (Figure 2.4D). These data support the idea that any CD8+ T cell activation by traversed cells is predominantly due to the immunogenicity of the cleaved form of CSP. Previous imaging studies in mice show that CD8+ T cells in close proximity to EEFs can eliminate parasites, presumably though direct recognition of peptides presented by infected cells [241]. We sorted live hepatocytes three days after infection with a GFP-expressing P. falciparum line [170] and determined that developing EEFs contain the cleaved form of CSP (Figure 2.4E). Transcriptome analysis determined that CSP transcript is detectable in EEFs [230]. The detection of only the cleaved form of the protein could be due to CSP being both translated and cleaved in the EEF or because CSP found in the EEF predominantly originated in the sporozoite. In either scenario the cleaved form of CSP predominates in EEFs and is potentially the antigen processed and presented if direct recognition of EEFs by CD8+ T cells does occur.

To assess the immunogenicity of these two forms, we generated a CSP expression system that allows for expression of either CSP-Full or CSP-Short and contains a chimeric

C-terminal sequence capable of being presented in the context of HLA-A2. This HLA allele is present across Africa [218] and has been used to identify the presence of naturally occurring reactivity to CSP epitopes [152]. Using this expression system in a HLA-A2 positive hepatocyte line, HC-04 and purified CD8+ T cells specific to viral epitopes (Figure

2.6), we determined that T cells are differentially activated by these two forms of CSP

(Figure 2.8). Three of the four T cell lines were capable of being activated by CSP expressing hepatocytes only if the relevant peptide epitope was present in the C-terminus of CSP (Figure 2.8). Of the T cell lines that were specifically activated, all three had higher percentages of IFNγ producing CD8+ T cells when activated by hepatocytes expressing

CSP-Full rather than CSP-Short. This difference was observed using two epitopes and three different T cell lines, supporting the assertion that the absence of the N-terminus of CSP hinders activation of T cells specific to a C-terminal peptide.

Next, we determined whether the difference in T cell activation of CSP-Full and

CSP-Short resulted in differential target cytotoxicity. Using release of lactate dehydrogenase as a metric for cytotoxicity we found that CSP-Full-expressing cells were killed at a higher percentage than those expressing CSP-Short (Figure 2.9A). This trend is consistent with the difference in T cell activation. One possibility is that different amounts of peptides are generated from CSP-Full and CSP-Short and that this leads to different numbers of specific peptide-MHC complexes reaching the hepatocyte surface. After considerable effort we were unable to obtain enough peptide from the cell surface to quantify our specific epitope by mass spectrometry. Instead we used an indirect measure of peptide by titrating synthetic peptide on the cell surface and generating a killing curve as a function of peptide (Figure 2.9B). Using this curve and simultaneous measurements of cytotoxicity of CSP expressing targets we estimated that over 2.5 fold more peptide is presented at the cell surface from CSP-Full than CSP-Short (Figure 2.9B).

However, we were unable to formally exclude the potential contribution that different forms of CSP might have on the inherent ability of hepatocytes to activate or to be killed by CD8+ T cells. CSP has been shown to alter host cell processes to promote EEF

61 development [153,156], but it is unclear if CSP alone can modulate hepatocyte sensitivity to cytotoxicity. We measured the levels of MHC class I and the adhesion molecule CD54 in CSP-transfected cells and found no difference in their surface staining phenotypes

(Figure 2.10A). To test the susceptibility of CSP expressing targets to cytotoxicity independent of antigen processing, we triggered death by crosslinking of TRAIL receptors or FAS. We did not observe any difference in the susceptibilities of CSP expressing targets to TRAIL- or FAS-induced lysis (Figure 2.10B). Since we did not observe any difference in surface phenotype or susceptibility to death receptor mediated cytotoxicity, we assessed if two forms of wild-type CSP could alter susceptibility to CD8+ mediated cytotoxicity when targets were pulsed with exogenous peptide. In this case we did not observe any difference in cytotoxicity (Figure 2.10C), supporting the conclusion that the two forms of

CSP do not differentially alter the intrinsic ability of targets to be killed by T cells. These data are in line with previous observations [242]. These data suggest that the differences in

T cell activation and cytotoxicity by CSP-Full or CSP-Short are due to differences in the processing and presentation of epitopes in CSP. To better understand this process we needed to determine what pathways are involved in CSP degradation leading to presentation on MHC class I.

First, by halting nascent translation of CSP and following CSP over time we determined that CSP-Full and CSP-Short do not have appreciable differences in their half- lives when ectopically expressed (Figure 2.11A). Since both forms of ectopically expressed

CSP have similar stabilities we aimed to determine what proteolytic systems degrade these proteins and have the ability to contribute to antigen presentation. Degradation of proteins by the proteasome is the major source of peptides loaded onto MHC class I. Using

62 lactacystin to inhibit degradation of proteins by the proteasome we determined that both

CSP-Full and CSP-Short presentation on MHC class I are completely dependent on the proteasome. If both forms of CSP require access to the proteasome for generation of peptides to be presented on MHC class I, we hypothesized that access to proteasomal degradation may influence the ultimate generation of antigenic peptides. Proteins are targeted to the proteasome after being covalently tagged with ubiquitin, which forms multi- subunit chains at lysine residues. To specifically identify ubiquitin chains on CSP we expressed a HA-tagged ubiquitin along with CSP, immunoprecipitated for HA, and blotted for CSP. We first asked which proteolytic pathways are responsible for degrading ubiquitinated CSP by chemically inhibiting their proteolytic activity. We observed accumulation of ubiquitinated CSP only with inhibition of the proteasome, suggesting that the lysosomal and autophagic pathways are not influencing access of ubiquitinated cytosolic CSP to the proteasome. However, upon inhibition of the proteasome we observed more ubiquitination of CSP-Full than CSP-Short (Figure 2.12A). This difference suggests that the two forms of CSP form ubiquitin linkages at different efficiencies and is consistent with CSP-Full presenting more peptide on MHC class I than CSP-Short.

Ubiquitin has the potential to form lysine chains in a variety of linkages [232]. The types of linkages formed have the potential to direct protein degradation through different pathways. For instance, linkages at lysine 48 (K48) direct proteins for proteasomal degradation and linkages at lysine 63 (K63) direct proteins to the lysosome [234,235]. To determine if CSP-Full and CSP-Short differ in the types of ubiquitin chain linkages they form we transfected with HA-tagged ubiquitin that is restricted to K48, K63, or lysine independent chain formation. We found that the amount of K48 and K63 chains was similar

63 between CSP-Full and CSP-Short. However, CSP-Full was able to form more lysine independent ubiquitin linkages than CSP-Short (Figure 2.12B). To our knowledge the contribution of lysine independent ubiquitin linkages to antigen presentation has not been previously investigated in any system.

Since CSP-Full forms more ubiquitin linkages and is more susceptible to cytotoxicity than CSP-Short we aimed to determine what characteristics of the N-terminus of CSP contribute to presentation of CSP-Full. We generated two intermediate N-terminal truncation mutants that were shorter than CSP-Full but longer than CSP-Short. We measured the ability of these mutants to be killed by CD8+ T cells and determined that both mutants were killed less than CSP-Full (Figure 2.13A). These data support the notion that amino acid residues in the N-terminus influence presentation of CSP. Both these mutants contain the region I putative cleavage site but have alterations in their presentation.

Since the difference in cytotoxicity correlates with differences in ubiquitination, we hypothesized that specific lysine residues in the N-terminus may be important for ubiquitin chain formation, subsequent proteasomal degradation, and antigen presentation. To test this hypothesis we mutated three lysine residues in the N-terminus of CSP to alanines. Of the single point mutation substitutions we found that K58 replacement significantly reduced cytotoxicity (Figure 2.13B). We confirmed that lysine at position 58 had reduced ubiquitin chain formation (Figure 2.13C), suggesting that the observed reduction in cytotoxicity by CD8+ T cells is due to the loss of ubiquitin chain formation at lysine 58 in the N-terminus. Even though individual mutations of lysines at position 66 and 67 did not significantly alter ubiquitination and antigen presentation, simultaneous substitution significantly decreased both ubiquitination and CTL mediated cytotoxicity (Figure

2.13B,C). These data support the hypothesis that specific lysines in the N-terminus influence CSP presentation through formation of ubiquitin chains, but to different extents depending on the specific position in the N-terminus.

The N-terminal cleavage of CSP has been well documented to be important for the establishment of EEFs in the liver. Yet, the contributions these two forms have to activation of the immune response has not been previously addressed. Using an ectopic expression system we are able to compare differences in the processing and presentation of two naturally occurring forms of CSP. We found CSP-Full activates CD8+ T cells more than

CSP-Short and can be killed to a greater degree. These data led us to examine how this mechanistically occurs, whether by alterations in presentation or modification of host cell biology. We found no evidence that CSP alters the overall susceptibility of hepatocytes to be killed; rather we found that ubiquitination of specific lysines in the N-terminus drives degradation by the proteasome and subsequent presentation of C-terminal peptides.

We found that CSP-Short predominates in traversed and infected hepatocytes. It is known that T cell responses against the C-terminus of CSP can be primed and recalled

[152] but do not appear protective in naturally exposed individuals [98]. It is possible that in addition to a functional role of CSP cleavage, the parasite benefits by abrogating the ability of CSP to be processed and to activate the effector arm of the immune response.

This is an important consideration because in animals models of vaccination peptides from the C-terminus of CSP are sufficient to protect mice [121,204] and therefore any modifications to their presentation may influence the protective nature of an effector immune response.

Current formulations of the RTS,S vaccine based on CSP do not include the N- terminal portion of CSP. Even though we have not specifically tested the ability of CSP-

Full or CSP-Short to prime an immune response, it is possible that processing and presentation may be enhanced through inclusion of the N-terminus and contribute to priming the immune response.

Chapter 3 : Specific detection and isolation of P. falciparum

EEFs in vitro

Portions of this chapter have been adapter form “Flow cytometry based detection and isolation of Plasmodium falciparum liver stages in vitro” (under review)

Introduction

Direct access to in vivo infected human hepatocytes is untenable due to ethical and logistic constraints. Biopsy of a heavily infected human liver was crucial for the original identification of the P. falciparum liver stage [45] but is unreasonable for routine experimentation. Consequently, studies of the liver stage of Plasmodium infection have relied mainly on the use of rodent parasites both in vivo and in vitro [159,243]. The rodent parasites Plasmodium berghei and Plasmodium yoelii complete full development in less than three days after mosquito bite and can fully develop into infectious merozoites in human hepatocellular carcinoma cell lines in vitro [81,82]. However, the human parasite

P. falciparum requires at least 144 hours for full EEF development in the liver and has a limited ability to infect human hepatocellular carcinoma cell lines [159]. Thus, the majority of scientific work concerning the biology of the Plasmodium liver stage has been accomplished using the tools of rodent parasites.

Multiple experimental models utilizing primary human hepatocytes for P. falciparum EEF development have been reported. Infection of primary hepatocytes in vitro by P. falciparum was first described almost thirty years ago [55]. Recent work using micropatterned primary hepatocytes surrounded by stromal cells has allowed for both complete development of P. falciparum EEFs and possibly generation of P. vivax hypnozoites in vitro [167]. The inability of primary hepatocytes to be continually propagated in culture adds to the cost of experimentation, as does the introduction of variability between donors and even individual cell preparations. The determinants of a robust in vitro P. falciparum liver stage infection have not been clearly defined. The use of

68 a standardized, immortalized hepatocyte cell line with slow growth kinetics and susceptibility to P. falciparum EEF formation would be ideal.

The first mouse models relying on the engraftment of human hepatocytes into immune-compromised animals capable of generating mature EEFs were reported more than two decades ago [244] and were further used to obtain isolated infected cells from fixed frozen liver tissues through micro-dissection [245]. Complete development of P. falciparum liver stages and liver-to-blood transmission were later demonstrated in vivo in immune-compromised and fumarylacetoacetate hydrolase-deficient animals backcrossed with NOD mice [85]. Recently, SCID mice with chimeric human livers were used to show the protective effect of parasite antigen-specific human monoclonal antibodies derived from RTS,S vaccine recipients [246]. These mice lack a functional immune system and therefore can only be used to test individual, reconstituted components of immune protection.

The in vivo and in vitro methods described above demonstrated the generation of

P. falciparum merozoites capable of infecting red blood cells. However, the technical complexity and the high associated costs restrict the widespread use of these methodologies for routine studies on P. falciparum liver stages. Additionally, these methods rely on immunofluorescence or quantification of total parasite biomass and are unable to isolate live, individual P.falciparum EEFs. Therefore, a technically reproducible and cost- effective experimental system for in vitro monitoring and purification of P. falciparum

EEFs is still needed.

Mouse models of the liver stage using rodent parasites suggest a role for both CD8+

T cells and sporozoite antigen-specific antibodies in sterilizing immunity [132]. However,

69 understanding the contributions of the humoral and cell-mediated immune responses directed against P. falciparum EEFs during the natural course of infection [83,247] or induced upon vaccination [248,249] requires a robust in vitro system.

Two modes of interaction between sporozoites and host hepatocytes are currently described in vitro [60,250] and in vivo [59,73]: (i) breaching of the host cell plasma membrane followed by intracellular movement and subsequent exit, referred to as traversal, and (ii) productive invasion and parasitophorous vacuole (PV) formation within hepatocytes. The influence of traversed cells on infection, the subsequent immune response, and parasite biology are largely unknown. Thus, an optimal experimental in vitro system recapitulating the liver stage of P. falciparum should allow for specific identification and isolation of traversed from non-traversed cells and infected from uninfected cells. Utilizing in vitro models of Plasmodium infection, non-traversed and uninfected cell populations are similarly exposed to a plethora of biological factors from the salivary glands of infected mosquitoes and serve as the most accurate control population to study the immunology and developmental biology of P. falciparum liver stage infection in vitro. However, in vivo, sporozoites are deposited into the host dermis and the direct impact of salivary gland factors on hepatocytes is therefore diminished [1].

Here we report a flow cytometry-based in vitro system to monitor P. falciparum liver stages that permits detection and isolation of P. falciparum EEFs. A previously reported method for detection of P. falciparum infection in vitro utilized an anti-CSP stain

[251] that was unable to differentiate between cells traversed by sporozoites and productively infected cells. Additionally, the use of an intracellular antigen to identify parasites required fixation and thus the inability to purify live samples.

Isolation of individual EEFs creates the potential to study both the metabolic and/or transcriptional activity of both the parasite and the host hepatocyte. Expression profile libraries of the liver stage have been generated using rodent parasites [252] but comparable libraries have not been created for the human parasite P. falciparum.

Results

Immortalized human hepatocyte cell lines are permissive for traversal by P. falciparum sporozoites

Traversal of hepatocytes by Plasmodium sporozoites has been previously documented in vitro [60,250] and in vivo [59,73]. The in vitro traversed cell population can be identified by flow cytometry by its’ ability to retain fluorescent dextran during migration of sporozoites through the host cell [250]. Detection of traversal was dependent on the number of sporozoites added (Figure 3.1) and could be reversed by pre-incubation of sporozoites with the actin inhibitor cytochalasin D (Figure 3.1B,C). This demonstrates the specificity of detection of traversal and the absence of any factors other than sporozoites in quantification of traversal in vitro.

Figure 3.1 Specific detection of traversal by P. falciparum sporozoites in vitro. Traversal was assessed by flow cytometry as an ability of cells to uptake and retain fluorescent high molecular weight dextran during sporozoite migration. (A) Gating strategy for detection of dextran retention. Cells gated on forward and side scatter. Dextran percentages determine by signal in FL-2 (B) Traversal is inhibited by incubation of sporozoites with the mycotoxin cytochalasin D (C) Traversal is dependent on the initial sporozoite to hepatocyte ratio and in all cases inhibited by cytochalasin D. Performed in biological triplicate, Mean±SD shown. Ratio and cytochalasin D p<0.0001 by two-way ANOVA, post-hoc Bonferroni adjusted comparisons shown ***p<0.001.

In search of cell lines that allow for efficient detection of traversal and invasion of

P. falciparum sporozoites, we turned to immortalized cell lines derived from human hepatocytes that included THLE-2 and THLE-3 [253], HepG2, HepG2-CD81 [75] and HC-

04. HC-04 is the only cell line previously reported to support progressive development of

P. falciparum liver stages [167,169]. However, the process of traversal occurs in several tissues and is likely not specific to any one cell type. We found that all five cell lines tested had a detectable population of hepatocytes traversed by P. falciparum 3D7HT-GFP sporozoites (Figure 3.2A). HC-04, HepG2 and HepG2-CD81 had comparable percentages of cells retaining fluorescent dextran, which was about 2-3 fold higher than in cultures of

THLE-2 and THLE-3 cell lines (Figure 3.2B). We concluded that each of the tested cell lines can be utilized to study traversal by P. falciparum sporozoites in vitro.

Figure 3.2 Human hepatocyte cell lines can be traversed by P. falciparum sporozoites.

(A) Representative plots for each cell line are shown. Numbers indicate percentage of dextran-positive cells. (B) Percentage of traversed cells normalized to HC-04 obtained from three sporozoite preparations each in triplicate. Mean±SD shown, ***p<0.001, *p<0.05.

Human hepatocyte cell lines exhibit differential ability to support development of P. falciparum EEFs

Next, to assess the ability of immortalized hepatocyte cell lines to support the development of P. falciparum 3D7HT-GFP parasites, we used a novel, quantitative, flow cytometry-based analysis of infected cultures. We utilized relative FL1 (530/30) to FL2

(585/42) fluorescence ratios (non-overlapping) to detect the weak GFP signal of the parasite in relation to background autofluorescence of the host cell while simultaneously excluding dead cells using propidium iodide. To validate our ability to specifically detect developing parasites we quantified parasite 18S expression using real-time PCR and visualized P. falciparum EEFs by immunofluorescence microscopy following flow cytometry-based sorting of infected cells.

Here we show that GFP+ events could be detected not only in HC-04, but also in

HepG2 and HepG2-CD81, which had been previously described as non-permissive for the development of P. falciparum EEFs (Figure 3.3A) [159]. However, the relative frequencies

(percentages) of GFP+ PI- events were 2.5-fold higher (Figure 3.3B) and the total number of GFP+ events per well were 8-fold higher (Figure 3.3C) in HC-04 as compared to HepG2 and HepG2-CD81 cells. Both THLE-2 and THLE-3 had significantly fewer total GFP+ events than the other cell lines tested (Figure 3.3C). In contrast, the fluorescence intensity

76 of the GFP+ populations, a probable indication of parasite development, was comparable in all cell lines tested but had a large within sample variability (Figure 3.3D).

Asynchronous development of rodent EEFs has been previously reported [254] and the broad range of the GFP signal intensity detected may be due to asynchronous development of P. falciparum parasites.

Figure 3.3 Identification of P. falciparum parasites by flow cytometry. Infected cells were identified by flow cytometry at 96 hours postinfection as GFP-positive events in PI-negative (viable) cell populations. Uninfected cultures propagated in parallel with infected cultures were used to define specificity of GFP-positive events. A similar number of total events were acquired from infected and uninfected cultures of each cell lines. (A) Data from one representative culture is shown for each cell line. Event number corresponding to the GFP-positive gate (middle panels) and the geometric mean of events

77 in the GFP-positive gate is indicated (right panels). (B) Percentage of GFP-positive PI- negative events. Mean+SD shown, ***p<.001.

The percentage of GFP positive events in HC-04 cultures at 48 hours postinfection is dependent on the initial sporozoite to hepatocyte ratio and is inhibited if sporozoite motility is abrogated by cytochalasin D prior to infection (Figure 3.4). Even though our sporozoite preparations involve purification from a density gradient and significantly removes contamination from mosquito material, it is important to establish the specificity of our detection method.

Figure 3.4 Specific detection of infection by P. falciparum sporozoites in HC-04 cultures in vitro (48 hours postinfection). Infection was assessed by flow cytometry. Detection of GFP events was dependent on sporozoite to hepatocyte ratio and inhibited by pretreatment of sporozoites with

78 cytochalasin D. Performed in biological triplicate, Mean±SD shown. Ratio and cytochalasin D p<0.0001 by two-way ANOVA, post-hoc Bonferroni adjusted comparisons shown ***p<0.001, *p<0.05.

Quantification of P. falciparum 18S rRNA at two time points postinfection supported our flow cytometry findings (Figure 3.5). The ratio of parasite 18S copy number relative to a hepatocyte housekeeping gene (GAPDH arbitrary units) was 2-fold higher in

HC-04 than in HepG2 and HepG2-CD81, and about 30-fold higher than in THLE-2 and

THLE-3 (Figure 3.5A). In HC-04, a 3-fold increase in the Pf18S rRNA copy numbers per well was observed between 48 and 96 hours postinfection (Figure 3.5B). However, a decrease in the 18S:GAPDH ratio was observed in the same cultures from 48 to 96 hours postinfection (Figure 3.5A). This can be explained by extensive proliferation of uninfected

HC-04 cells (Figure 3.5C) resulting in the relative drop in percentage of infected hepatocytes.

Figure 3.5 Quantification of parasite-encoded 18S rRNA in cultures of human hepatocytes infected with P. falciparum. Real-time PCR analysis of 18S rRNA expression was done at 48 and 96 hours postinfection. Each culture (n=3) was analyzed in technical triplicate. (A) 18S copy number normalized using CT values from GAPDH (B) 18S copy number per culture. (C) Growth curve of uninfected hepatocyte cell lines (n=3 per time point). Mean±SD shown for all graphs, ***p<0.001.

P. falciparum-infected HC-04 hepatocytes can be specifically isolated

Infected hepatocytes from mice have been sorted ex vivo using P. yoelii parasites expressing GFP [164]. We have previously demonstrated that HC-04 cells infected with P. berghei ANKA-GFP can be isolated by flow cytometry-based cell sorting based on GFP expression in vitro [242]. Utilizing a similar approach we performed infection of HC-04 with P. falciparum 3D7HT-GFP sporozoites and sought to verify that GFP+ HC-04 cells contain developing parasites. Hepatocytes were sorted based on a GFP positivity and cytospun, followed by immunofluorescence analysis of GFP+ cell populations. This process is particularly harsh and may disrupt native host and parasite morphology, but it is necessary due to the low number of GFP+ cells and the rapid outgrowth of uninfected HC-

04 during the 96 hours pi (Figure 3.5C).

As shown in Figure 3.6, GFP+ cells isolated at 96 hours pi contained P. falciparum parasites. We used three fluorescent markers to identify the presence of EEFs: parasite- encoded GFP, DAPI to stain both host and parasite DNA, and a monoclonal antibody against PfHsp70. We observed three patterns in sorted GFP+ cells (Figure 3.6): (a, b) a

GFP-positive, Hsp70 positive parasite located perinuclear and morphologically similar to parasites in primary hepatocytes [55,167] (c, d) a GFP-positive Hsp70-positive parasite

81 adjacent to a dividing or fragmented host nucleus and (e, f) a GFP-positive Hsp70-positive cell with indistinguishable host DNA that was either devoid of a host nucleus or that had a host nucleus containing the parasite. Punctate DNA staining within the parasites is indicative of development. We do not exclusively observe P. falciparum localized in the nucleus of HC-04 cells as has been previously reported for HepG2 [75], however, we cannot formally exclude that this may occur in a subset of GFP+ HC-04 cells.

Figure 3.6 Visualization of parasites in HC-04 cells infected with P. falciparum.

GFP-positive PI-negative events were isolated by flow cytometry-based cell sorting 96 hours postinfection and cytospun. Representative micrographs of EEFs show DNA staining with DAPI, GFP expression and immunofluorescent detection of PfHsp70 by anti- Hsp70 monoclonal antibody (Blue = DAPI, Green = GFP, Red = PfHsp70. Scale bars indicate 10 μm).

Primary human hepatocytes support P. falciparum 3D7HT-

GFP EEFs in vitro

We next asked if P. falciparum infection of HC-04 is comparable to that of primary human hepatocytes and if our flow cytometry based method can identify parasites. Higher levels of autofluorescence observed in primary hepatocyte cultures (Figure 3.7) required adjustments of flow cytometer voltages prior to acquisition of primary hepatocytes versus

HC-04 cells.

Figure 3.7 Primary hepatocytes have a high degree of autofluorescence and require adjustment to voltages. HC-04 and primary hepatocytes (donor 4051) have different levels of background autofluorescence in live cells (PI negative). Acquisition of HC-04 required use of the “high” voltage settings listed and primary hepatocytes were acquired using the “low” voltage settings.

Infected hepatocyte cultures from three donors had viable infected (GFP+PI-) events detectable by flow cytometry at 96 hours pi (Figure 3.8A). Direct comparisons of GFP

85 mean fluorescence intensity between HC-04 and primary hepatocyte cultures are not feasible or relevant due to the necessary voltage adjustment. Unlike HC-04, GFP+ events detected in primary hepatocyte cultures were within the range of background autofluorescence in the FL1 fluorescence channel (530/30) (Figure 3.8A). This highlights the necessity of using a ratio of closely related fluorescent emission channels to distinguish

GFP+ events from the autofluorescence of primary hepatocytes. The relative frequencies of GFP+ events detected in primary hepatocytes at 96 hours pi were 20-fold higher (Figure

3.8A-B) than HC-04. However, the numbers of GFP+PI- cells in each individual culture

(Figure 3.8C) did not significantly differ between HC-04 and two of the primary hepatocyte cultures (donors 4055A and 4059). Infected hepatocyte cultures from donor 4051 supported 3-fold more GFP+ cells than HC-04 and primary cells from the two other donors.

The drastic difference in percentages of infected cells found in HC-04 versus primary hepatocyte cultures at 96 hours pi (Figure 3.8A,B) was due to extensive proliferation of uninfected HC-04 cells (Figure 3.5C). To confirm that GFP+ primary hepatocytes contained parasites we sorted and stained cells with an Hsp70-specific antibody (Figure

3.8D) in a similar fashion to GFP+ HC-04.

Figure 3.8 Comparison of detection of P. falciparum infection between primary human hepatocytes and HC-04. Infected cells were identified by flow cytometry at 96 hours postinfection as GFP-positive events in PI-negative (viable) cell populations (n=3). Uninfected cultures were used to define the positive gates. (A) Representative gating for detection of GFP events using three human hepatocyte donors and HC-04. Number of GFP-positive PI-negative events indicated (middle panels). Geometric mean of GFP-positive PI-negative events indicated (right panels). (B) Comparisons of the percentage of GFP-positive events and (C) total number of GFP events obtained per well. Mean±SD shown. (D) Representative examples of GFP-positive PI-negative events isolated by flow cytometry-based cell sorting 96 hours postinfection and cytospun (Scale bars indicate 10 μm).

Similar to HC-04, the percentage of GFP positive events is specific to the initial sporozoite to hepatocyte ratio and is inhibited by treatment of sporozoites with cytochalasin

D (Figure 3.9).

Figure 3.9 Specific detection of infection by P. falciparum sporozoites in primary hepatocytes in vitro (96 hours postinfection). Infection is dependent on the initial sporozoite to hepatocyte ratio and inhibited by cytochalasin D. Performed in biological triplicate, Mean±SD shown. *p<0.05, **p<0.01.

The lack of proliferation of primary hepatocytes allowed for visualization of unsorted EEFs maintained on coverslips throughout infection (Figure 3.10A). This technique has been used by others to visualize EEFs and allows us to demonstrate the presence of EEFs in our lab by a well-established protocol. We defined parasites by Hsp70 staining (red) and observed no significant differences in parasite size between hepatocytes

88 from different donors at 96 hours pi (Figure 3.10B, C). Similar to previous studies [55,167],

Pf3D7-HTGFP parasites were either adjacent to or touching the host nucleus (Figure

3.10D). A distance of >10μm was observed between some parasites and the closest host nucleus (Figure 3.10E). However, unlike GFP+ HC-04 cells, the high background of green autofluorescence, as seen by flow cytometry, obscured the parasite GFP signal.

Figure 3.10 Parasite morphology and size in cryopreserved human hepatocytes from three donors. Cultures were fixed and stained directly on coverslips 96 hours postinfection. (A) Representative micrographs of EEFs observed from each donor. DAPI (blue) represents host and Plasmodium nuclei and PfHsp70 (red) denotes EEF. Arrows indicate EEF in phase contrast micrographs (Scale bar = 10 μM). EEF size was measured by (B) surface area and (C) maximum diameter. (D) Proportion of EEFs observed to be directly adjacent to host

90 nucleus. (E) Of EEFs distant from host nucleus, the distance to the closest nucleus is indicated. Mean shown (horizontal line) for individual dot plots, ***p<0.001, **p<0.01.

Dynamics of P. falciparum EEF detection by flow cytometry differ in HC-04 and primary hepatocytes

Our prior experiments were performed at 96 pi to allow for detection of developing parasites. However, the overall ability to detect GFP+ parasites by flow cytometry is a function of their brightness over background and attrition over time. To determine the progression of PfEEF development as well as the sensitivity of flow cytometry-based detection of Pf3D7HT-GFP EEFs at different time points post infection, we monitored two parameters: the number of viable infected (GFP+PI-) cells and the intensity of GFP fluorescence in these cells as a marker of parasite development (Figure 3.11).

The maximal numbers of parasites developing in HC-04 cells were detectable starting at 48 hours pi followed by a steady decrease in the number of GFP+ events per culture over time (Figure 3.11A). In contrast, at 48 hours pi detection of parasites in primary hepatocytes was partially obscured by background autofluorescence, even when using the ratio of closely related fluorescence channels. Beginning at 72 hours pi a higher number of parasite-infected cells were detectable in primary hepatocyte cultures by flow cytometry (Figure 3.11B). The relative parasite survival rate between 96-144 hours pi was

29% for HC-04 and 65% in primary hepatocytes (Figure 3.11). The parasite-specific GFP fluorescence intensity increased steadily in both HC-04 (Figure 3.11A) and primary hepatocytes (Figure 3.11B) and reached its maximum at 144 hours pi, suggesting development of the remaining persisting parasites. Since HC-04 and primary hepatocytes required adjustments to the voltages prior to acquisition (Figure 3.7) direct comparison of

91 the geometric MFI (Figure 3.11 y-axis) is not valid. Based on these data, for all subsequent experiments we chose to monitor Pf3D7HT-GFP development at 48 hours pi in HC-04 and at 96 hours pi in primary hepatocytes, when the maximal numbers of parasite-infected cells were observed in their respective cell cultures.

Figure 3.11 Parasite detection and persistence over time. Cultures were infected and collected for flow cytometry starting at 48 hours postinfection in duplicate. Plots are shown for the number of GFP-positive events acquired at each time point and the geometric MFI (acquired at voltages specific to the cell type) of these events for (A) HC-04 and (B) primary donor 4051 (shading indicates time when a portion of the GFP population is obscured). Mean±SD shown.

Discussion

The liver stage of P. falciparum infection, a leading target for generation of protective vaccines, still remains the least characterized part of the parasite lifecycle. Since the demonstration of P. falciparum EEF development in primary human hepatocytes, a number of alternative methodologies relying on microscopy have been identified to study

P. falciparum liver stages in vitro [159,167]. The generation of fluorescent rodent parasite strains along with a wide range of host cell tropisms has allowed for routine identification and sorting of rodent Plasmodium EEFs [164], though such an approach has not been described for P. falciparum. Using a recently developed strain of P. falciparum expressing

GFP [170] we show that live P. falciparum-infected hepatocytes are identifiable by flow cytometry and, therefore, can be quantitatively assessed and isolated. This approach allows for sensitive and rapid screening of hepatocyte cell lines and primary human hepatocytes from different donors for their ability to support PfEEF development in vitro.

Prior to EEF formation in the liver, Plasmodium sporozoites traverse through several host cell types including hepatocytes [59,60,67,72]. We found that in vitro, the amount of cells retaining dextran after parasite traversal differed depending on the hepatocyte cell line used (Figure 3.2). These differences in detection of traversed cells could be a function of inherent susceptibilities to traversal by sporozoites or differences in the cells to retain dextran.

No correlation was observed between the numbers of traversed cells detected 6 hours pi and the frequencies of infected cells detected 96 hours pi (Figure 3.3). These data suggest that in the cell lines tested, the process of invasion and development is distinct from traversal. CSP is shed from the sporozoite surface and is found in both cells

93 permissive or non-permissive to EEF formation [255]. We recently found that flow cytometry-based detection of CSP in P. falciparum-infected hepatocyte cultures does not represent an accurate measure of hepatocyte infection, but rather reflects the retention of

CSP shed during parasite traversal (Trop S. et al., submitted). Therefore, we used parasite- encoded GFP as a marker for specific identification of developing parasites by flow cytometry. Using a ratio of two fluorescent channels, a FL1 (530/30) for detection of parasite GFP and FL2 (585/42) for detection of propidium iodide (PI) positive cells, we compared five cell lines (Figure 3.3) and three primary hepatocyte cultures (Figure 3.8) for their ability to support PfEEF formation and development.

Intrinsic autofluorescence from cells during flow cytometry has the potential to generate false positives (bright events classified as positive) and false negative (dim events obscured in the background). To avoid improper classification of artifacts as true positive or true negatives, techniques using multiple fluorescent channels have been utilized [256].

Previous attempts at identifying fluorescent parasites by flow cytometry may have been obscured due to the presence of false negatives [172] resulting from high autofluorescence.

The use of two channels allows for discrimination between the dead/PI-positive

(FL1FL2) cells in infected hepatocyte cultures. We are not able to quantify directly the individual contributions of autofluorescence to each channel but can adjust the voltage characteristics to allow for detection of autofluorescence as shown previously [256]. This setup allows for the exclusion of autofluorescent false positive while detecting weak GFP signals that would be considered false negative if only a single parameter was used.

Among all cell lines tested, HC-04 [169] supported the highest absolute number of

PfEEFs at 96 hours pi (Figure 3.3A,C). Surprisingly the HepG2 cell line, previously described as being non-permissive for P. falciparum sporozoite infection [159], had GFP+ events detectable in the viable hepatocyte gate (Figure 3.3A). The SV40-immortalized hepatocyte lines THLE-2 and THLE-3 [253] had previously not been tested for their ability to support P. falciparum infection. Though these lines had a low proliferation rate, making them suitable to study the late stages of PfEEFs, both THLE-2 and THLE-3 showed a minimal ability to support Pf liver stages in vitro (Figure 3.3A,C). In addition to the numbers of GFP+ events detectable in infected hepatocyte cultures, we compared the fluorescent intensity of these events as a measure of parasite development. The geometric mean fluorescence intensity (MFI) of these GFP+ populations in each well were not significantly different (Figure 3.3D). To further verify the presence of developing P. falciparum EEFs we used quantitative real-time PCR for detection of Pf18S at two time points postinfection (Figure 3.5). Of the five cell lines tested, a time-dependent increase in

Pf18S gene copy number was detected only in HC-04 cultures (Figure 3.5B), indicating that P. falciparum parasites can develop in these cells. It is unclear if the failure to increase

Pf18s copy number in the other hepatoma cell lines is a function of parasites failing to develop or a decrease in the overall parasite number.

Primary human hepatocytes remain the gold standard for generation of PfEEFs in vitro. However, recent work has questioned the susceptibility of all primary hepatocytes to

P. falciparum infection, at least in vitro [167]. Using flow cytometry we demonstrated, that cryopreserved, commercially available human hepatocytes obtained from 3 different donors were permissive for infection with P. falciparum 3D7HT-GFP sporozoites. Though

95 efficient detection of parasite-specific GFP signal at the early stages of infection was hampered due to high autofluorescence in normal hepatocytes combined with the weak fluorescence of infected cells in which parasites have not yet multiplied [257,258]. Primary hepatocytes infected with Pf3D7HT-GFP were reproducibly detectable by flow cytometry between 72-144 hours pi (Figure 3.11B). It needs to be seen if flow cytometry based detection can be applied to the ex vivo detection of the liver stage parasites in the recently developed humanized mouse models of P. falciparum infection [85]. This process could conceivable circumvent the need for mosquito dissection and sporozoite purification by directly feeding mosquitoes on humanized mice. It will be interesting to see if this could provide an even larger number of P. falciparum EEFs than in vitro infections.

To address the dynamics of 3D7HT-GFP parasite detection and development in hepatocytes in vitro we used flow cytometry to monitor changes in parasite numbers and parasite-specific GFP fluorescence (MFI) as a reflection of parasite expansion within infected hepatocytes. The maximum numbers of GFP+ events in HC-04 cultures were detected at 48 hours pi followed by a rapid decline (Figure 3.11A). In contrast, a larger proportion of infected cells persisted from 96 to 144 hours pi in primary hepatocytes

(Figure 3.11B). A similar trend has been observed by others [167]. Importantly, an increase in GFP fluorescence over time was seen in infected cells that persisted in both HC-04 and primary hepatocyte cultures, indicative of parasite development. Taken together, these results show that P. falciparum parasites can develop in both HC-04 and primary hepatocytes.

To visualize the presence of parasites in GFP+ cells and to demonstrate the ability to selectively isolate live parasites from bulk infected cultures we sorted GFP+ HC-04 cells

96 and primary hepatocytes (Figure 3.6). It does not appear that PfEEFs developing in HC-04 cells exclusively localize to the host nucleus, a phenomenon that has been previously observed in HepG2 [75]. Using GFP and PfHsp70 as parasite-specific markers, we identified three patterns of P. falciparum parasites in HC-04 cells at 96 hours pi. Parasites were found either adjacent to an intact host nucleus, surrounded by a fragmenting or dividing host nuclei or without an apparent host nucleus distinct from the parasite DNA

(Figure 3.6). Additional studies are needed to describe phenotypes of parasites detected in

HC-04 cells at the later time points of infection. In contrast to parasites developing in HC-

04, PfEEFs detected in sorted GFP+ primary hepatocytes were mainly adjacent to the intact host nucleus easily distinguishable from the parasite nuclei (Figure 3.8D).

In the context of low levels of infection, contrary to the rapidly dividing and vertically growing HC-04 cells, non-proliferating primary hepatocytes permit identification of EEFs by fluorescent microscopy without prior sorting (Figure 3.10).

Unlike sorting and cytospin procedures, direct fixation of infected cells on coverslips better preserve both parasite and host hepatocyte morphology. This technique has been used by other groups and was used to compare our EEFs to previously published data. In all three primary infected hepatocyte cultures, PfHsp70-specific antibody identified developing parasites (Figure 3.10), but GFP fluorescence was not detectable in these cells by fluorescence microscopy (data not shown). As discussed above, the inability of GFP signal to be detected by fluorescence microscopy is likely due to high autofluorescence in normal hepatocytes [257]. At 96 hours pi PfEEFs developing in primary hepatocyte cultures were mainly adjacent to the host nucleus and comparable in size to parasites described in vivo

[85] (Figure 3.10).

Previously, detection of PfEEFs by flow cytometry has only been possible using rodent Plasmodium species [159,164,242,250]. Although the use of rodent parasites and mouse models of Plasmodium infection will continue to be a cornerstone of malaria research, advancements in methods for studying PfEEFs in vitro are required to strengthen our understanding of basic parasite biology and to provide objective and quantifiable protocols to inform translational research. HC-04 and primary hepatocytes constitute complementary in vitro models to study PfEEF formation. Among five cell lines tested,

HC-04 is able to support the greatest number of developing parasites. However, uninfected cells in HC-04 cultures rapidly proliferate, complicating parasite detection after 72-96 hours pi. On the other hand, parasite traversal can be quantified in these cells early after infection and fluorescent parasites are clearly detectable by flow cytometry starting at 48 hours pi. Consequently, HC-04 is a suitable host cell to screen for the efficacy of interventions aimed at blocking the motility of sporozoites, such as anti-parasite antibodies, but is not fully suitable for study of late Pf liver stages.

In contrast to HC-04 cells, primary hepatocytes do not proliferate in vitro, however, the PfEEFs can be clearly detected in these cells by flow cytometry only beginning at 72 hours pi. Though primary hepatocytes will continue to be the standard for study of PfEEFs, de novo generation or identification of additional human hepatocyte cell lines with low doubling times and limited autofluorescence still remains a priority. The development of a

P. falciparum line expressing GFP through the parasite’s lifecycle [170] was paramount in our ability to reproducibly detect and quantify PfEEFs by flow cytometry. In both HC-04 and primary hepatocytes detection of parasite GFP is specific, as shown by influence of sporozoite number on GFP percentages and inhibition by cytochalasin D. However, the

98 generation of a novel P. falciparum strain expressing a fluorophore brighter than GFP and/or use of a stronger liver stage promoter will facilitate flow cytometry-based detection and isolation of P. falciparum parasites in human hepatocytes even before 48 hours postinfection.

Chapter 4 : Flow cytometry based assays of P. falciparum liver stages in vitro: from sporozoite infectivity to EEF development

Portions of this chapter have been adapter form “Flow cytometry based detection and isolation of Plasmodium falciparum liver stages in vitro” (under review)

100

Introduction

Study of the blood stages of P. falciparum has been greatly facilitated by the development of continual in vitro blood cultures [157]. This method has contributed to a detailed understanding of merozoites invasion ligands [259], in vitro assays to screen for antibody-mediated growth inhibition [260], and high-throughput drug screening [261].

Prior to in vitro cultivation of the blood stages, human malaria parasites could only be propagated in susceptible primate hosts. In contrast, study of the liver stage of Plasmodium has benefited from the use of alternative Plasmodium species capable of infecting laboratory rodents [57,58]. These parasites have been instrumental in studies of parasite liver stage biology including global transcriptional profiling [252] and gene disruptions

[161] to determine essential components of sporozoite infectivity [67,68,70] or EEF development [103]. In addition to routine infection of mice and rats, rodent Plasmodium species have a wide variety of susceptible cell lines in vitro [159], of both hepatocytic and non-hepatocytic origin. The specific determinants that allow for P. falciparum growth in one particular hepatocyte donor’s cells over another are enigmatic [167]. Understandably, data obtained from rodent-trophic parasites do not always correlate with those from human parasites [105] and the results require confirmation in P. falciparum [107].

For instance, the human tetraspanin protein CD81 has been found to be necessary but not sufficient for invasion of P. falciparum sporozoites into hepatocytes [74] but is not required for rodent-trophic P. berghei EEF formation [75]. The necessity of CD81 in P. falciparum EEF formation has recently been confirmed in vivo [79]. Therefore, the choice of parasite and host is important when studying the nuances of the liver stage.

101

A variety of assays both in vitro and in vivo have been used to evaluate components of a protective immune response and the requirements for parasite development in the liver.

In general these assays have aimed to measure inhibition of sporozoite invasion (ISI) or inhibition of liver stage development (ILSDA). Both of these assays have been limited by low invasion or infection rates and in the case of P. falciparum often rely on manual counting of parasites by an immunofluorescent assay (IFA).

The general concept of the ISI assay is to measure interventions that target sporozoite motility, such as antibodies. Due to the wide variety of cultured cell lines that are susceptible to infection by rodent Plasmodium parasites [159], invasion assays have been used routinely to examine the inhibitory effect of antibodies against the sporozoite

[262,263]. Similar assays were then developed for use with P. falciparum, which unfortunately used cell types (WI-38 and HepG2-A16) unsuitable for complete P. falciparum EEF development [264]. Consequently, the use of P. falciparum sporozoite inhibition in these assays is likely a measure of parasite traversal and not productive invasion rates. Both traversal and productive invasion are dependent on parasite motility, but the mechanics and dynamics greatly differ between the two processes. Therefore even though ISI assays can be performed with P. falciparum sporozoites using a non-permissive cell type, they lack the ability to identify any inhibition of initial productive invasion and subsequent PV formation in hepatocytes.

Several methods have been developed to address this issue and to determine an assay more suitable for P. falciparum sporozoites and EEFs. Primary hepatocytes have been used as the gold standard for in vitro P. falciparum EEF evaluation [114]. These techniques originally relied on microscopic identification of parasites and recently, on

102 parasite RNA [166]. However, quantification of parasite development using 18S rRNA is unable to differentiate between delays in parasite growth or abrogation of the total number of parasites in the liver.

Alternatively, humanized mice have been used in tandem with the passive transfer of antibody to establish their inhibitory capacity [171,244,246]. Interestingly, purified IgG from chemoprophylaxis-immunized humans was inhibitory in an in vitro traversal assay and in an in vivo humanized mouse model, but measures of sporozoite gliding motility were inconclusive [171]. However, the use of humanized mice requires large amounts of purified monoclonal antibodies or pooled, purified IgG and still relies on the microscopic identification of parasites in liver sections or detection of luciferase [85,265].

Modification of rodent parasites to express specific antigens such as CSP from P. falciparum [266] or P. vivax [267] allows for the evaluation of responses directed toward candidate antigens. These systems provide a simplified and standardized method with which to evaluate immune responses in vivo but are limited in the scope of targets that can be studied.

Ideally, any system that studies the inhibition of Plasmodium sporozoites or the inhibition of EEF development would have an unbiased method of detection with a high sensitivity in order to detect even low levels of hepatocyte infection. Our flow-cytometry- based detection protocol has the capability to detect parasite number (invasion, ISI) and parasite development (geoMFI, ISLDA). However, an individual assay is not suitable for all experiments. The specific scientific question being asked will determine the strengths and weaknesses of each approach.

103

Results

CD81 is required for P. falciparum infection of primary human hepatocytes, but is not essential for the infection of HC-04

It has been previously shown that functional antibody-based neutralization of CD81 on the surface of primary human hepatocytes abolishes infection by P. falciparum sporozoites in vitro, as measured by the numbers of P. falciparum EEFs detected by immunofluorescence [74]. Antibodies against CD81 were also capable of blocking P. falciparum sporozoite invasion in vivo, using liver chimeric mice [79]. To address the requirement of CD81 using our experimental model utilizing 3D7-HTGFP parasite infection of human hepatocytes in vitro, we first verified the expression of CD81 on the surface of hepatocyte cell lines and primary human hepatocytes. Primary hepatocytes from three donors as well as THLE-2 and -3 cells expressed surface CD81, whereas neither HC-

04 nor HepG2 had detectable expression by flow cytometry (Figure 4.1A). These data were further confirmed by RT-PCR (data not shown), ruling out our inability to detect low CD81 expression by flow cytometry. These observations in combination with the differential ability of hepatocyte cell lines to support development of P. falciparum EEFs (see Chapter

3) reveal no correlation between the expression of CD81 and the susceptibility of human hepatocyte cell lines to P. falciparum infection or to traversal in vitro. A lack of CD81 did not preclude infection of HC-04, and endogenous expression of CD81 on THLE-2 and -3 was not sufficient to allow for efficient infection. Accordingly, ectopic expression of CD81 on HepG2 cells did not result in more efficient P. falciparum infection. Similarly, transient

104 expression of CD81 in HC-04 cells did not significantly change the numbers and development of EEFs in these cultures (Figure 4.1C, D).

Figure 4.1 Expression of surface CD81 by human hepatocyte cell lines and primary hepatocyte donors. Surface CD81 was stained using specific antibodies or an isotype control followed by flow cytometry for detection. (A) Representative plots for all cells are shown and geometric MFI is indicated for both isotype (black) and anti-CD81 staining (red). (B) Surface staining of a mock and transient transfection of HC-04. Transiently transfected HC-04 were infected and run on flow cytometry 96 hours postinfection; (C) GFP-positive number and (D) geometric MFI shown. Mean±SD shown.

Using our experimental model, we recapitulated previously described conditions

[74] for antibody-based neutralization of CD81 (Figure 4.2). We found that the CD81- blocking antibody (clone 1D6) had no effect on P. falciparum 3D7HT-GFP infection of

CD81-negative HC-04, as assessed by percentages and the numbers of infected (GFP+PI-) cells detected by flow cytometry at 48 hours pi (Figure 4.2A, B). In contrast, the same neutralizing antibody blocked P. falciparum infection in primary human hepatocytes

105

(Figure 4.2C, D). Moreover, 1D6 did not alter parasite numbers when added 6 hours pi, suggesting that CD81 is critical only during parasite invasion. These data are in agreement with the original observation by Silvie et al., demonstrating that CD81 blocking significantly reduces invasion of primary human hepatocytes by P. falciparum sporozoites

[74]. Therefore, though invasion of HC-04 appears to be independent of CD81, we confirm that invasion of primary hepatocytes is CD81-dependent.

Figure 4.2 Influence of CD81 blocking by mAb 1D6 on P. falciparum hepatocyte infection. 1D6 or isotype were added to cultures at 10µg/ml prior to infection (-2 to 0 hrs), during invasion (0 to 6 hours) or after invasion (6 to 24 hours). Representative flow plots shown for (A) HC-04 48 hours postinfection and (B) number and percentage of GFP-positive events in duplicate. (C) Flow plots for donor 4051 96 hours postinfection and (D) graphs indicated the number and percentage of GFP-positive events in duplicate. Mean±SD shown on all graphs, **p<0.01, *p<0.05.

106

Humanized CSP-specific antibody 2A10 inhibits traversal and reduces the number of P. falciparum EEFs in human hepatocytes in vitro

The major surface protein of Plasmodium sporozoites, the circumsporozoite protein

(CSP), is a well-established target for humoral immune responses aimed at neutralization of sporozoite infectivity [268]. To determine the potential utility of our system for the screening of sera for the presence of sporozoite-specific antibodies we characterized the potency of a recently described humanized CSP-specific monoclonal antibody h2A10, produced in a vectored immunoprophylaxis (VIP)-vector transduced mouse [269] (Figure

4.3). We found that serial dilutions of mouse serum initially containing 1.93 mg/ml of human IgG inhibited parasite traversal in an antibody concentration-dependent manner

(Figure 4.3A). Moreover, h2A10 reduced the relative frequencies and total numbers of

GFP+PI- cells in both HC-04 and primary hepatocyte cultures (Figure 4.3B,C), although overall inhibition of infection was less prominent in primary cells. Even at a concentration of 3.86 μg/ml (1:500 dilution of h2A10 antibody-containing serum) infection was not completely abolished, suggesting that higher titers of sporozoite antigen-specific antibodies will be required to completely prevent infection. The intensity of GFP fluorescence in hepatocyte cultures infected along with h2A10 was not significantly different from cultures infected with control sera (Figure 4.3B,C). This suggests that parasites not inhibited by h2A10 during invasion develop normally in infected hepatocytes.

107

Figure 4.3 Influence of a humanized anti-CSP mAb 2A10 on P. falciparum traversal and invasion. Cells were infected in the presence of h2A10 or pre-2A10-AAV serum at varying dilutions. (A) Percentage of traversed cells measured by dextran uptake 6 hours postinfection. Representative flow plots for (B) HC-04 48 hours postinfection and (C) donor 4051 96 hours postinfection. Graphs indicated the number, percentage and geometric MFI of GFP- positive events in duplicate. Mean±SD shown on all graphs.

108

Discussion

After establishing a model of in vitro PfEEF detection and verifying the presence of parasites in both primary hepatocyte and HC-04 cell cultures, we designed several experiments to explore potential applications of our model for both basic and translational studies. Ligands on the human hepatocyte cell surface facilitating invasion by P. falciparum sporozoites are still poorly understood. We re-examined the necessity of CD81 expression for invasion by P. falciparum sporozoites. Previous studies determined that blocking CD81 during P. falciparum infection reduces EEF numbers in primary hepatocytes [74], however expression of CD81 on non-permissive cell types was insufficient to confer invasion [75]. We showed that endogenous surface expression of

CD81 (Figure 4.1A) did not correlate with P. falciparum EEF numbers in the five cell lines tested. Primary hepatocytes derived from 3 human donors were permissive for P. falciparum infection and had expression of CD81 on the cell surface (Figure 4.1A). HC-

04, the most permissive to P. falciparum infection among all cell lines tested, lacks CD81 and transient ectopic expression of CD81 did not alter susceptibility of HC-04 cells to invasion (Figure 4.1B, C). Taking advantage of the ability to quantitatively assess EEF development by flow cytometry, we re-examined the requirement for CD81 expression during P. falciparum sporozoite invasion (Figure 4.2). We found that the CD81-blocking monoclonal antibody 1D6 did not alter P. falciparum infection of HC-04 cells (Figure

4.2A,B). However, in agreement with the original finding by Silvie et al. [74], the same antibody decreased the PfEEF numbers in primary human hepatocyte cultures when used during sporozoite invasion (Figure 4.2C,D). The inability of CD81-specific antibody 1D6 to alter sporozoite invasion of CD81-negative HC-04 suggests that it has no unspecific

109 effect on sporozoites. In agreement, pre-incubation of primary hepatocytes with 1D6 followed by its removal prior to infection blocked sporozoite invasion. Additionally, no effect of 1D6 was observed when added six hours after infection in either HC-04 or primary hepatocyte cultures. Our findings suggest that different host cell surface receptors and/or distinct parasite invasion pathways can be utilized by P. falciparum sporozoites to establish liver stage infection. One possibility is that differences in heparan sulfate proteoglycans

(HSPG) between HC-04 and primary hepatocytes mediate CD81 dependence. It has been established that CSP binds to HSPGs and influences sporozoite invasion [62,65,270] but do so more prominently under dynamic flow conditions [66]. We propose that our experimental approach can facilitate the discovery of additional human host factors or further characterize the importance of HSPGs during P. falciparum sporozoite invasion.

Next we demonstrated the utility of these methods to quantify the effects of anti- sporozoite antibodies on traversal and invasion. Sterilizing immunity to the liver stage of

Plasmodium infection does not occur during natural infection [98]. It has been well established that antibodies directed against the sporozoite are capable to blocking parasite motility in vitro and in vivo and can consequently reduce parasite invasion into hepatocytes

[262,271,272]. The RTS,S vaccine is capable of inducing anti-CSP antibodies able to protect against P. falciparum infection in a dose dependent manner [246]. Inhibition of parasite motility as measured by traversal in vitro has been used to characterize humoral responses against the sporozoite [171]. However, to establish a correlation between the effects exerted by parasite-specific antibodies on traversal with effects on formation of

PfEEFs has required a humanized mouse model [171,246]. Using serum from a vectored immunoprophylaxis (VIP)-vector transduced mouse producing a humanized PfCSP-

110 specific monoclonal antibody [269], we demonstrate an inhibition of both traversal and infection by P. falciparum sporozoites in vitro (Figure 4.3). The variable and constant regions of antibodies have classically been considered as independent domains. This modular view of antibody functionality may be an oversimplification. Several studies have detailed alterations in the Fc portion that influence affinity and specificity [273–275].

Therefore, it is possible that alteration of the Fc portion of 2A10 to “humanize” the antibody changed the affinity or specificity of the molecule. Since our approach is not limited to a specific parasite antigen, it may be applied to screening of sera from human cohorts immunized with radiation attenuated (RAS), genetically attenuated (GAS) sporozoites, or sporozoites combined with chloroquine chemoprophylaxis (CPS) based protection [83,276]. In vitro methods enabling objective evaluation of anti-sporozoite humoral immune responses are particularly advantageous because they require considerably less material than humanized mouse models and can include multiple biological replicates. However, it should be stated that in vitro models of infection lack the complex hepatic architecture and cannot entirely recapitulate the natural infection.

Previously, detection of EEFs by flow cytometry has only been possible using rodent Plasmodium species [159,164,242,250]. Although the use of rodent parasites and mouse models of Plasmodium infection will continue to be a cornerstone of malaria research, advancements in methods for studying PfEEFs in vitro are required to strengthen our understanding of basic parasite biology and to provide objective and quantifiable protocols to inform translational research. HC-04 and primary hepatocytes constitute complementary in vitro models to study PfEEF formation. Among five cell lines tested,

HC-04 is able to support the greatest number of developing parasites. However, uninfected

111 cells in HC-04 cultures rapidly proliferate, complicating parasite detection after 72-96 hours pi. On the other hand, parasite traversal can be quantified in these cells early after infection and fluorescent parasites are clearly detectable by flow cytometry starting at 48 hours pi. Consequently, HC-04 is a suitable host cell to screen for the efficacy of interventions aimed at blocking the motility of sporozoites, such as anti-parasite antibodies, but is not fully suitable for study of late Pf liver stages.

P. falciparum line expressing GFP through the parasite’s lifecycle [170] was paramount in our ability to reproducibly detect and quantify PfEEFs by flow cytometry. The generation of a novel P. falciparum strain expressing a fluorophore brighter than GFP and/or use of a stronger liver stage promoter will facilitate flow cytometry-based detection and isolation of P. falciparum parasites in human hepatocytes even before 48 hours postinfection.

112

Chapter 5 : Future Directions

113

The ability of T cells to recognize peptides from CSP presented in the context of

MHC class I in vaccinated and naturally exposed individuals shows that immune responses to CSP can be primed and recalled [152]. Since we show that degradation of ectopically expressed CSP for presentation on MHC class I is primarily achieved through the proteasome, prediction of peptides generated from CSP by the proteasome could identify additional potentially presented epitopes. Methods exist for the in silico prediction of protein degradation by the proteasome [277,278] that can be validated by in vitro reconstitution of the proteasome [279,280]. Use of an in vitro proteasomal degradation allows for the generation of peptides that can be identified by mass spectrometry [281]. In this system, peptides generated by the proteasome can be identified independent of loading onto MHC class I. The ability to produce full-length recombinant CSP [282] can be used in conjunction with in vitro proteasomal degradation to identify peptides generated by CSP outside of the context of a specific HLA allele.

Use of a chimeric P. falciparum CSP has allowed us to monitor processing and presentation of ectopically expressed CSP on MHC class I in vitro. Using specific lysine mutations in the N-terminus of CSP we were able to demonstrate the importance of ubiquitination at these sites for presentation of CSP-derived peptides on MHC class I.

Individual lysine mutations reduced the susceptibility of transfected targets to be killed by

CD8+ T cells, but did not completely recapitulate the phenotype observed with targets transfected with CSP-Short. It would be beneficial to generate a chimeric CSP with mutations in all three residues concurrently and/or to mutate residues more proximal to region I to further characterize the contribution of N-terminal lysines to presentation of

CSP on MHC class I. The original mutation of lysine was achieved by substitution to

114 alanine. To address the importance of charge and potential structural changes on presentation, mutations could be done with substitutions of arginine instead. Presumably these N-terminal lysine residues have a functional and conserved role in parasite biology.

Detection of polymorphisms in P. falciparum CSP has historically focused on the C- terminus, particularly in Th2R and Th3R [23]. It would be interesting to investigate the population diversity within a single Plasmodium species at the N-terminus of CSP and the potential conservation-specific lysine residues. It is possible that aside from a functional role during sporozoite invasion into hepatocytes, shedding of the N-terminus of CSP also alters the antigenicity of CSP in favor of the parasite.

In support of this notion, we have been able to show the presence of predominantly

CSP-Short in both traversed and infected hepatocytes, suggesting their potential to be processed and presented by these cell populations during infection. Using a rodent parasite deficient in traversal (spect -/-) [68] but not infection in vitro, it was shown that activation of CD8+ T cells was abrogated, suggesting a contribution of antigen in traversed cells to T cell activation [283]. Ideally, populations of traversed/non-traversed and infected/non- infected cells could be isolated and assessed for their ability to activate and to be killed by

CD8+ T cells specific to CSP. These experiments are limited by the requirement of CD8+

T cells to recognize wild-type CSP epitopes and by the ability to acquire a sufficient number of effector hepatocytes. Cloned CD8+ T cells specific to a native epitope in CSP are well characterized in rodent parasites [121] but are difficult to generate in relation to P. falciparum [284]. Additionally, isolation and identification of traversed and infected cells is more efficient when using rodent parasites [159,163,242]. Therefore, initial studies on

115 the contribution of CSP presentation by traversed and infected cells could utilize an in vitro rodent model and could then be confirmed utilizing our in vitro system using P. falciparum.

The ability to separate out cells traversed or infected by P. falciparum sporozoites could also be utilized to study the impact on the host cell, similar to what has been done using rodent parasites [163]. This type of an experiment is particularly appealing for use with RNA-sequencing to quantify transcript abundance without a priori template selection and with the ability to sequence both host and parasite transcripts simultaneously [285].

However, our infection rates using P. falciparum in vitro are much lower than those reported for rodent parasites, thus limiting the potential amount of RNA obtainable to analysis. Low-input and single cell techniques have been developed for RNA-sequencing but introduce additional bias and cost [286,287]. These techniques could be employed in our in vitro model of infection and the isolation of P. falciparum EEFs. It may also be beneficial to explore the use of our gating strategy (FL1 vs. FL2) on P. falciparum-infected livers from humanized mice [85,172]. Humanized mice potentially have a higher efficiency of infection by P. falciparum sporozoites and could potentially generate more EEFs than are easily obtainable in vitro at this time.

It is unclear why the efficiency of infection (EEF per sporozoite) in vitro is relatively low when using P. falciparum sporozoites and primary human hepatocytes.

Several possibilities exist to explain this inefficiency, including improper activation of sporozoites, early abortion of parasites due to metabolic pressures, or innate immune activation of hepatocytes. Detection of live P. falciparum EEFs in vitro is an important ability, and an enhancement of this technique could aid in future experiments by increasing the amount of obtainable parasite material. We have not tested the influence of heparin

116 sulfates on sporozoite invasion, a possible explanation for the CD81-independent invasion seen with HC-04. Additionally, our experiments remove media from culture plates when adding sporozoites. Since conditioned medium, particularly from hepatocytes, can influence other cell types, it would be interesting to test the influence of hepatocyte- conditioned media on sporozoite infectivity [288,289].

Even at low efficiencies of infection we were able to demonstrate potential uses for in vitro detection of both parasite number and development simultaneously by flow cytometry that have advantages over single parameter methods of detection such as RT-

PCR or bioluminescence. Quantification of parasite traversal was also shown to be useful to examine the inhibitory ability of antibodies aimed at the surface of the sporozoite.

Detection of traversal is more rapid (2-6 hours postinfection) than detection of EEFs, and can be detected in a variety of cell lines. This method would allow for rapid screening of many monoclonal antibodies or sera and for the comparison of the potential influence of isotype on inhibitory activity [273–275]. However, it is important to correlate in vitro inhibition with in vivo protection. Determining a protective threshold for in vitro assays that could be used during a preclinical screen is necessary since our data show inhibition of traversal is not a bimodal phenotype and a single EEF can seed a blood stage infection.

Growth inhibition assays have been used to evaluate the activity of serum from vaccinated or naturally exposed individuals but a consensus on their relation to in vivo phenotypes is lacking [260,290].

The ability to detect P. falciparum EEFs by flow cytometry is a function of the background fluorescence of the host cell and the brightness of the expressed fluorophore over time. Autofluorescence from hepatocytes did not appear to change over the course of

117 infection, but the parasite-specific signal gradually increased after being obscured at early time points postinfection. Expression of GFP in the 3D7HT-GFP parasite is under control of the EF1α promoter which was selected for non-stage-specific expression and for strength of expression [170]. A new parasite could potentially utilize a stronger promoter that would be determined experimentally and/or the use of a brighter fluorophore [291]. Such a parasite could allow earlier detection of EEFs in vitro but it is likely that the same principles and gating strategies described here will apply. Consequently, we have demonstrated a significant proof of principle that will ideally be expanded upon to increase the potential ease and breadth of these techniques.

118

Chapter 6 : Materials and Methods

119

Human hepatocyte culture

HC-04 [169] was obtained from the ATCC (Manassas, VA, USA). HepG2 and

HepG2-CD81 [75] were kindly provided by Dr. I. Coppens (Johns Hopkins Bloomberg

School of Public Health). These cell lines were maintained in Iscove's Modified Dulbecco's

Medium (IMDM) supplemented with 2.5% fetal calf serum (FCS), 100 IU/ml penicillin,

100 μg/ml streptomycin and 2 mM L-glutamine. THLE-2 and THLE-3 cell lines [253] were kindly provided by Dr. C. Harris (NCI, Center for Cancer Research) and maintained using BEGM BulletKit medium (Lonza, Walkersville, MD) supplemented with 5 ng/ml

EGF, 70 ng/ml phosphoethanolamine and 10% FCS. All cell lines were routinely tested for the presence of mycoplasma [292]. Original culture containing mycoplasma were treated with Plasmocure at 50µg/ml (Invivo Gen, San Diego, USA) for a minimum of two weeks and tested until negative by PCR.

Cryopreserved human primary hepatocytes, hepatocyte thawing, plating and maintenance medium were obtained from Triangle Research Labs (TRL, North Carolina).

Primary human hepatocyte plating and maintenance media consisted of a base of William’s

E without the additional of phenol red. Human hepatocyte plating medium was supplemented with fetal bovine serum (FBS), GlutaMax, dexamethasone, penicillin- streptomycin and insulin. Hepatocyte maintenance medium was supplemented with

HEPES, GlutaMax, ITS+, dexamethasone and penicillin-streptomycin. Human hepatocyte thawing medium was based on William’s E and supplemented with FBS, HEPES,

GlutaMax, dexamethasone, penicillin-streptomycin, insulin, percoll and phenol red. Exact concentrations of the supplements for human primary hepatocytes media are proprietary

(TRL, North Carolina).

120

Hepatocytes were thawed in a 37°C water bath for 2 minutes in thawing medium, and spun at 100xg for 8 minutes. The resulting pellet was re-suspended in plating medium and seeded on collagen-coated wells or coverslips. Plating medium was replaced with maintenance medium supplemented with 10% FCS 6 hours after plating and replaced every

24 hours.

Collagen coating and hepatocyte plating densities

Plates were coated with rat tail collagen type I in PBS at a final concentration of

54.9μg/ml (BD Pharmingen, San Diego, USA) overnight at 4°C. Subsequently, plates were washed 3 times with PBS, allowed to dry for 10 minutes and washed with plating media before addition of cells.

Prior to additional of sporozoites, HC-04, HepG2 and HepG2-CD81 were seeded at densities of 400,000, 200,000 or 100,000 cells/well in 12, 24 or 48 well plates respectively to achieve confluence. THLE-2 and THLE3 were seeded at 200,000, 100,000 or 50,000 cells/well in 12, 24 or 48 well plates to achieve confluence. Primary hepatocytes were seeded at 300,000 hepatocytes per well in a 24 well plate 48 hours prior to addition of sporozoites. Prior to transfection of HC-04, cell were seeded at a density of 200,000 cells/well in a 12 well plate and allowed to adhere overnight. Cells were subconfluent at the time of transfection.

Infection with P. falciparum sporozoites

As mentioned previously, hepatoma cell lines were seeded 24 hours prior to infection and primary hepatocytes were seed 48 hours prior to infection, all to confluence.

All experiments using sporozoite utilized the GFP expressing P. falciparum stain 3D7HT-

GFP [170]. Seventeen days after feeding mosquitos with a blood meal containing

121 gametocytes, sporozoites were isolated and purified by density gradient as described previously [242,293].

Briefly, sporozoite isolation began with gross dissection of the mid-thorax from

200-300 mosquitoes. Tissue was collected and stored on ice in complete medium until dissection was complete. All dissected tissues were pooled and mechanically homogenized using a mortar and pestle. The resulting suspension was pipetted into 15ml tubes and spun at 500rpm for 5 minutes at 4°C. Supernatant was collected and the pellet was resuspended in complete medium and spun at 500rpm for 5 minutes at 4°C. The supernatants from both spins were combined and spun at 10,000rpm for 10 minutes at 4°C with the brake on. The supernatant was discarded and the pellet was suspended in 1ml of complete medium. This suspension was added to the top of an OptiPrep (Sigma-Aldrich, St. Louis, USA) density gradient (5.8ml medium + 1.19 OptiPrep, bottom = 5.2ml medium + 1.8ml OptiPrep) and spun at 10,000rpm for 10 minutes at 4°C without break. The interface was collected and resuspended in 10-12ml of medium. This suspension was spun at 10,000rpm for 10 minutes at 4°C with brake on. The supernatant was then removed and the pellet was resuspended in 1ml medium. Sporozoite yield was quantified using a chilled glass hemocytometer.

Sporozoites were then diluted to the necessary volume and added to cells. Plates were then spun at 1500rpm for 5 minutes without brake. Plates were moved to standard CO2 incubators at 37°C and were allowed to traverse and invade hepatocytes for 6 hours.

There was considerable variability in infectivity between sporozoite preparations; consequently, relevant comparisons are made only between sporozoites from the same preparations or are normalized where relevant.

122

Detection of hepatocyte traversal

In vitro detection of hepatocyte traversal by flow cytometry has been previously described [250]. 10,000 MW dextran-tetramethylrhodamine (Life Technologies, Grand

Island, NY) at a concentration of 0.2 mg/ml was added with sporozoites or in complete medium as mock infection. Infected and uninfected cells were incubated for 6 hours then washed three times with cold PBS, collected by trypsinization and resuspended in

PBS/0.1% BSA. Quantification of the percentage of cells retaining dextran was made using a using FACSCalibur and CellQuest acquisition software (Becton Dickinson, New Jersey,

USA). Data analyses were performed using FlowJo software (TreeStar Inc., Ashland

Oregon, USA). Uninfected cultures with dextran were used to establish gates and control for spontaneous dextran uptake by hepatocytes. Pre-incubation of sporozoites with cytochalasin D to inhibit motility or addition of material from mosquito salivary glands were used to establish specificity.

Detection of EEFs by flow cytometry

Individual wells were washed three times with PBS and hepatocytes were trypsinized. Pellets were resuspended in PBS/0.1% BSA and analyzed using a Becton

Dickinson LSR II or FACSCalibur (Becton Dickinson, New Jersey, USA). Propidium iodide (PI) was added prior to acquisition to allow for exclusion of dead cells by fluorescence in FL2 (585/42). GFP-positive PI-negative events were identified using a ratio of the FL1 (530/30) and FL2 (585/42) fluorescence channels. Data analyses were performed using FlowJo software (TreeStar Inc., Ashland Oregon, USA).

123

Quantification of parasite development by RT- PCR

Cells were trypsanized and washed in PBS. Pellets were stored at -20°C Hepatocyte pellets were lysed using the Power SYBR Green Cells-to-CT kit (Ambion, Grand Island,

NY) at the density of 2000 cells/μl lysis buffer. Lysis was stopped with a proportional volume of 10x Stop Solution. Reverse transcription was performed using 22.5 μl of cell lysate in a total volume of 50 μl. Polymerase chain reaction was performed using 4μl of cDNA template per reaction. Amplification used the following primer pairs: Human

GAPDH 5′-gcaaattccatggcaccgt-3′/5′-tcgccccacttgattttgg-3′ P. falciparum 18S 5′- tcagataccgtcgtaatctta-3′/5'-aactttctcgcttgcgcgaa-3′ [294]. Samples were amplified in triplicate using an Applied Biosystems StepOnePlus. SYBR green was used for detection and quantification of double stranded DNA. Thermal cycling proceeded for 2 minutes at

50°C, 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 1 minute at

60°C. The copy number of Pf18S in each reaction was calculated using a standard curve generated from serial dilution of a plasmid template containing the Pf18S gene. Each sample was normalized to their own GAPDH CT values (arbitrary units).

Immunofluorescence of EEFs in HC-04 and primary human hepatocytes

HC-04 cells and primary hepatocytes were sorted based on GFP-positivity and PI- negativity 96 hours pi directly into 4% paraformaldehyde using a Beckman Coulter MoFlo

Cell Sorter (Johns Hopkins Bloomberg School of Public Health, Flow Cytometry and Cell

Sorting Core Facility). These fixed cells were cytospun at 1500 rpm for 5 minutes onto cytoslides (Thermo Scientific, Astmoor, UK). Subsequently, cells were blocked and

124 permeabilized in PBS containing 10% goat serum, 1% BSA, and 0.1% Triton X-100, and then stained with anti-PfHsp70 (cl. 4C9, F. Zavala, JHU) [295]. Secondary anti-mouse

IgG-Alexa Fluor 594 (Molecular Probes, Grand Island, NY) was used to visualize Hsp70 staining. ProLong Gold Antifade containing DAPI (Molecular Probes, Eugene, OR) was used to mount slides. Slides were viewed on a widefield Nikon 90i microscope. Data were acquired and processed with Volocity software (Perkin Elmer, Waltham, MA).

Primary hepatocytes were also grown on the collagen-coated glass coverslips to directly visualize parasites without sorting. Hepatocytes were infected on coverslips in the same manner as previously described. Cells were, fixed and stained with an anti-PfHsp70 antibody at 96 hours pi as described. Parasite size and distance from host nucleus were quantified using Volocity software. Parasite margins were delineated by PfHsp70-specific fluorescence signal for surface area and longest axis calculations. To determine distance between the parasite and nearest nucleus, the edge of PfHsp70 signal to the edge of the nearest hepatocyte nucleus was measured.

CD81 detection and surface neutralization

Hepatocytes were trypsinized and washed. Single cell suspensions were stained on ice with either anti-CD81 (clone JS-81, BD Pharmingen, San Diego, CA) or IgG1 isotype control antibody. Antibodies were directly conjugated to allophycocyanin (APC) for detection. Functional grade (azide-free) anti-CD81 (cl.1D6) (Abcam, Cambridge, MA) mouse monoclonal or a functional grade IgG1 isotype control antibody (eBioscience, San

Diego, CA) were used in CD81 blocking experiments in vitro. Both antibodies were added in three conditions; 2 hours prior to infection and washed away, added with sporozoites or added 6 hours after addition of sporozoites.

125

Evaluation of the efficacy of mouse serum containing humanized anti-CSP mAb 2A10 on parasite motility

Total mouse serum containing a humanized version of a CSP-specific monoclonal antibody 2A10 [150,296] (h2A10) was kindly provided by Dr. Gary Ketner and Dr. Cailin

Deal (Johns Hopkins Bloomberg School of Public Health) [269]. As a control, serum was collected from the same mouse prior to intra-muscular administration of 1x1011 genome copies of AAV-expressing humanized 2A10 and 11 weeks post AAV administration. At

11 weeks post AAV administration 1.93 mg/ml human IgG was present in the serum. In both experiments measuring traversal percentages and infection efficiencies sera was added at the same time as sporozoites and allowed to incubate for 10min at room temperature before the plate was spun.

Generation and maintenance of CTLs

Leukocyte enriched whole blood was purchased from the New York Blood Center

(New York Blood Center, NY). Red blood cells were removed by Ficoll gradient.

Peripheral blood mononuclear cells (PBMCs) were stained for surface HLA-A2. Donors that stained positive for HLA-A2 were then stained with a CD8-specific antibody (Becton

Dickinson, NJ, USA) and HLA A2-pentamers containing GLCTLVAML or GILGFVFTL peptides (ProImmune Inc., FL, USA). Cultures that contained CD8+ T cells labeled with pentamers were expanded on PHA-activated, irradiated allogenic feeders. After expansion pentamer-positive CD8-positive T-cells were isolated by flow cytometry and further expanded. Cultures were propagated in RPMI containing 10% FCS, 100 IU/ml penicillin,

126

100 μg/ml streptomycin, 2 mM L-glutamine and 100 units/ml human recombinant IL-2

(PeproTech, NJ, USA).

Construction of CSP containing mammalian expression plasmids

Mammalian expression vectors were generated using the pVITRO2-neo-mcs

(Invivo Gen, CA, USA) backbone. The plasmid allows for simultaneous expression from two multiple cloning sites using the human ferritin heavy and light promoters without the inclusion of the 5’ iron-responsive element (IRE). Cloning of P. falciparum 3D7 CSP sequences and murine CD8 alpha chain were described previously [242]. Modifications of the native CSP sequence, such as lysine to alanine point mutations and generation of C- terminal chimeric sequences were performed using the Phusion Site-Directed Mutagenesis

Kit (Thermo Scientific, PA, USA) in a pJET1.2 plasmid containing the full coding sequence of CSP (Fermentas, MA, USA). CSP was PCR amplified from a pJET1.2 plasmid template and the PCR product was digested and ligated into pVITRO2-neo-mcs containing the murine CD8 alpha chain. Amplification of CSP-Full omitted the first 16 amino acids that constitute the parasite signal sequence and used forward primer 5’- tataggatccatggaggccttattccaggaata-3’ and reverse primer 5’-attagctagcctgatgaggt-3’. CSP-

Short was amplified with the following forward primer 5’-tataggatccatggcggatggtaatcc-3’ and the same reverse primer as CSP-Full. Intermediate N-terminal truncation mutants were generated using the forward primers 5’-tataggatccatgcaggaaaattggtatagtct-3’ and 5’- tataggatccatgaatagtagatcacttggaga-3. All PCR amplified CSP products were digested with

BamHI/NheI and ligated into pVITRO2-neo-mcs containing the murine CD8 alpha chain digested with BglII/NheI. All plasmid were produced in Dh5α.

127

Transfection of hepatocytes and purification by MACS

24 hours prior to transfection, HC-04 cells were seeded in 12-well plates to reach

80% confluence at the time of transfection. Each well was transfected with 600 ng of DNA using Lipofectamine LTX with Plus Reagent (Life Technologies, NY, USA). Five hours after transfection cells were extensively washed and maintained in complete medium. 24 hours after transfection cultures were trypsinized, washed and labeled with Ly-2

MicroBeads (Miltenya Biotec Inc., CA, USA). To magnetically purify transfected cells, labeled cells were loaded onto a MACS Large Cell Separation Column (Miltenya Biotec

Inc., CA, USA) and immobilized in columns. Columns were washed and retained cells were mechanically eluted.

Detection of CSP by western blot

Native CSP or CSP generated from mammalian expression constructs was detected by western blot using the mouse monoclonal antibody 2A10 [150,296] and an HRP conjugated secondary (GE Healthcare, UK) for visualization by chemiluminescence. Cells were lysed using 1x RIPA buffer (Cell Signaling Technology, MA, USA) and one volume

2x Laemmli sample buffer was added. Samples were sonicated for 1 minute and then boiled for 10 minutes. Samples were resolved on a 7.5% TRIS-HCL, 1.0MM gel (Bio-Rad, CA,

USA). Protein was transferred to PVDF membrane using the Trans-Blot TURBO system

(Bio-Rad, CA, USA) for 10 minutes at a constant 2.5A up to 25V. Membranes were blocked with 0.1% milk/PBS for one hour. Subsequently, membranes were probed overnight at 4°C with 1 µg/ml 2A10 [150,296]. Quantification of band intensity was done using ImageJ software [297].

128

Detection of ectopically expressed CSP

Transfected HC-04 were fixed with 4% paraformaldehyde 24 hours post- transfection. Cells were blocked and permeabilized in PBS containing 10% goat serum,

1% BSA, and 0.1% Triton X-100 and subsequently stained with 2A10 [150] and secondary anti-mouse IgG-Alexa Fluor 594 (Molecular Probes, Grand Island, NY). Slides were mounted with ProLong Gold Antifade (Molecular Probes, Eugene, OR) and imaged using a widefield Nikon 90i microscope. Data were acquired and processed with Volocity (Perkin

Elmer, Waltham, MA).

T-cell activation assay: IFNγ release

CTL activation was measured by the ability of CTLs to produce IFNγ after co- incubation with MACS purified HC-04 targets. Effectors and targets were incubated at a

1:1 ratio in the presence of 1 µl/ml of GolgiPlug (Becton Dickinson, NJ, USA) for eight hours. Cells were collected, washed and stained with anti-CD8-FITC (Becton Dickinson,

NJ, USA) for 30 minutes on ice. Cells were subsequently washed, incubated in

Cytofix/CytoPerm (Becton Dickinson, NJ, USA) for 30 minutes on ice and washed twice with Perm/wash buffer (Becton Dickinson, NJ, USA). Production of IFNγ by CD8 T-cells was detected by flow cytometry (FACS Calibur, Becton Dickinson, NJ, USA) using anti-

IFNγ-APC antibody (Becton Dickinson, NJ, USA).

Cytotoxicity lactate dehydrogenase assay

Cytotoxicity and cell lysis were measured by quantifying the release of enzymatically active lactate dehydrogenase (LDH) following addition of a cytotoxic agent.

Measurements were made using a LDH detection kit (Roche Diagnostics, IN, USA)

129 according to manufacturer’s instructions and read at 492nm in an ELISA plate reader

(Thermo Scientific, Waltham, MA). A phenol red free DMEM supplemented with BSA to

1% was used for the reaction (Life Technologies, NY, USA). Total LDH values for each target were calculated in triplicate following TRITON-X 100 target lysis. Cytotoxicity was calculated in triplicates using the formula (treatment LDH – target spontaneous LDH – effector spontaneous LDH)/(total target LDH – target spontaneous LDH)*100. The reaction was allowed to progress for 30 minutes.

For evaluation of cytotoxicity mediated by CD8 T-cells, 100µl containing 10,000 transfected HC-04 cells purified by MACS were used as targets in each well of a V-bottom

96-well plate (Corning Incorporated, MA, USA). T-cells were added at various ratios in

100 µl per well. Cells were co-incubated for 8 hours and 100 µl was removed per well to measure LDH values in a clear flat bottom 96-well plate (Corning Incorporated, MA,

USA).

Chemical inhibitor treatments

Chemical inhibition of protein degradation pathways was performed 24 hours after transfection. Cells were treated in complete medium with 10 µM clasto-Lactacystin β- lactone (Sigma-Aldrich, MO, USA), 2 µM spautin (Sigma-Aldrich, MO, USA) or 50 µM chloroquine (Sigma-Aldrich, MO, USA) to inhibit the proteasome, autophagy, or lysosomal degradation respectively.

Immunoprecipitation of HA-ubiquitin complexes

Transfection was done as described above with CSP and a separate plasmid containing HA-tagged ubiquitin. Ubiquitin plasmids based on pRK5-HA-Ubiquitin was a

130 gift from Ted Dawson (Addgene plasmids # 17608, 17605, 17606, 17603) [233].

Transfected cells were lysed in CelLytic M Cell Lysis Reagent (Sigma-Aldrich, MO, USA) for 15 minutes on ice and spun to remove cellular debris. A portion of sample was removed as a loading control and the remaining supernatants were placed in a new pre-chilled tube.

Samples were incubated with anti-HA-Agarose (Sigma-Aldrich, MO, USA) at 4ºC overnight with rotation. Agarose was washed six times in 1xIP Buffer (Sigma-Aldrich,

MO, USA), with a final wash in .1xIP Buffer and re-suspended in 2x Laemmli sample buffer (Bio-Rad, CA, USA). Samples were boiled for 10 minutes and the supernatant was used for western blot as described. Ubiquitin chains were detected using an anti-HA antibody (Life Technologies, NY, USA).

Peptide synthesis and target pulsing

Generation of synthetic peptides was done by The Synthesis & Sequencing Facility,

Johns Hopkins University. Peptides were reconstituted in DMSO at 10^-2 M. For cytotoxicity experiments targets were pulsed with peptide in complete medium at 37°C for one hour and washed prior to addition to effectors.

Statistical analysis

Comparisons of multiple groups were performed with a one way ANOVA. A post- hoc Tukey’s test was used for comparisons between individual groups. Adjusted p-values are shown where relevant (*p<0.05, **p<0.01, ***p<0.001).

When multiple factors were tested a two way ANOVA was used. Bonfferoni corrected post-tests were used to compare individual groups (p-values indicated).

131

References

1. Sidjanski S, Vanderberg JP (1997) Delayed Migration of Plasmodium Sporozoites from the Mosquito Bite Site to the Blood. Am J Trop Med Hyg 57: 426–429.

2. Yamauchi Lucy M. LM, Coppi A, Snounou G, Sinnis P (2007) Plasmodium sporozoites trickle out of the injection site. Cell Microbiol 9: 1215–1222. doi:10.1111/j.1462- 5822.2006.00861.x.

3. Vaughan AM, Aly ASI, Kappe SHI (2008) Malaria Parasite Pre-Erythrocytic Stage Infection: Gliding and Hiding. Cell Host Microbe 4: 209–218. doi:http://dx.doi.org/10.1016/j.chom.2008.08.010.

4. Boddey JA, Cowman AF (2013) Plasmodium Nesting: Remaking the Erythrocyte from the Inside Out. Annu Rev Microbiol 67: 243–269. doi:10.1146/annurev-micro-092412- 155730.

5. Prudêncio M, Rodriguez A, Mota MM (2006) The silent path to thousands of merozoites: the Plasmodium liver stage. Nat Rev Microbiol 4: 849–856. doi:10.1038/nrmicro1529.

6. Sturm A, Amino R, van de Sand C, Regen T, Retzlaff S, et al. (2006) Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids. Science 313: 1287– 1290. doi:10.1126/science.1129720.

7. The malaria parasite life cycle (n.d.). Available: http://www.cdc.gov/malaria/about/biology/. Accessed 3 January 2015.

8. Jongwutiwes S, Putaporntip C, Iwasaki T, Sata T, Kanbara H (2004) Naturally acquired Plasmodium knowlesi malaria in human, Thailand. Emerg Infect Dis 10: 2211–2213. doi:10.3201/eid1012.040293.

9. Cox-Singh J, Davis TME, Lee K-S, Shamsul SSG, Matusop A, et al. (2008) Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening. Clin Infect Dis 46: 165–171. doi:10.1086/524888.

10. World Health Organization (2013) World Malaria Report 2013. 284 p. doi:ISBN: 9 789241 56469 4.

11. Gallup JL, Sachs JD (2001) The economic burden of malaria . Am J Trop Med Hyg 64 : 85– 96.

12. Bonds MH, Keenan DC, Rohani P, Sachs JD (2010) Poverty trap formed by the ecology of infectious diseases. Proc Biol Sci 277: 1185–1192. doi:10.1098/rspb.2009.1778.

132

13. Coluzzi M (1999) The clay feet of the malaria giant and its African roots: Hypotheses and inferences about origin, spread and control of Plasmodium falciparum. Parassitologia 41: 277–283.

14. Miller LH, Baruch DI, Marsh K, Doumbo OK (2002) The pathogenic basis of malaria. Nature 415: 673–679. doi:10.1038/415673a.

15. Marsh K, Forster D, Waruiru C, Mwangi I, Winstanley M, et al. (1995) Indicators of life- threatening malaria in African children. N Engl J Med 332: 1399–1404. doi:10.1056/NEJM199505253322102.

16. Taylor TE, Borgstein A, Molyneux ME (1993) Acid-base status in paediatric Plasmodium falciparum malaria. Q J Med 86: 99–109.

17. English M, Waruiru C, Amukoye E, Murphy S, Crawley J, et al. (1996) Deep breathing in children with severe malaria: Indicator of metabolic acidosis and poor outcome. Am J Trop Med Hyg 55: 521–524.

18. Maier AG, Cooke BM, Cowman AF, Tilley L (2009) Malaria parasite proteins that remodel the host erythrocyte. Nat Rev Microbiol 7: 341–354. doi:10.1038/nrmicro2110.

19. Buffet PA, Safeukui I, Deplaine G, Brousse V, Prendki V, et al. (2011) The pathogenesis of Plasmodium falciparum malaria in humans: Insights from splenic physiology. Blood 117: 381–392. doi:10.1182/blood-2010-04-202911.

20. Takala SL, Smith DL, Thera MA, Coulibaly D, Doumbo OK, et al. (2007) Short report: Rare Plasmodium falciparum merozoite surface protein 1 19-KDA (MSP-119) haplotypes identified in Mali using high-throughput genotyping methods. Am J Trop Med Hyg 76: 855–859.

21. Escalante AA, Lal AA, Ayala FJ (1998) Genetic polymorphism and natural selection in the malaria parasite Plasmodium falciparum. Genetics 149: 189–202.

22. Branch OH, Udhayakumar V, Hightower AW, Oloo AJ, Hawley WA, et al. (1998) A longitudinal investigation of IgG and IgM antibody responses to the merozoite surface protein-1 19-kilodalton domain of Plasmodium falciparum in pregnant women and infants: Associations with febrile illness, parasitemia, and anemia. Am J Trop Med Hyg 58: 211–219.

23. Gandhi K, Thera MA, Coulibaly D, Traoré K, Guindo AB, et al. (2014) Variation in the circumsporozoite protein of Plasmodium falciparum: Vaccine development implications. PLoS One 9: 1–9. doi:10.1371/journal.pone.0101783.

24. Laurens MB, Thera MA, Coulibaly D, Ouattara A, Kone AK, et al. (2013) Extended safety, immunogenicity and efficacy of a blood-stage malaria vaccine in Malian children: 24- Month follow-up of a randomized, double-blinded phase 2 trial. PLoS One 8. doi:10.1371/journal.pone.0079323.

133

25. Tembo DL, Nyoni B, Murikoli R V, Mukaka M, Milner DA, et al. (2014) Differential PfEMP1 Expression Is Associated with Cerebral Malaria Pathology. 10. doi:10.1371/journal.ppat.1004537.

26. Swamy L, Amulic B, Deitsch KW (2011) Plasmodium falciparum var gene silencing is determined by cis DNA elements that form stable and heritable interactions. Eukaryot Cell 10: 530–539. doi:10.1128/EC.00329-10.

27. Diagne N, Rogier C, Cisse B, Trape JF (1997) Incidence of clinical malaria in pregnant women exposed to intense perennial transmission. Trans R Soc Trop Med Hyg 91: 166– 170. doi:10.1016/S0035-9203(97)90209-1.

28. Mohanty S, Taylor TE, Kampondeni S, Potchen MJ, Panda P, et al. (2014) Magnetic resonance imaging during life: the key to unlock cerebral malaria pathogenesis? Malar J 13: 276. doi:10.1186/1475-2875-13-276.

29. Ampomah P, Stevenson L, Ofori MF, Barfod L, Hviid L (2014) Kinetics of B Cell Responses to Plasmodium falciparum Erythrocyte Membrane Protein 1 in Ghanaian Women Naturally Exposed to Malaria Parasites. J Immunol 192: 5236–5244. doi:10.4049/jimmunol.1400325.

30. Liu W, Li Y, Learn GH, Rudicell RS, Robertson JD, et al. (2010) Origin of the human malaria parasite Plasmodium falciparum in gorillas. Nature 467: 420–425. doi:10.1038/nature09442.

31. Prugnolle F, Durand P, Ollomo B, Duval L, Ariey F, et al. (2011) A fresh look at the origin of Plasmodium falciparum, the most malignant malaria agent. PLoS Pathog 7. doi:10.1371/journal.ppat.1001283.

32. Mu J, Duan J, Makova KD, Joy DA, Huynh CQ, et al. (2002) Chromosome-wide SNPs reveal an ancient origin for Plasmodium falciparum. Nature 418: 323–326. doi:10.1038/nature01168.

33. Hedrick PW (2011) Population genetics of malaria resistance in humans. Heredity (Edinb) 107: 283–304. doi:10.1038/hdy.2011.16.

34. Pappas G, Kiriaze IJ, Falagas ME (2008) Insights into infectious disease in the era of Hippocrates. Int J Infect Dis 12: 347–350. doi:10.1016/j.ijid.2007.11.003.

35. West JB (2014) Galen and the beginnings of Western physiology. Am J Physiol Lung Cell Mol Physiol 0623: 121–128. doi:10.1152/ajplung.00123.2014.

36. Hawgood BJ (2003) Francesco Redi (1626-1697): Tuscan philosopher, physician and poet. J Med Biogr 11: 28–34.

37. La Berge A (1999) The history of science and the history of microscopy. Perspect Sci 7: 111–142.

134

38. Pasteur L (1880) On the extension of the germ theory to the etiology of certain common diseases. Comptes rendus Acad des Sci.

39. Laveran A (1880) Note sur un nouveau parasite trouve dans le sang de plusieurs malades atteints de fievre palustre. Bull Acad Medicale 9: 1235–1236.

40. MacCallum WG (1897) On the Flagellated form of the malarial parasite. Lancet 2: 1240– 1241.

41. Ross R, Smyth J (1897) On some peculiar pigmented cells found in two mosquitos fed on malarial blood. Wilderness Environ Med 10: 29–33.

42. Cox FE (2010) History of the discovery of the malaria parasites and their vectors. Parasit Vectors 3: 5. doi:10.1186/1756-3305-3-5.

43. Garnham P (1988) History of discoveries of malaria parasites and of their life cycles. Hist Philos Life Sci 10: 93–108.

44. Grassi B, Bignami A BG (1899) Ulteriore ricerche sul ciclo dei parassiti malarici umani sul corpo del zanzarone. Atti R Accad Lincei 8: 21–28.

45. Shortt HE, Fairley NH, Covell G, Shute PG, Garnham PCC (1949) The pre-erythrocytic stage of Plasmodium falciparum. Trans R Soc Trop Med Hyg 44: 405–419.

46. Grassi GB (1900) Studi di uno Zoologo sulla Malaria. Mem Rend Accad Lincei 53: 299– 502.

47. Schaudinn F (1903) Studien über krankheitserregende Protozoen. II. Plasmodium vivax (Grassi & Feletti), der Erreger des Tertianfiebers beim Menschen. Arb Kaiserl Gesundheits 19: 169–250.

48. Paraense W (2004) A fragment of malaria history. Mem Inst Oswaldo Cruz 99: 439–442.

49. Huff C, Coulston F (1944) The Development of Plasmodium gallinaceum from Sporozoite to Etythrocytic Trophozoite. J Infect Dis 75: 231–249.

50. Raffaele G (1934) Un ceppo italiano di Plasmodium elongatum. Riv Malariol 15: 3–11.

51. Garnham P (1948) The developmental cycle of Hepatocystes (Plasmodium) kochi in the monkey host. Trans R Soc Trop Med Hyg 41: 601–616. doi:10.1016/S0035- 9203(48)90418-0.

52. Shortt HE, Garnham PCC (1948) Pre-erythrocytic stage in mammalian malaria parasites. Nature 161: 126.

53. Shortt HE, Garnham PCC, Covell G, Shute PG (1948) Pre-Erythrocytic Stage of Human Malaria, Plasmodium Vivax. Br Med J 1: 547.

135

54. Herrera S, Perlaza BL, Bonelo A, Arévalo-Herrera M (2002) Aotus monkeys: Their great value for anti-malaria vaccines and drug testing. Int J Parasitol 32: 1625–1635. doi:10.1016/S0020-7519(02)00191-1.

55. Mazier D, Beaudoin RL, Mellouk S, Druilhe P, Texier B, et al. (1985) Complete development of hepatic stages of Plasmodium falciparum in vitro. Science 227: 440–442. doi:10.1126/science.3880923.

56. Hayton K, Gaur D, Liu A, Takahashi J, Henschen B, et al. (2008) Erythrocyte Binding Protein PfRH5 Polymorphisms Determine Species-Specific Pathways of Plasmodium falciparum Invasion. Cell Host Microbe 4: 40–51. doi:10.1016/j.chom.2008.06.001.

57. Yoeli M, Most H (1965) Pre-Erythrocytic Development Of Plasmodium berghei. Nature 205: 715–716.

58. Vincke I, Lips M (1948) Un nouveau Plasmodium d’un rongeur sauvage du Congo, Plasmodium berghei n.sp. Ann Soc Belg Med Trop (1920): 97–104.

59. Amino R, Giovannini D, Thiberge S, Gueirard P, Boisson B, et al. (2008) Host Cell Traversal Is Important for Progression of the Malaria Parasite through the Dermis to the Liver. Cell Host Microbe 3: 88–96. doi:http://dx.doi.org/10.1016/j.chom.2007.12.007.

60. Mota MM, Pradel G, Vanderberg JP, Hafalla JCR, Frevert U, et al. (2001) Migration of Plasmodium Sporozoites Through Cells Before Infection. Science (80- ) 291: 141–144. doi:10.1126/science.291.5501.141.

61. Vanderberg JP, Chew S, Stewart MJ (1990) Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis. J Protozool 37: 528–536.

62. Frevert U, Sinnis P, Cerami C, Shreffler W, Takacs B, et al. (1993) Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes. J Exp Med 177: 1287–1298. doi:10.1084/jem.177.5.1287.

63. Cerami C, Frevert U, Sinnis P, Takacs B, Clavijo P, et al. (1992) The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites. Cell 70: 1021–1033. doi:10.1016/0092- 8674(92)90251-7.

64. Coppi A, Tewari R, Bishop JR, Bennett BL, Lawrence R, et al. (2007) Heparan Sulfate Proteoglycans Provide a Signal to Plasmodium Sporozoites to Stop Migrating and Productively Invade Host Cells. Cell Host Microbe 2: 316–327. doi:10.1016/j.chom.2007.10.002.

65. Frevert U, Sinnis P, Esko JD, Nussenzweig V (1996) Cell surface glycosaminoglycans are not obligatory for Plasmodium berghei sporozoite invasion in vitro. Mol Biochem Parasitol 76: 257–266. doi:http://dx.doi.org/10.1016/0166-6851(95)02563-4.

136

66. Pinzon-Ortiz C, Friedman J, Esko J, Sinnis P (2001) The Binding of the Circumsporozoite Protein to Cell Surface Heparan Sulfate Proteoglycans is Required for Plasmodium Sporozoite Attachment to Target Cells. J Biol Chem 276: 26784–26791. doi:10.1074/jbc.M104038200.

67. Ishino T, Chinzei Y, Yuda M (2005) A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection. Cell Microbiol 7: 199–208. doi:10.1111/j.1462-5822.2004.00447.x.

68. Ishino T, Yano K, Chinzei Y, Yuda M (2004) Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer. PLoS Biol 2: E4. doi:10.1371/journal.pbio.0020004.

69. Frevert U, Usynin I, Baer K, Klotz C (2006) Nomadic or sessile: Can Kupffer cells function as portals for malaria sporozoites to the liver? Cell Microbiol 8: 1537–1546. doi:10.1111/j.1462-5822.2006.00777.x.

70. Kariu T, Ishino T, Yano K, Chinzei Y, Yuda M (2006) CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts. Mol Microbiol 59: 1369–1379. doi:10.1111/j.1365-2958.2005.05024.x.

71. Tavares J, Formaglio P, Thiberge S, Mordelet E, Van Rooijen N, et al. (2013) Role of host cell traversal by the malaria sporozoite during liver infection. J Exp Med 210: 905–915. doi:10.1084/jem.20121130.

72. Tavares J, Formaglio P, Medvinsky A, Ménard R, Amino R (2013) Imaging Sporozoite Cell Traversal in the Liver of Mice. In: Ménard R, editor. Malaria SE - 28. Methods in Molecular Biology. Humana Press, Vol. 923. pp. 401–410. doi:10.1007/978-1-62703-026- 7_28.

73. Frevert U, Engelmann S, Zougbédé S, Stange J, Ng B, et al. (2005) Intravital Observation of Plasmodium berghei Sporozoite Infection of the Liver. PLoS Biol 3: e192. doi:10.1371/journal.pbio.0030192.

74. Silvie O, Rubinstein E, Franetich J-F, Prenant M, Belnoue E, et al. (2003) Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity. Nat Med 9: 93–96. doi:10.1038/nm808.

75. Silvie O, Greco C, Franetich J-F, Dubart-Kupperschmitt A, Hannoun L, et al. (2006) Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species. Cell Microbiol 8: 1134–1146. doi:10.1111/j.1462-5822.2006.00697.x.

76. Sincock PM, Mayrhofer G, Ashman LK (1997) Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: comparison with CD9, CD63, and alpha5beta1 integrin. J Histochem Cytochem 45: 515–525. doi:10.1177/002215549704500404.

137

77. Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, et al. (1998) Binding of hepatitis C virus to CD81. Science 282: 938–941. doi:10.1126/science.282.5390.938.

78. Rodrigues CD, Hannus M, Prudêncio M, Martin C, Gonçalves LA, et al. (2008) Host scavenger receptor SR-BI plays a dual role in the establishment of malaria parasite liver infection. Cell Host Microbe 4: 271–282. doi:10.1016/j.chom.2008.07.012.

79. Foquet L, Hermsen CC, Verhoye L, van Gemert G-J, Cortese R, et al. (2015) Anti-CD81 but not anti-SR-BI blocks Plasmodium falciparum liver infection in a humanized mouse model. J Antimicrob Chemother: 4–7. doi:10.1093/jac/dkv019.

80. Bano N, Romano JD, Jayabalasingham B, Coppens I (2007) Cellular interactions of Plasmodium liver stage with its host mammalian cell. Int J Parasitol 37: 1329–1341. doi:10.1016/j.ijpara.2007.04.005.

81. Hollingdale MR, Leland P, Schwartz AL (1983) In vitro cultivation of the exoerythrocytic stage of Plasmodium berghei in a hepatoma cell line. Am J Trop Med Hyg 32: 682–684.

82. Mota MM, Rodriguez A (2000) Plasmodium yoelii: Efficient in Vitro Invasion and Complete Development of Sporozoites in Mouse Hepatic Cell Lines. Exp Parasitol 96: 257–259. doi:http://dx.doi.org/10.1006/expr.2000.4570.

83. Roestenberg M, McCall M, Hopman J, Wiersma J, Luty AJF, et al. (2009) Protection against a Malaria Challenge by Sporozoite Inoculation. N Engl J Med 361: 468–477. doi:10.1056/NEJMoa0805832.

84. Rennenberg A, Lehmann C, Heitmann A, Witt T, Hansen G, et al. (2010) Exoerythrocytic Plasmodium parasites secrete a cysteine protease inhibitor involved in sporozoite invasion and capable of blocking cell death of host hepatocytes. PLoS Pathog 6: e1000825. doi:10.1371/journal.ppat.1000825.

85. Vaughan AM, Mikolajczak SA, Wilson EM, Grompe M, Kaushansky A, et al. (2012) Complete Plasmodium falciparum liver-stage development in liver-chimeric mice. 122. doi:10.1172/JCI62684.3618.

86. Baer K, Klotz C, Kappe SHI, Schnieder T, Frevert U (2007) Release of hepatic Plasmodium yoelii merozoites into the pulmonary microvasculature. PLoS Pathog 3: 1651–1668. doi:10.1371/journal.ppat.0030171.

87. Fenner F (1980) The global eradication of smallpox. Med J Aust 1: 455–456. doi:10.1016/0196-6553(82)90003-7.

88. Gross CP, Sepkowitz KA (1998) The myth of the medical breakthrough: smallpox, vaccination, and Jenner reconsidered. Int J Infect Dis 3: 54–60. doi:10.1016/S1201- 9712(98)90096-0.

138

89. Riedel S (2005) Edward Jenner and the history of smallpox and vaccination. Proc (Bayl Univ Med Cent) 18: 21–25.

90. Fulginiti VA, Papier A, Lane JM, Neff JM, Henderson DA (2003) Smallpox vaccination: a review, part I. Background, vaccination technique, normal vaccination and revaccination, and expected normal reactions. Clin Infect Dis 37: 241–250. doi:10.1086/375824.

91. Henderson DA, Klepac P (2013) Lessons from the eradication of smallpox: an interview with D. A. Henderson. Philos Trans R Soc Lond B Biol Sci 368: 20130113. doi:10.1098/rstb.2013.0113.

92. Nussenzweig RS, Vanderberg J, Most H, Orton C (1967) Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. Nature 216: 160–162. doi:10.1038/216160a0.

93. Vanderberg J, Nussenzweig RS MH (1970) Protective immunity produced by the bite of x- irradiated mosquitoes infected with Plasmodium berghei. J Parasitol 56: 350–351.

94. Clyde DF, Most H, McCarthy VC, Vanderberg JP (1973) Immunization of man against sporozite-induced falciparum malaria. 169-177 p. doi:10.1097/00000441-197309000- 00002.

95. McCarthy VC, Clyde DF (1977) Plasmodium vivax: correlation of circumsporozoite precipitation (CSP) reaction with sporozoite-induced protective immunity in man. Exp Parasitol 41: 167–171. doi:10.1016/0014-4894(77)90142-4.

96. Nussenzweig V, Nussenzweig RS (1986) Development of a sporozoite malaria vaccine. Am J Trop Med Hyg 35: 678–688. doi:10.1590/S0074-02761986000600008.

97. Clyde DF, McCarthy VC, Miller RM, Hornick RB (1973) Specificity of protection of man immunized against sporozoite-induced falciparum malaria. Am J Med Sci 266: 398–403. doi:10.1097/00000441-197312000-00001.

98. Tran TM, Li S, Doumbo S, Doumtabe D, Huang CY, et al. (2013) An Intensive Longitudinal Cohort Study of Malian Children and Adults Reveals No Evidence of Acquired Immunity to Plasmodium falciparum Infection. Clin Infect Dis 57: 40–47. doi:10.1093/cid/cit174.

99. Hoffman SL, Goh LML, Luke TC, Schneider I, Le TP, et al. (2002) Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis 185: 1155–1164. doi:10.1086/339409.

100. Alger NE, Harant J (1976) Plasmodium berghei: heat-treated sporozoite vaccination of mice. Exp Parasitol 40: 261–268. doi:10.1016/0014-4894(76)90089-8.

101. Luke TC, Hoffman SL (2003) Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine. J Exp Biol 206: 3803–3808. doi:10.1242/jeb.00644.

139

102. Vercruysse J, Schetters TPM, Knox DP, Willadsen P, Claerebout E (2007) Control of parasitic disease using vaccines: an answer to drug resistance? Rev Sci Tech 26: 105–115.

103. Mueller A-K, Labaied M, Kappe SHI, Matuschewski K (2005) Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature 433: 164– 167. doi:10.1038/nature05642.

104. Butler NS, Schmidt NW, Vaughan AM, Aly AS, Kappe SHI, et al. (2011) Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites. Cell Host Microbe 9: 451–462. doi:10.1016/j.chom.2011.05.008.

105. Khan SM, Janse CJ, Kappe SHI, Mikolajczak SA (2012) Genetic engineering of attenuated malaria parasites for vaccination. Curr Opin Biotechnol 23: 908–916. doi:10.1016/j.copbio.2012.04.003.

106. Murphy SC, Kas A, Stone BC, Bevan MJ (2013) A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations. Proc Natl Acad Sci U S A 110: 6055–6060. doi:10.1073/pnas.1303834110.

107. Mikolajczak SA, Lakshmanan V, Fishbaugher M, Camargo N, Harupa A, et al. (2014) A Next-generation Genetically Attenuated Plasmodium falciparum Parasite Created by Triple Gene Deletion. Mol Ther 22: 1707–1715. doi:10.1038/mt.2014.85.

108. Annoura T, Ploemen IHJ, van Schaijk BCL, Sajid M, Vos MW, et al. (2012) Assessing the adequacy of attenuation of genetically modified malaria parasite vaccine candidates. Vaccine 30: 2662–2670. doi:10.1016/j.vaccine.2012.02.010.

109. Vanderberg J, Nussenzweig R MH (1969) Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. V. In vitro effects of immune serum on sporozoites. Mil Med 134: 1183–1190.

110. Yoshida N, Nussenzweig RS, Potocnjak P, Nussenzweig V, Aikawa M (1980) Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science 207: 71–73. doi:10.1126/science.6985745.

111. Potocnjak P, Yoshida N, Nussenzweig RS, Nussenzweig V (1980) Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection. J Exp Med 151: 1504–1513. doi:10.1084/jem.151.6.1504.

112. Lindner SE, Swearingen KE, Harupa A, Vaughan AM, Sinnis P, et al. (2013) Total and putative surface proteomics of malaria parasite salivary gland sporozoites. Mol Cell Proteomics 12: 1127–1143. doi:10.1074/mcp.M112.024505.

113. Lasonder E, Janse CJ, Van Gemert GJ, Mair GR, Vermunt AMW, et al. (2008) Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity. PLoS Pathog 4. doi:10.1371/journal.ppat.1000195.

140

114. Mazier D, Mellouk S, Beaudoin RL, Texier B, Druilhe P, et al. (1986) Effect of antibodies to recombinant and synthetic peptides on P. falciparum sporozoites in vitro. Science 231: 156–159. doi:10.1126/science.3510455.

115. Hoffman SL, Oster CN, Plowe C V, Woollett GR, Beier JC, et al. (1987) Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications. Science 237: 639–642. doi:10.1126/science.3299709.

116. Chen DH, Tigelaar RE, Weinbaum FI (1977) Immunity to sporozoite-induced malaria infeciton in mice. I. The effect of immunization of T and B cell-deficient mice. J Immunol 118: 1322–1327.

117. Verhave JP, Meuwissen JH, Golenser J (1978) Cell-mediated reactions and protection after immunization with sporozoites. Isr J Med Sci 14: 611–616.

118. Egan JE, Weber JL, Ballou WR, Hollingdale MR, Majarian WR, et al. (1987) Efficacy of murine malaria sporozoite vaccines: implications for human vaccine development. Science 236: 453–456. doi:10.1126/science.3551073.

119. Schofield L, Villaquiran J, Ferreira A, Schellekens H, Nussenzweig R, et al. (1987) Gamma interferon, CD8+ T cells and antibodies required for immunity to malaria sporozoites. Nature 330: 664–666. doi:10.1038/330664a0.

120. Weiss WR, Sedegah M, Beaudoin RL, Miller LH, Good MF (1988) CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites. Proc Natl Acad Sci U S A 85: 573–576.

121. Romero P, Maryanski JL, Corradin G, Nussenzweig RS, Nussenzweig V, et al. (1989) Cloned cytotoxic T cells recognize an epitope in the circumsporozoite protein and protect against malaria. Nature 341: 323–326. doi:10.1038/341323a0.

122. Schmidt NW, Podyminogin RL, Butler NS, Badovinac VP, Tucker BJ, et al. (2008) Memory CD8 T cell responses exceeding a large but definable threshold provide long-term immunity to malaria. Proc Natl Acad Sci U S A 105: 14017–14022. doi:10.1073/pnas.0805452105.

123. Doolan DL, Hoffman SL (1997) Pre-erythrocytic-stage immune effector mechanisms in Plasmodium spp. infections. Philos Trans R Soc Lond B Biol Sci 352: 1361–1367. doi:10.1098/rstb.1997.0121.

124. Doolan DL, Hoffman SL (2000) The complexity of protective immunity against liver-stage malaria. J Immunol 165: 1453–1462. doi:ji_v165n3p1453 [pii].

125. Weiss WR, Good MF, Hollingdale MR, Miller LH, Berzofsky JA (1989) Genetic control of immunity to Plasmodium yoelii sporozoites. J Immunol 143: 4263–4266.

141

126. Crompton PD, Pierce SK, Miller LH (2010) Advances and challenges in malaria vaccine development. J Clin Invest 120: 4168–4178. doi:10.1172/JCI44423.4168.

127. Kumar KA, Sano G, Boscardin S, Nussenzweig RS, Nussenzweig MC, et al. (2006) The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites. Nature 444: 937–940. doi:10.1038/nature05361.

128. Hoffman SL (2006) Malaria: a protective paradox. Nature 444: 824–827. doi:10.1038/nature05409.

129. Cockburn IA, Amino R, Kelemen RK, Kuo SC, Tse S-W, et al. (2013) In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages. Proc Natl Acad Sci U S A 110: 9090– 9095. doi:10.1073/pnas.1303858110.

130. Cockburn IA, Tse SW, Zavala F (2014) CD8+ T cells eliminate liver-stage Plasmodium berghei parasites without detectable bystander effect. Infect Immun 82: 1460–1464. doi:10.1128/IAI.01500-13.

131. Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, et al. (2004) Efficacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: Randomised controlled trial. Lancet 364: 1411–1420. doi:10.1016/S0140-6736(04)17223-1.

132. Hafalla JC, Silvie O, Matuschewski K (2011) Cell biology and immunology of malaria. Immunol Rev 240: 297–316. doi:10.1111/j.1600-065X.2010.00988.x.

133. Scheller LF, Azad AF (1995) Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites. Proc Natl Acad Sci U S A 92: 4066–4068. doi:10.1073/pnas.92.9.4066.

134. Cockburn IA, Chen YC, Overstreet MG, Lees JR, van Rooijen N, et al. (2010) Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites. PLoS Pathog 6: 1–13. doi:10.1371/journal.ppat.1000877.

135. Mueller A-K, Deckert M, Heiss K, Goetz K, Matuschewski K, et al. (2007) Genetically attenuated Plasmodium berghei liver stages persist and elicit sterile protection primarily via CD8 T cells. Am J Pathol 171: 107–115. doi:10.2353/ajpath.2007.060792.

136. Chatterjee S, Ngonseu E, Van Overmeir C, Correwyn A, Druilhe P WM (2001) Rodent malaria in the natural host - irradiated sporozoites of Plasmodium berghei induce liver- stage specific immune responses in the natural host Grammomys surdaster and protect immunized Grammomys against P. berghei sporozoite challenge. Afr J Med Med Sci: 25– 33.

137. Seder RA, Chang L-J, Enama ME, Zephir KL, Sarwar UN, et al. (2013) Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science 341: 1359–1365. doi:10.1126/science.1241800.

142

138. Kaushansky A, Metzger PG, Douglass AN, Mikolajczak SA, Lakshmanan V, et al. (2013) Malaria parasite liver stages render host hepatocytes susceptible to mitochondria- initiated apoptosis. Cell Death Dis 4: e762. doi:10.1038/cddis.2013.286.

139. Good MF, Berzofsky JA, Miller LH (1988) The T cell response to the malaria circumsporozoite protein: an immunological approach to vaccine development. Annu Rev Immunol 6: 663–688. doi:10.1146/annurev.iy.06.040188.003311.

140. Sinden RE (2010) A biologist’s perspective on malaria vaccine development. Hum Vaccin 6: 3–11. doi:10.4161/hv.6.1.9604.

141. Ellis J, Ozaki LS, Gwadz RW, Cochrane AH, Nussenzweig V, et al. (1983) Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi. Nature 302: 536–538. doi:10.1038/302536a0.

142. Godson GN, Ellis J, Svec P, Schlesinger DH, Nussenzweig V (1983) Identification and chemical synthesis of a tandemly repeated immunogenic region of Plasmodium knowlesi circumsporozoite protein. Nature 305: 29–33. doi:10.1038/305029a0.

143. Bowman NM, Congdon S, Mvalo T, Patel JC, Escamilla V, et al. (2013) Comparative population structure of Plasmodium falciparum circumsporozoite protein NANP repeat lengths in Lilongwe, Malawi. Sci Rep 3: 1990. doi:10.1038/srep01990.

144. McCutchan TF, Hansen JL, Dame JB, Mullins JA (1984) Mung bean nuclease cleaves Plasmodium genomic DNA at sites before and after genes. Science 225: 625–628. doi:10.1126/science.6330899.

145. Dame J, Williams J, McCutchan T, Weber J, Wirtz R, et al. (1984) Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science (80- ) 225: 593–599. doi:10.1126/science.6204383.

146. Scheiblhofer S, Chen D, Weiss R, Khan F, Mostböck S, et al. (2001) Removal of the circumsporozoite protein ( CSP ) glycosylphosphatidylinositol signal sequence from a CSP DNA vaccine enhances induction of CSP- specific Th2 type immune responses and improves protection against malaria infection. Gene: 692–698.

147. Sinnis P, Clavijo P, Fenyö D, Chait BT, Cerami C, et al. (1994) Structural and functional properties of region II-plus of the malaria circumsporozoite protein. J Exp Med 180: 297– 306. doi:10.1084/jem.180.1.297.

148. Coppi A, Natarajan R, Pradel G, Bennett BL, James ER, et al. (2011) The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host. J Exp Med 208: 341–356. doi:10.1084/jem.20101488.

143

149. Coppi A, Pinzon-Ortiz C, Hutter C, Sinnis P (2005) The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion. J Exp Med 201: 27–33. doi:10.1084/jem.20040989.

150. Zavala F, Tam JP, Hollingdale MR, Cochrane AH, Quakyi I, et al. (1985) Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria. Science 228: 1436–1440. doi:10.1126/science.2409595.

151. Good MF, Pombo D, Quakyi IA, Riley EM, Houghten RA, et al. (1988) Human T-cell recognition of the circumsporozoite protein of Plasmodium falciparum: immunodominant T-cell domains map to the polymorphic regions of the molecule. Proc Natl Acad Sci U S A 85: 1199–1203. doi:10.1073/pnas.85.4.1199.

152. Doolan DL, Hoffman SL, Southwood S, Wentworth PA, Sidney J, et al. (1997) Degenerate cytotoxic T cell epitopes from P. falciparum restricted by multiple HLA-A and HLA-B supertype alleles. Immunity 7: 97–112.

153. Frevert U, Galinski MR, Hügel FU, Allon N, Schreier H, et al. (1998) Malaria circumsporozoite protein inhibits protein synthesis in mammalian cells. EMBO J 17: 3816–3826. doi:10.1093/emboj/17.14.3816.

154. Usynin I, Klotz C, Frevert U (2007) Malaria circumsporozoite protein inhibits the respiratory burst in Kupffer cells. Cell Microbiol 9: 2610–2628. doi:10.1111/j.1462- 5822.2007.00982.x.

155. Klotz C, Frevert U (2008) Plasmodium yoelii sporozoites modulate cytokine profile and induce apoptosis in murine Kupffer cells. Int J Parasitol 38: 1639–1650. doi:10.1016/j.ijpara.2008.05.018.

156. Singh AP, Buscaglia CA, Wang Q, Levay A, Nussenzweig DR, et al. (2007) Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite. Cell 131: 492–504. doi:10.1016/j.cell.2007.09.013.

157. Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 193: 673–675.

158. Wu Y, Sifri CD, Lei HH, Su XZ, Wellems TE (1995) Transfection of Plasmodium falciparum within human red blood cells. Proc Natl Acad Sci U S A 92: 973–977. doi:10.1073/pnas.92.4.973.

159. Prudêncio M, Mota MM, Mendes AM (2011) A toolbox to study liver stage malaria. Trends Parasitol 27: 565–574. doi:10.1016/j.pt.2011.09.004.

160. De Koning-Ward TF, Janse CJ, Waters AP (2000) The development of genetic tools for dissecting the biology of malaria parasites. Annu Rev Microbiol 54: 157–185. doi:10.1146/annurev.micro.54.1.157.

144

161. Janse CJ, Franke-Fayard B, Mair GR, Ramesar J, Thiel C, et al. (2006) High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol Biochem Parasitol 145: 60–70. doi:10.1016/j.molbiopara.2005.09.007.

162. Franke-Fayard B, Trueman H, Ramesar J, Mendoza J, Van Der Keur M, et al. (2004) A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle. Mol Biochem Parasitol 137: 23–33. doi:10.1016/j.molbiopara.2004.04.007.

163. Albuquerque SS, Carret C, Grosso AR, Tarun AS, Peng X, et al. (2009) Host cell transcriptional profiling during malaria liver stage infection reveals a coordinated and sequential set of biological events. BMC Genomics 10: 270. doi:10.1186/1471-2164-10- 270.

164. Tarun AS, Baer K, Dumpit RF, Gray S, Lejarcegui N, et al. (2006) Quantitative isolation and in vivo imaging of malaria parasite liver stages. Int J Parasitol 36: 1283–1293. doi:10.1016/j.ijpara.2006.06.009.

165. Mwakingwe A, Ting L-M, Hochman S, Chen J, Sinnis P, et al. (2009) Noninvasive real-time monitoring of liver-stage development of bioluminescent Plasmodium parasites. J Infect Dis 200: 1470–1478. doi:10.1086/606115.

166. Zou X, House BL, Zyzak MD, Richie TL, Gerbasi VR (2013) Towards an optimized inhibition of liver stage development assay (ILSDA) for Plasmodium falciparum. Malar J 12: 394. doi:10.1186/1475-2875-12-394.

167. March S, Ng S, Velmurugan S, Galstian A, Shan J, et al. (2013) A Microscale Human Liver Platform that Supports the Hepatic Stages of Plasmodium falciparum and vivax. Cell Host Microbe 14: 104–115. doi:http://dx.doi.org/10.1016/j.chom.2013.06.005.

168. Karnasuta C, Pavanand K, Chantakulkij S, Luttiwongsakorn N, Rassamesoraj M, et al. (1995) Complete development of the liver stage of Plasmodium falciparum in a human hepatoma cell line. Am J Trop Med Hyg 53: 607–611.

169. Sattabongkot J, Yimamnuaychoke N, Leelaudomlipi S, Rasameesoraj M, Jenwithisuk R, et al. (2006) Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax. Am J Trop Med Hyg 74: 708–715. doi:74/5/708 [pii].

170. Talman AM, Blagborough AM, Sinden RE (2010) A Plasmodium falciparum Strain Expressing GFP throughout the Parasite’s Life-Cycle. PLoS One 5: e9156. doi:10.1371/journal.pone.0009156.

171. Behet MC, Foquet L, van Gemert G-J, Bijker E, Meuleman P, et al. (2014) Sporozoite immunization of human volunteers under chemoprophylaxis induces functional antibodies against pre-erythrocytic stages of Plasmodium falciparum. Malar J 13: 136. doi:10.1186/1475-2875-13-136.

145

172. Vaughan AM, Mikolajczak SA, Camargo N, Lakshmanan V, Kennedy M, et al. (2012) A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle. Mol Biochem Parasitol 186: 143–147. doi:10.1016/j.molbiopara.2012.10.004.

173. Miller JF, Osoba D (1967) Current concepts of the immunological function of the thymus. Physiol Rev 47: 437–520. doi:10.1016/0167-5699(91)90111-6.

174. Cerottini JC, Nordin AA, Brunner KT (1970) Specific in vitro cytotoxicity of thymus-derived lymphocytes sensitized to alloantigens. Nature 228: 1308–1309. doi:10.1038/2281308a0.

175. Claman HN, Chaperon EA, Triplett RF (1966) Thymus-Marrow Cell Combinations. Synergism in Antibody Production. Exp Biol Med 122: 1167–1171. doi:10.3181/00379727-122-31353.

176. Cantor H, Boyse EA (1975) Functional subclasses of T-lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T-cell subclasses is a differentiative process independent of antigen. J Exp Med 141: 1376–1389. doi:10.1084/jem.141.6.1376.

177. Zinkernagel RM, Doherty PC (1974) Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 248: 701–702. doi:10.1038/248701a0.

178. Zinkernagel RM, Doherty PC (1974) Immunological surveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis. Nature 251: 547–548. doi:10.1038/251547a0.

179. Neefjes J, Jongsma MLM, Paul P, Bakke O (2011) Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol 11: 823–836. doi:10.1038/nri3084.

180. Kägi D, Ledermann B, Bürki K, Seiler P, Odermatt B, et al. (1994) Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369: 31–37. doi:10.1038/369031a0.

181. Walsh CM, Glass AA, Chiu V, Clark WR (1994) The role of the Fas lytic pathway in a perforin-less CTL hybridoma. J Immunol 153: 2506–2514.

182. Kägi D, Vignaux F, Ledermann B, Bürki K, Depraetere V, et al. (1994) Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265: 528–530. doi:10.1126/science.7518614.

183. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, et al. (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3: 673–682. doi:10.1016/1074-7613(95)90057-8.

146

184. LeBlanc HN, Ashkenazi A (2003) Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 10: 66–75. doi:10.1038/sj.cdd.4401187.

185. Guidotti LG, Chisari F V (2001) Noncytolytic control of viral infections by the innate and adaptive immune response. Annu Rev Immunol 19: 65–91. doi:10.1146/annurev.immunol.19.1.65.

186. Harty JT, Tvinnereim AR, White DW (2000) CD8+ T cell effector mechanisms in resistance to infection. Annu Rev Immunol 18: 275–308. doi:10.1146/annurev.immunol.18.1.275.

187. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, et al. (1987) Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329: 506–512. doi:10.1038/329506a0.

188. Fremont DH, Matsumura M, Stura EA, Peterson PA, Wilson IA (1992) Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb. Science 257: 919–927. doi:10.1126/science.1323877.

189. Townsend A, Ohlén C, Bastin J, Ljunggren HG, Foster L, et al. (1989) Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 340: 443–448. doi:10.1038/340443a0.

190. York IA, Rock KL (1996) Antigen processing and presentation by the class I major histocompatibility complex. Annu Rev Immunol 14: 369–396. doi:10.1146/annurev.immunol.14.1.369.

191. Yewdell JW, Bennink JR (1992) Cell biology of antigen processing and presentation to major histocompatibility complex class I molecule-restricted T lymphocytes. Adv Immunol 52: 1–123.

192. Rock KL, Gramm C, Rothstein L, Clark K, Stein R, et al. (1994) Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78: 761–771. doi:10.1016/S0092-8674(94)90462-6.

193. Rechsteiner M (1987) Ubiquitin-mediated pathways for intracellular proteolysis. Annu Rev Cell Biol 3: 1–30. doi:10.1146/annurev.cellbio.3.1.1.

194. Vyas JM, Van der Veen AG, Ploegh HL (2008) The known unknowns of antigen processing and presentation. Nat Rev Immunol 8: 607–618. doi:10.1038/nri2368.

195. Rock KL, Goldberg AL (1999) Degradation of cell proteins and the generation of MHC classI-presented peptides. Annu Rev Immunol 17: 739–779.

196. Traherne JA (2008) Human MHC architecture and evolution: Implications for disease association studies. Int J Immunogenet 35: 179–192. doi:10.1111/j.1744- 313X.2008.00765.x.

147

197. Vandiedonck C, Knight JC (2009) The human Major Histocompatibility Complex as a paradigm in genomics research. Brief Funct Genomic Proteomic 8: 379–394. doi:10.1093/bfgp/elp010.

198. Robinson J, Waller MJ, Parham P, Groot N De, Bontrop R, et al. (2003) IMGT / HLA and IMGT / MHC : sequence databases for the study of the major histocompatibility complex. Most 31: 311–314. doi:10.1093/nar/GKG070.

199. Van Valen L (1973) A new evolutionary theory. Evol Theory 1: 1–30.

200. Hewitt EW (2003) The MHC class I antigen presentation pathway: Strategies for viral immune evasion. Immunology 110: 163–169. doi:10.1046/j.1365-2567.2003.01738.x.

201. Hansen TH, Bouvier M (2009) MHC class I antigen presentation: learning from viral evasion strategies. Nat Rev Immunol 9: 503–513. doi:10.1038/nri2575.

202. Rodrigues EG, Zavala F, Eichinger D, Wilson JM, Tsuji M (1997) Single immunizing dose of recombinant adenovirus efficiently induces CD8+ T cell-mediated protective immunity against malaria. J Immunol 158: 1268–1274.

203. Reece WHH, Pinder M, Gothard PK, Milligan P, Bojang K, et al. (2004) A CD4(+) T-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural Plasmodium falciparum infection and disease. Nat Med 10: 406– 410. doi:10.1038/nm1009.

204. Sano G, Hafalla JC, Morrot A, Abe R, Lafaille JJ, et al. (2001) Swift development of protective effector functions in naive CD8(+) T cells against malaria liver stages. J Exp Med 194: 173–180. doi:10.1084/jem.194.2.173.

205. Chakravarty S, Cockburn IA, Kuk S, Overstreet MG, Sacci JB, et al. (2007) CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes. Nat Med 13: 1035–1041. doi:10.1038/nm1628.

206. Tse S-W, Radtke AJ, Espinosa DA, Cockburn IA, Zavala F (2014) The Chemokine Receptor CXCR6 Is Required for the Maintenance of Liver Memory CD8+ T Cells Specific for Infectious Pathogens. J Infect Dis 210: 1–9. doi:10.1093/infdis/jiu281.

207. Chakravarty S, Baldeviano GC, Overstreet MG, Zavala F (2008) Effector CD8+ T lymphocytes against liver stages of Plasmodium yoelii do not require gamma interferon for antiparasite activity. Infect Immun 76: 3628–3631. doi:10.1128/IAI.00471-08.

208. Lalvani A, Hurt N, Aidoo M, Kibatala P, Tanner M, et al. (1996) Cytotoxic T lymphocytes to Plasmodium falciparum epitopes in an area of intense and perennial transmission in Tanzania. Eur J Immunol 26: 773–779. doi:10.1002/eji.1830260408.

148

209. Kusi KA, Bosomprah S, Dodoo D, Kyei-Baafour E, Dickson EK, et al. (2014) Anti-sporozoite antibodies as alternative markers for malaria transmission intensity estimation. Malar J 13: 103. doi:10.1186/1475-2875-13-103.

210. Takala SL, Coulibaly D, Thera MA, Batchelor AH, Cummings MP, et al. (2009) Extreme polymorphism in a vaccine antigen and risk of clinical malaria: implications for vaccine development. Sci Transl Med 1: 2ra5. doi:10.1126/scitranslmed.3000257.

211. Zarling S, Berenzon D, Dalai S, Liepinsh D, Steers N, et al. (2013) The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria. J Immunol 190: 5128–5141. doi:10.4049/jimmunol.1203396.

212. Hill A V, Elvin J, Willis AC, Aidoo M, Allsopp CE, et al. (1992) Molecular analysis of the association of HLA-B53 and resistance to severe malaria. Nature 360: 434–439. doi:10.1038/360434a0.

213. Hill A V, Allsopp CE, Kwiatkowski D, Anstey NM, Twumasi P, et al. (1991) Common west African HLA antigens are associated with protection from severe malaria. Nature 352: 595–600. doi:10.1038/352595a0.

214. Aidoo M, Lalvani A, Gilbert SC, Hu JT, Daubersies P, et al. (2000) Cytotoxic T-lymphocyte epitopes for HLA-B53 and other HLA types in the malaria vaccine candidate liver-stage antigen 3. Infect Immun 68: 227–232. doi:10.1128/IAI.68.1.227-232.2000.

215. Cattamanchi A, Kyabayinze D, Hubbard A, Rosenthal PJ, Dorsey G (2003) Distinguishing recrudescence from reinfection in a longitudinal antimalarial drug efficacy study: Comparison of results based on genotyping of MSP-1, MSP-2, and GLURP. Am J Trop Med Hyg 68: 133–139.

216. Liljander A, Bejon P, Mwacharo J, Kai O, Ogada E, et al. (2011) Clearance of asymptomatic P. falciparum infections interacts with the number of clones to predict the risk of subsequent malaria in Kenyan children. PLoS One 6: 1–8. doi:10.1371/journal.pone.0016940.

217. Coulibaly D, Diallo DA, Thera MA, Dicko A, Guindo AB, et al. (2002) Impact of preseason treatment on incidence of falciparum malaria and parasite density at a site for testing malaria vaccines in Bandiagara, Mali. Am J Trop Med Hyg 67: 604–610.

218. Cao K, Moormann AM, Lyke KE, Masaberg C, Sumba OP, et al. (2004) Differentiation between African populations is evidenced by the diversity of alleles and haplotypes of HLA class I loci. Tissue Antigens 63: 293–325.

219. Abdulla S, Oberholzer R, Juma O, Kubhoja S, Machera F, et al. (2008) Safety and immunogenicity of RTS,S/AS02D malaria vaccine in infants. 2533-2544 p. doi:10.1056/NEJMoa0807773.

149

220. Olotu A, Lusingu J, Leach A, Lievens M, Vekemans J, et al. (2011) Efficacy of RTS,S/AS01E malaria vaccine and exploratory analysis on anti-circumsporozoite antibody titres and protection in children aged 5-17 months in Kenya and Tanzania: a randomised controlled trial. Lancet Infect Dis 11: 102–109. doi:10.1016/S1473-3099(10)70262-0.

221. Agnandji ST, Lell B, Fernandes JF, Abossolo BP, Methogo BGNO, et al. (2012) A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants. N Engl J Med 367: 2284–2295. doi:10.1056/NEJMoa1208394.

222. White MT, Bejon P, Olotu A, Griffin JT, Riley EM, et al. (2013) The Relationship between RTS,S Vaccine-Induced Antibodies, CD4+ T Cell Responses and Protection against Plasmodium falciparum Infection. PLoS One 8. doi:10.1371/journal.pone.0061395.

223. Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, et al. (2007) A phase I/IIa safety, immunogenicity, and efficacy bridging randomized study of a two-dose regimen of liquid and lyophilized formulations of the candidate malaria vaccine RTS,S/AS02A in malaria-naïve adults. Vaccine 25: 5359–5366. doi:10.1016/j.vaccine.2007.05.005.

224. Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, et al. (2001) Efficacy of recombinant circumsporozoite protein vaccine regimens against experimental Plasmodium falciparum malaria. 640-647 p. doi:10.1086/318534.

225. Pinder M, Reece WHH, Plebanski M, Akinwunmi P, Flanagan KL, et al. (2004) Cellular immunity induced by the recombinant Plasmodium falciparum malaria vaccine, RTS,S/AS02, in semi-immune adults in The Gambia. Clin Exp Immunol 135: 286–293. doi:10.1111/j.1365-2249.2004.02371.x.

226. Sun P, Schwenk R, White K, Stoute JA, Cohen J, et al. (2003) Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma. 6961-6967 p. doi:10.4049/jimmunol.171.12.6961.

227. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanović S (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213–219.

228. Mota MM, Hafalla JCR, Rodriguez A (2002) Migration through host cells activates Plasmodium sporozoites for infection. Nat Med 8: 1318–1322. doi:10.1038/nm785.

229. Tewari R, Spaccapelo R, Bistoni F, Holder AA, Crisanti A (2002) Function of region I and II adhesive motifs of Plasmodium falciparum circumsporozoite protein in sporozoite motility and infectivity. J Biol Chem 277: 47613–47618. doi:10.1074/jbc.M208453200.

230. Tarun AS, Peng X, Dumpit RF, Ogata Y, Silva-Rivera H, et al. (2008) A combined transcriptome and proteome survey of malaria parasite liver stages. Proc Natl Acad Sci U S A 105: 305–310. doi:10.1073/pnas.0710780104.

150

231. Schumacher TN, De Bruijn ML, Vernie LN, Kast WM, Melief CJ, et al. (1991) Peptide selection by MHC class I molecules. Nature 350: 703–706. doi:10.1038/350703a0.

232. Komander D (2009) The emerging complexity of protein ubiquitination. Biochem Soc Trans 37: 937–953. doi:10.1042/BST0370937.

233. Lim KL, Chew KCM, Tan JMM, Wang C, Chung KKK, et al. (2005) Parkin mediates nonclassical, proteasomal-independent ubiquitination of synphilin-1: implications for Lewy body formation. J Neurosci 25: 2002–2009. doi:10.1523/JNEUROSCI.4474-04.2005.

234. Jacobson AD, Zhang N-Y, Xu P, Han K-J, Noone S, et al. (2009) The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26S proteasome. J Biol Chem 284: 35485–35494. doi:10.1074/jbc.M109.052928.

235. Tan JMM, Wong ESP, Kirkpatrick DS, Pletnikova O, Ko HS, et al. (2008) Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases. Hum Mol Genet 17: 431–439. doi:10.1093/hmg/ddm320.

236. Kravtsova-Ivantsiv Y, Ciechanover A (2012) Non-canonical ubiquitin-based signals for proteasomal degradation. J Cell Sci 125: 539–548. doi:10.1242/jcs.093567.

237. Tasaki T, Sriram SM, Park KS, Kwon YT (2012) The N-End Rule Pathway. Annu Rev Biochem 81: 261–289. doi:10.1146/annurev-biochem-051710-093308.

238. Tartz S, Deschermeier C, Retzlaff S, Heussler V, Sebo P, et al. (2013) Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8(+) memory T cells. Eur J Immunol 43: 693–704. doi:10.1002/eji.201142262.

239. Daily JP (2012) Malaria vaccine trials--beyond efficacy end points. N Engl J Med 367: 2348–2349. doi:10.1056/NEJMe1212457.

240. Good MF, Doolan DL (2010) Malaria vaccine design: immunological considerations. Immunity 33: 555–566. doi:10.1016/j.immuni.2010.10.005.

241. Cockburn IA, Tse S-W, Zavala F (2014) CD8+ T cells eliminate liver-stage Plasmodium berghei parasites without detectable bystander effect. Infect Immun 82: 1460–1464. doi:10.1128/IAI.01500-13.

242. Ma J, Trop S, Baer S, Rakhmanaliev E, Arany Z, et al. (2013) Dynamics of the Major Histocompatibility Complex Class I Processing and Presentation Pathway in the Course of Malaria Parasite Development in Human Hepatocytes: Implications for Vaccine Development. PLoS One 8: e75321. doi:10.1371/journal.pone.0075321.

151

243. Duffy PE, Sahu T, Akue A, Milman N, Anderson C (2012) Pre-erythrocytic malaria vaccines: identifying the targets. Expert Rev Vaccines 11: 1261–1280. doi:10.1586/erv.12.92.

244. Sacci JB, Schriefer ME, Resau JH, Wirtz RA, Detolla LJ, et al. (1992) Mouse model for exoerythrocytic stages of Plasmodium falciparum malaria parasite. Proc Natl Acad Sci U S A 89: 3701–3705.

245. Sacci Jr. JB, Alam U, Douglas D, Lewis J, Tyrrell DLJ, et al. (2006) Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers. Int J Parasitol 36: 353–360. doi:http://dx.doi.org/10.1016/j.ijpara.2005.10.014.

246. Foquet L, Hermsen CC, van Gemert G-J, Van Braeckel E, Weening KE, et al. (2014) Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection. J Clin Invest 124: 140–144. doi:10.1172/JCI70349.

247. Spence PJ, Langhorne J (2012) T cell control of malaria pathogenesis. Curr Opin Immunol 24: 444–448. doi:http://dx.doi.org/10.1016/j.coi.2012.05.003.

248. Schwenk RJ, Richie TL (2011) Protective immunity to pre-erythrocytic stage malaria. Trends Parasitol 27: 306–314. doi:http://dx.doi.org/10.1016/j.pt.2011.02.002.

249. Tse S-W, Radtke AJ, Zavala F (2011) Induction and maintenance of protective CD8+ T cells against malaria liver stages: implications for vaccine development. Mem Inst Oswaldo Cruz 106 Suppl : 172–178.

250. Prudêncio M, Rodrigues CD, Ataíde R, Mota MM (2008) Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry. Cell Microbiol 10: 218–224. doi:10.1111/j.1462-5822.2007.01032.x.

251. Kaushansky A, Rezakhani N, Mann H, Kappe SHI (2012) Development of a quantitative flow cytometry-based assay to assess infection by Plasmodium falciparum sporozoites. Mol Biochem Parasitol 183: 100–103. doi:10.1016/j.molbiopara.2012.01.006.

252. Zhou Y, Ramachandran V, Kumar KA, Westenberger S, Refour P, et al. (2008) Evidence- based annotation of the malaria parasite’s genome using comparative expression profiling. PLoS One 3. doi:10.1371/journal.pone.0001570.

253. Pfeifer AM, Cole KE, Smoot DT, Weston A, Groopman JD, et al. (1993) Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens. Proc Natl Acad Sci U S A 90: 5123– 5127.

254. Vanderberg JP (1982) Asynchronous maturation of Plasmodium berghei exo-erythrocytic forms in rats. Trans R Soc Trop Med Hyg 76: 251–252. doi:10.1016/0035-9203(82)90290- 5.

152

255. Hügel F-U, Pradel G, Frevert U (1996) Release of malaria circumsporozoite protein into the host cell cytoplasm and interaction with ribosomes. Mol Biochem Parasitol 81: 151– 170. doi:10.1016/0166-6851(96)02701-6.

256. Li F, Yang M, Wang L, Williamson I, Tian F, et al. (2012) Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: A lesson learned from flow cytometry. J Immunol Methods 386: 101–107. doi:http://dx.doi.org/10.1016/j.jim.2012.08.014.

257. Croce AC, Ferrigno A, Vairetti M, Bertone R, Freitas I, et al. (2004) Autofluorescence properties of isolated rat hepatocytes under different metabolic conditions. Photochem Photobiol Sci 3: 920–926. doi:10.1039/B407358D.

258. Croce AC, De Simone U, Freitas I, Boncompagni E, Neri D, et al. (2010) Human liver autofluorescence: An intrinsic tissue parameter discriminating normal and diseased conditions. Lasers Surg Med 42: 371–378. doi:10.1002/lsm.20923.

259. Wright GJ, Rayner JC (2014) Plasmodium falciparum Erythrocyte Invasion: Combining Function with Immune Evasion. PLoS Pathog 10: e1003943. doi:10.1371/journal.ppat.1003943.

260. Bergmann-Leitner ES, Duncan EH, Burge JR, Spring M, Angov E (2008) Short report: Miniaturization of a high-throughput pLDH-based Plasmodium falciparum growth inhibition assay for small volume samples from preclinical and clinical vaccine trials. Am J Trop Med Hyg 78: 468–471.

261. Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M (2004) Simple and Inexpensive Fluorescence-Based Technique for High-Throughput Antimalarial Drug Screening. Antimicrob Agents Chemother 48: 1803–1806. doi:10.1128/AAC.48.5.1803- 1806.2004.

262. Stewart MJ, Nawrot RJ, Schulman S, Vanderberg JP (1986) Plasmodium berghei sporozoite invasion is blocked in vitro by sporozoite-immobilizing antibodies. Infect Immun 51: 859–864.

263. Hollingdale MR, Zavala F, Nussenzweig RS, Nussenzweig V (1982) Antibodies to the protective antigen of Plasmodium berghei sporozoites prevent entry into cultured cells. J Immunol 128: 1929–1930.

264. Hollingdale MR, Nardin EH, Tharavanij S, Schwartz AL, Nussenzweig RS (1984) Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured cells; an in vitro assay of protective antibodies. J Immunol 132: 909–913.

265. Kaushansky A, Mikolajczak S a., Vignali M, Kappe SHI (2014) Of men in mice: The success and promise of humanized mouse models for human malaria parasite infections. Cell Microbiol 16: 602–611. doi:10.1111/cmi.12277.

153

266. Persson C, Oliveira GA, Sultan AA, Bhanot P, Nussenzweig V, et al. (2002) Cutting edge: a new tool to evaluate human pre-erythrocytic malaria vaccines: rodent parasites bearing a hybrid Plasmodium falciparum circumsporozoite protein. J Immunol 169: 6681–6685. doi:10.4049/jimmunol.169.12.6681.

267. Espinosa DA, Yadava A, Angov E, Maurizio PL, Ockenhouse CF, et al. (2013) Development of a chimeric Plasmodium berghei strain expressing the repeat region of the P. vivax circumsporozoite protein for in vivo evaluation of vaccine efficacy. Infect Immun 81: 2882–2887. doi:10.1128/IAI.00461-13.

268. Macete E, Sacarlal J, Aponte J, Leach A, Navia M, et al. (2007) Evaluation of two formulations of adjuvanted RTS, S malaria vaccine in children aged 3 to 5 years living in a malaria-endemic region of Mozambique: a Phase I/IIb randomized double-blind bridging trial. Trials 8: 11. doi:10.1186/1745-6215-8-11.

269. Deal C, Balazs AB, Espinosa DA, Zavala F, Baltimore D, et al. (2014) Vectored antibody gene delivery protects against Plasmodium falciparum sporozoite challenge in mice. Proc Natl Acad Sci 111: 12528–12532. doi:10.1073/pnas.1407362111.

270. Pradel G, Garapaty S, Frevert U (2002) Proteoglycans mediate malaria sporozoite targeting to the liver. Mol Microbiol 45: 637–651. doi:10.1046/j.1365- 2958.2002.03057.x.

271. Vanderberg JP (1974) Studies on the motility of Plasmodium sporozoites. J Protozool 21: 527–537. doi:10.1111/j.1550-7408.1974.tb03693.x.

272. Vanderberg JP, Frevert U (2004) Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int J Parasitol 34: 991–996. doi:http://dx.doi.org/10.1016/j.ijpara.2004.05.005.

273. Tudor D, Yu H, Maupetit J, Drillet A-S, Bouceba T, et al. (2012) Isotype modulates epitope specificity, affinity, and antiviral activities of anti-HIV-1 human broadly neutralizing 2F5 antibody. Proc Natl Acad Sci 109: 12680–12685. doi:10.1073/pnas.1200024109.

274. Cooper LJ, Shikhman a R, Glass DD, Kangisser D, Cunningham MW, et al. (1993) Role of heavy chain constant domains in antibody-antigen interaction. Apparent specificity differences among streptococcal IgG antibodies expressing identical variable domains. J Immunol 150: 2231–2242.

275. Casadevall A, Janda A (2012) Immunoglobulin isotype influences affinity and specificity. Proc Natl Acad Sci 109: 12272–12273. doi:10.1073/pnas.1209750109.

276. Butler NS, Vaughan AM, Harty JT, Kappe SHI (2012) Whole parasite vaccination approaches for prevention of malaria infection. Trends Immunol 33: 247–254. doi:http://dx.doi.org/10.1016/j.it.2012.02.001.

154

277. Keşmir C, Nussbaum AK, Schild H, Detours V, Brunak S (2002) Prediction of proteasome cleavage motifs by neural networks. Protein Eng 15: 287–296. doi:10.1093/protein/15.4.287.

278. Nielsen M, Lundegaard C, Lund O, Keşmir C (2005) The role of the proteasome in generating cytotoxic T-cell epitopes: insights obtained from improved predictions of proteasomal cleavage. Immunogenetics 57: 33–41. doi:10.1007/s00251-005-0781-7.

279. Mishto M, Luciani F, Holzhütter H-G, Bellavista E, Santoro A, et al. (2008) Modeling the in vitro 20S proteasome activity: the effect of PA28-alphabeta and of the sequence and length of polypeptides on the degradation kinetics. J Mol Biol 377: 1607–1617. doi:10.1016/j.jmb.2008.01.086.

280. Kopp F, Dahlmann B, Kuehn L (2001) Reconstitution of hybrid proteasomes from purified PA700–20 S complexes and PA28αβ activator: ultrastructure and peptidase activities1. J Mol Biol 313: 465–471. doi:http://dx.doi.org/10.1006/jmbi.2001.5063.

281. Textoris-Taube K, Keller C, Kuckelkorn U, Kloetzel PM (2013) Analysis of proteasome generated antigenic peptides by mass spectrometry. Methods Mol Biol 960: 15–29. doi:10.1007/978-1-62703-218-6-2.

282. Noe AR, Espinosa D, Li X, Coelho-dos-Reis JGA, Funakoshi R, et al. (2014) A Full-Length Plasmodium falciparum Recombinant Circumsporozoite Protein Expressed by Pseudomonas fluorescens Platform as a Malaria Vaccine Candidate. PLoS One 9: e107764. doi:10.1371/journal.pone.0107764.

283. Bongfen SE, Torgler R, Romero JF, Renia L, Corradin G (2007) Plasmodium berghei- infected primary hepatocytes process and present the circumsporozoite protein to specific CD8+ T cells in vitro. J Immunol 178: 7054–7063. doi:10.4049/jimmunol.178.11.7054.

284. Bonelo A, Valmori D, Triponez F, Tiercy JM, Mentha G, et al. (2000) Generation and characterization of malaria-specific human CD8+ lymphocyte clones: Effect of natural polymorphism on T cell recognition and endogenous cognate antigen presentation by liver cells. Eur J Immunol 30: 3079–3088. doi:10.1002/1521- 4141(200011)30:11<3079::AID-IMMU3079>3.0.CO;2-7.

285. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10: 57–63. doi:10.1038/nrg2484.

286. Shalek AK, Satija R, Adiconis X, Gertner RS, Gaublomme JT, et al. (2013) Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells. Nature 498: 236–240. doi:10.1038/nature12172.

287. Adiconis X, Borges-Rivera D, Satija R, DeLuca DS, Busby M a, et al. (2013) Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat Methods 10: 623–629. doi:10.1038/nmeth.2483.

155

288. Bordoni V, Alonzi T, Zanetta L, Khouri D, Conti A, et al. (2007) Hepatocyte-conditioned medium sustains endothelial differentiation of human hematopoietic-endothelial progenitors. Hepatology 45: 1218–1228. doi:10.1002/hep.21568.

289. Parkes JG, Templeton DM (2003) Effects of retinol and hepatocyte-conditioned medium on cultured rat hepatic stellate cells. Ann Clin Lab Sci 33: 295–305.

290. Dent AE, Bergmann-Leitner ES, Wilson DW, Tisch DJ, Kimmel R, et al. (2008) Antibody- mediated growth inhibition of Plasmodium falciparum: relationship to age and protection from parasitemia in Kenyan children and adults. PLoS One 3: e3557. doi:10.1371/journal.pone.0003557.

291. Snapp EL (2009) Fluorescent Proteins: A Cell Biologist’s User Guide. Trends Cell Biol 19: 649–655. doi:10.1016/j.tcb.2009.08.002.

292. Young L, Sung J, Stacey G, Masters JR (2010) Detection of Mycoplasma in cell cultures. Nat Protoc 5: 929–934. doi:10.1038/nprot.2010.43.

293. Pacheco N, Strome C (1979) Rapid, large-scale isolation of Plasmodium berghei sporozoites from infected mosquitoes. J Parasitol 65: 414–417.

294. Schoone GJ, Oskam L, Kroon NCM, Schallig HDFH, Omar SA (2000) Detection and Quantification of Plasmodium falciparum in Blood Samples Using Quantitative Nucleic Acid Sequence-Based Amplification. J Clin Microbiol 38: 4072–4075.

295. Tsuji M, Mattei D, Nussenzweig R, Eichinger D, Zavala F (1994) Demonstration of heat- shock protein 70 in the sporozoite stage of malaria parasites. Parasitol Res 80: 16–21. doi:10.1007/BF00932618.

296. Anker R, Zavala F, Pollok BA (1990) VH and VL region structure of antibodies that recognize the (NANP) 3 dodecapeptide sequence in the circumsporozoite protein of Plasmodium falciparum. Eur J Immunol 20: 2757–2761. doi:10.1002/eji.1830201233.

297. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671–675. doi:10.1038/nmeth.2089.

156

Curriculum Vitae

Peter C. Dumoulin 615 North Wolfe Street, Baltimore, MD, 21205 Cell phone: (518) 527-9285 [email protected] Citizenship: United States of America

Education

Johns Hopkins University: Bloomberg School of Public Health, Baltimore (JHSPH), MD Ph.D. Candidate, Department of Molecular Microbiology and Immunology September 2009 – estimated graduation spring 2015

McGill University, Montréal, QC, Canada Bachelor of Science with First Class Honors, Spring 2008 Honors Thesis: Genetic study of a System A amino acid transporter in the human brain with relation to depression and suicide. Major: Biology Cumulative GPA: 3.69 out of 4.0

Research Experience

Johns Hopkins University, Bloomberg School of Public Health May 2010 - present Advisor: Jelena Levitskaya, M.D., Ph.D.

Novel detection methods of live, developing Plasmodium falciparum parasites in hepatocytic cell lines and primary human donors ▪Developed a technique capable of detecting live P. falciparum parasites in hepatocytic cell lines and cryopreserved human hepatocyte donors. ▪Determined the necessity of a host cell receptor for sporozoite invasion into hepatocytes. ▪Collaborated with researchers that generated humanized monoclonal, vectored immunoprophylaxis-targeting sporozoites. We designed and developed rapid screens to quantify the activity of immune sera on P.falciparum sporozoite motility and invasion. * Manuscript is under review

Presentation of two forms of the circumsporozoite protein (CSP) on MHC-I ▪Created a set of mammalian expression plasmids capable of expressing a full length or cleaved CSP with a chimeric MHC-I epitome. ▪Sorted and propagated four human CD8+ T-cell lines capable of recognizing expressed CSP in vitro. ▪Determined that full length and cleaved CSP have differential processing and presentation on MHC-I by hepatocytes

157

▪Utilized immunoprecipitation of HA-tagged ubiquitin chains to demonstrate that full length and cleaved CSP are differentially ubiquitinated. ▪Generated specific lysine mutations using site-directed mutagenesis and identified critical residues for ubiquitination of CSP. * Manuscript currently being prepared

National Institutes of Health, National Institute for Allergy and Infectious Disease September 2008 – August 2009 Malarial Genetics Section Postbaccalaureate – IRTA Fellow Dr. Thomas E. Wellems Generation of allelic replacements of PfRh5 alleles using parasite transfection and selection for single crossovers. Using these lines we investigated the contribution of these alleles to red blood cell invasion.

McGill University, Meakins-Christie Laboratories May 2008 – August 2008 Laboratory Research Assistant Dr. Christina Haston, Ph.D. Investigation of a genomic region found in linkage disequilibrium with mouse models of lung fibrosis. We used TaqMan probes to quantify and compare differences in candidate gene expression across the linkage region.

McGill University, Douglas Hospital May 2007 – April 2008 Laboratory Research Assistant, McGill Group for Suicide Studies Dr. Gustavo Turecki, M.D., Ph.D. Identified candidate genes with altered expression associated with suicide completion using a repository of human brain samples

Canadian Field Studies in Africa (with McGill University) January - March 2007 This three-month field study program combined students majoring in environmental science, political science and molecular biology. In addition to course work we designed and undertook a research project aimed at investigating local perceptions of malaria across Kenya, Uganda and Tanzania.

Publications

[1] J. Ma, S. Trop, S. Baer, E. Rakhmanaliev, Z. Arany, P. Dumoulin, H. Zhang, J. Romano, I. Coppens, V. Levitsky, and J. Levitskaya, “Dynamics of the Major Histocompatibility Complex Class I Processing and Presentation Pathway in the Course of Malaria Parasite Development in Human Hepatocytes: Implications for Vaccine Development,” PLoS One, vol. 8, no. 9, p. e75321, Sep. 2013. [2] K. Hayton, P. Dumoulin, B. Henschen, A. Liu, J. Papakrivos, and T. E. Wellems, “Various PfRH5 polymorphisms can support Plasmodium falciparum invasion into the erythrocytes of owl monkeys and rats,” Mol. Biochem. Parasitol., vol. 187, no. 2, pp. 103–10, Feb. 2013.

158

[3] C. Ernst, P. Dumoulin, S. Cabot, J. Erickson, and G. Turecki, “SNAT1 and a family with high rates of suicidal behavior,” Neuroscience, vol. 162, no. 2, pp. 415–22, Aug. 2009.

Conferences

Molecular Parasitology Meeting, Woods Hole Oceanographic Institute, MA, 2013 “Transient expression of CSP in human hepatocytes reveals distinct immunogenicity of two natural variants of CSP antigen” Authors: Peter Dumoulin, Stefanie Trop, Jinxia Ma, Samantha Baer, Jelena Levitskaya

American Society of Tropical Medicine and Hygiene 58th Annual Meeting, Washington DC, 2009 "The Role of Polymorphisms in PfRH5 in Species Specific Plasmodium falciparum Erythrocyte Invasion" poster: abstract #3096 Authors: Karen Hayton, Peter Dumoulin, Bruce Henschen, Anna Liu and Thomas E. Wellems

Teaching Experience

Biology of Parasitism (3rd term) teaching assistant, JHSPH, 2012 This graduate level course combines lecture, literature and laboratory components. Responsibilities included organization of lectures, leading literature group discussion, running live experiments and examination grading. Instructors: Dr. David Sullivan and Dr. Clive Shiff

Malariology (4th term) teaching assistant, JHSPH, 2011 Responsibilities included organization of lectures, preparation of examination questions and grading. Instructors: Dr. David Sullivan and Dr. Clive Shiff

Introduction to Biomedical Sciences (summer) teaching assistant, JHSPH, 2010, 2011 Graduate level lecture series designed for public health students with little or no background in biological sciences. Responsibilities included lecturing, grading and leading group discussion. Instructor: Dr. Noel Rose

Mentoring

University of Maryland Baltimore County (UMBC) Diversity Summer Internship Program, 2011 Supervision of Oseogie Okojie, an undergraduate student from UMBC. The project focused on expression of plasmepsins in Plasmodium liver stages and ended in a poster presentation of relevant findings by the student. After several weeks Oseogie was able to independently run complete western blots to detect plasmepsin protein expression in infected hepatocytes. Coordinator: Dr. Peter Agre

Funding

Johns Hopkins Malaria Research Institute (JHMRI) Pre-Doctoral Grant, September 2013 - present

159

The Johns Hopkins Malaria Research Institute offers two-year grants to support Ph.D. students studying malaria. Grant applications are reviewed and selected by the JHMRI board. I successfully competed for this grant. Title: Immunogenicity of two forms of Plasmodium falciparum CSP by MHC-I and development of an in vitro model of the human liver stage

Dr. J. Harold Drudge Scholarship, 2011 This scholarship was established by Dr. Harold Drudge and Caran C. St. John to support students in their study of parasitology.

160