WHO Immunological Basis for Immunization Series

Module 21: Rotavirus Update 2019

Department of Immunization, Vaccines and Biologicals

World Health Organization 20, Avenue Appia CH-1211 Geneva 27, Switzerland [email protected] http://www.who.int/immunization/en/

Immunization, Vaccines and Biologicals WHO Immunological Basis for Immunization Series

Module 21: Rotavirus Update 2019

Immunization, Vaccines and Biologicals The immunological basis for immunization series: module 21: Rotavirus Vaccines (Immunological basis for immunization series; module 21)

ISBN 978-92-4-000235-7

© World Health Organization 2020

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Abbreviations and acronyms...... v Preface...... vii Acknowledgements...... viii Conflict of interest...... ix

1. Introduction...... 1 2. Rotavirus structure and genome organization...... 3 3. Classification methods for rotaviruses ...... 7 4. Rotavirus epidemiology...... 9 4.1 Burden of disease...... 9 4.2 Rotavirus strain distribution...... 9 4.3 Seasonality of rotavirus disease ...... 10 4.4 Age at infection...... 11 5. Rotavirus replication ...... 12 6. Rotavirus pathogenesis...... 15 6.1 Clinical presentation...... 15 6.2 Extraintestinal infections...... 16 6.3 Pathophysiology and mechanisms of disease...... 17 7. Rotavirus immunity...... 19 7.1 Methods for detection of rotavirus...... 19 7.2 Rotavirus vaccines ...... 19 8. References...... 35

iii iv Abbreviations and acronyms

BRV-PV Bovine (UK) reassortant pentavalent vaccine BRV-TV Tetravalent bovine-human reassortant rotavirus vaccine CD Cluster of differentiation CI Confidence interval CSF Cerebrospinal fluid DLP Double-layered particle dsRNA Double-stranded RNA eIF4G Eukaryotic translation initiation factor 4 G ELISA Enzyme linked immunosorbent assays EM Electron microscopy ENS Enteric nervous system ER Endoplasmic reticulum FUT2 Fucosytranferase 2 G Glycoprotein HBGAs Histo-blood group antigens HSP Heat shock protein HT Hydroxytryptamine IFN-γ Interferon gamma Ig Immunoglobulin IL Interleukin JAMA Junctional adhesion molecule A LLR Lanzhou lamb rotavirus vaccine NCDV Nebraska Calf Diarrhea Virus NDP Nucleoside-diphosphate NEC Necrotizing enterocolitis NSP Non-structural proteins OPV Oral poliovirus vaccine P Protease-sensitive PABP Poly(A)-binding protein PAGE Polyacrylamide gel electrophoresis PFU Plaque forming units

v RER Rough endoplasmic reticulum RNA Ribonucleic acid RRV Rhesus rotavirus vaccine RRV-TV Rhesus–human reassortant tetravalent vaccine RTPase RNA triphosphatase RT-PCR Reverse transcription polymerase chain reaction SCID Severe combined immunodeficiency SG Subgroups SS single-stranded TGF Tumour growth factor TLP Triple-layered particle VP Viral proteins WC3 Wistar Calf 3

vi Preface

This module is part of the WHO series The immunological basis for immunization, which was initially developed in 1993 as a set of eight modules, comprising one module on general immunology and seven modules each devoted to one of the vaccines recommended for the Expanded Programme on Immunization – i.e. vaccines against diphtheria, measles, pertussis, polio, tetanus, tuberculosis and yellow fever. Since then, this series has been updated and extended to include other vaccines of international importance. The main purpose of the modules is to provide national immunization managers and vaccination professionals with an overview of the scientific basis of vaccination against a range of important infectious diseases. The modules developed since 1993 continue to be vaccine-specific, reflecting the biological differences in immune responses to the individual pathogens and the differing strategies employed to create the best possible level of protection that can be provided by vaccination. The modules also serve as a record of the immunological basis for the WHO recommendations on vaccine use, as published in the WHO vaccine position papers1.

vii Acknowledgements

The preparation of this publication was coordinated by the World Health Organization (WHO) Department of Immunization, Vaccines and Biologicals. WHO thanks the donors whose unspecified financial support has made the production of this document possible.

This module was updated for WHO by Sasirekha Ramani, Assistant Professor of Molecular Virology and Microbiology at Baylor College of Medicine, Houston, USA and Mary K. Estes, Cullen Endowed Chair of Human and Molecular Virology, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, USA.

WHO also expresses its thanks to those who provided expert and technical reviews for the initial preparation of the module and the 2018 update: Richard L Ward and David I Bernstein (Children’s Hospital Medical Center, Division of Infectious Diseases, Cincinnati, USA), Jon Gentsch (United States Centers for Disease Control and Prevention (CDC)), Gagandeep Kang (The Wellcome Trust Research Laboratory, India), Carl Kirkwood (Bill & Melinda Gates Foundation), Ikechukwu Udo Ogbuanu (Immunization, Vaccines and Biologicals Department, WHO), and Fatima Serhan (Immunization, Vaccines and Biologicals Department, WHO).

viii Conflict of interest

Sasirekha Ramani´s research unit received grants from Baylor College of Medicine through the Alkek Center for Microbiome and Metagenomics Research, from the National Institutes of Health and from the Bill & Melinda Gates Foundation for research on rotavirus vaccine-related topics. This interest was assessed as nonpersonal, specific and financially not significant.2

Mary K. Estes´ research unit received grants from the National Institutes of Health and from the Bill & Melinda Gates Foundation for research on rotavirus vaccine- related topics. This interest was assessed as nonpersonal, specific and financially not significant. Additionally she is listed as an inventor for a patent which belongs to Baylor College of Medicine on the Synthesis and Immunogenicity of Rotavirus Genes using a Baculovirus Expression system. This interest was assessed as nonpersonal, specific and financially significant.2

ix x 1. Introduction

Rotaviruses are a leading cause of severe, dehydrating gastroenteritis in children under the age of 5 years worldwide (Tate et al., 2012; Kotloff et al., 2013; Operario et al., 2017). Since the first description of rotavirus in humans in 1973 (Flewett et al., 1973; Bishop et al., 1973), and its subsequent recognition as a major human pathogen, many studies have documented the burden of disease, the epidemiology and the pathogenesis of rotaviruses. Rotaviruses are ubiquitous, and almost every child in the world is infected by the age of 5 years (Parashar et al., 1998). The spectrum of clinical presentations range from subclinical infection and mild diarrhoea to severe diarrhoea with vomiting, fever and sometimes fatal dehydration (Parashar et al. , 2013). Transmission of rotavirus occurs predominantly by the faecal-oral route, mainly by close person-to-person contact and through contaminated fomites. The virus is highly stable in the environment and few virions are needed to cause infections in susceptible hosts (Estes and Greenberg, 2013).

Decades of studies on rotaviruses have demonstrated a substantial disease burden worldwide and a need for the prevention of severe gastroenteritis associated with the virus. Unlike with bacterial pathogens, improvements in hygiene and sanitation alone are insufficient to prevent rotavirus disease. However, it has been noted that, while natural infections did not provide complete protection against subsequent infection and disease, the severity of disease was substantially reduced following primary infections (Bishop et al., 1983; Gladstone et al., 2011; Velazquez et al., 1996). This has provided a rationale for the development of vaccines as an effective strategy to reduce morbidity and mortality associated with rotavirus infections.

Four live-attenuated, oral vaccines against severe rotavirus disease (Rotarix from GlaxoSmithKline Biologicals; RotaTeq, from Merck & Co.; Rotavac from Bharat Biotech; and Rotasiil from Serum Institute of India) have been licensed for global use. Other live-attenuated, oral rotavirus vaccines are being used in specific countries – such as Rotavin in Viet Nam and Lanzhou lamb rotavirus vaccine (LLR) in China. A number of other vaccine candidates are in different stages of clinical development (Kirkwood et al., 2017). Although WHO recommends all countries to introduce rotavirus vaccines into their national immunization programmes (WHO, 2013), as of June 2018 rotavirus vaccines had been introduced in 94 countries and were planned for an additional six countries. Prior to the introduction of vaccines, rotavirus-associated gastroenteritis resulted in nearly half a million deaths in children under 5 years of age annually (Tate et al., 2012). Dramatic reductions in clinical visits, hospitalization and rotavirus-associated deaths have been reported in many high-income and middle-income countries following vaccine introduction (Burnett et al., 2017). However, the performance of the vaccines remains suboptimal in low-income developing countries in Asia and sub-Saharan Africa which have the greatest burden of disease (Jonesteller et al., 2017). Efforts are underway to improve the efficacy of current rotavirus vaccines and to develop more effective vaccines for the future.

1 The purpose of this module is to discuss the epidemiology and pathophysiology of rotavirus disease, to provide an understanding of the rationale behind today’s rotavirus vaccines, their mechanisms of protection, and to explain the expectations regarding their effects on human health as well as the potential need for alternative or improved candidate vaccines.

2 WHO immunological basis for immunization series - Module 21: Rotavirus 2. Rotavirus structure and genome organization

Rotaviruses are non-enveloped, double-stranded RNA (dsRNA) viruses belonging to the family Reoviridae (Estes and Greenberg, 2013). Electron micrographic studies reveal that the complete virus particle resembles a wheel (Latin: rota), with short spikes and a smooth outer rim (Figure 1). The infectious virion is an icosahedral, triple-layered particle (TLP) with a diameter of approximately 100 nm that encloses a genome of 11 dsRNA segments (Prasad and Chiu, 1994). The genome segments encode 6 structural viral proteins (VP1, VP2, VP3, VP4, VP6 and VP7) and 6 nonstructural proteins (NSP1−NSP6). The structural proteins are found in virus particles while nonstructural proteins are found in infected cells but not in the mature virion.

Rotavirus TLPs are composed of an inner core, an intermediate layer and an outer shell, with spikes extending from the smooth surface of the outer shell. Cryo-electron microscopic reconstruction images (Figure 2) and X-ray crystal studies show that 120 molecules of the structural protein VP2, arranged as 60 dimers, forms the core of the TLP (Lawton et al., 1997; McClain et al., 2010). The core encloses the dsRNA genome and replication enzyme complexes, including the viral RNA-dependent RNA polymerase VP1 and the capping enzyme VP3. A middle layer comprising 260 trimers of VP6 surrounds the core (Mathieu et al., 2001). The VP6 layer provides stability and morphological homogeneity to the virus particles and determines group and subgrouping specificities. The outer capsid layer is composed of 260 trimers of glycoprotein VP7 and 60 spikes of VP4 trimers (Shaw et al., 1993; Yeager et al., 1994). Both outer capsid proteins induce neutralizing antibodies and are required for the infectivity of rotaviruses (Hoshino et al., 1985; 1988; Sabara et al., 1985). VP4 is a protease-sensitive protein and is cleaved to VP5* and VP8* to initiate infection (Estes et al., 1981) (Figure 2, inset); these proteins remain noncovalently attached to the virion. The nonstructural proteins are essential for virus replication and morphogenesis, as well as for antagonism of innate immune responses, and include the viral enterotoxin NSP4 (Hu et al., 2012b). A number of structural and biochemical studies have provided crucial insights into the role of the viral and nonstructural proteins of rotaviruses (Estes and Greenberg, 2013) (summarized in Table 1).

Rotavirus dsRNA segments range in size from approximately 660 to 3300 base pairs, with a total genome size of about 18 500 base pairs (Estes and Cohen, 1989; Rixon et al., 1984). Each gene segment is monocistronic and codes for one protein, with the exception of segment 11 which encodes two proteins (NSP5 and NSP6) in some rotavirus strains (Mattion et al., 1991). Rotavirus RNA has been proposed to form conical cylinders around the replication complexes in the core, but detailed structures are yet to be elucidated (Pesavento et al., 2001; Prasad et al., 1996). The dsRNA genome segments, when extracted from viral particles, can be separated by polyacrylamide gel electrophoresis into 11 distinct bands and visualized by ethidium bromide or silver

3 staining. Prior to the widespread use of sequencing methods, the electrophoretic migration profile of the 11 segments was extensively used for classification of rotaviruses (Chanock et al., 1983).

Figure 1: Electron micrograph of rotavirus particles

Triple-layer particles and a few double-layer particles (DLPs, indicated by black arrows). Image provided by MK Estes.

4 WHO immunological basis for immunization series - Module 21: Rotavirus Figure 2: Cryo-electron microscopic reconstruction of a rotavirus particle

A cut-away of a cryo-electron microscopic reconstruction of a rotavirus triple-layer particle. The core layer is formed by VP2 and the intermediate layer is made of the protein VP6. The outer capsid is made of the glycoprotein VP7. Sixty spikes made of the protease-sensitive protein VP4 extend from the VP7 layer, and contain two domains, VP5* and VP8* (inset)

Source: Dormitzer et al., 2002b; Prasad et al., 1996; Settembre et al., 2011. Figure provided by Drs. Liya Hu and B.V.V. Prasad.

5 Specific genotypes are assigned to each of the 11 dsRNA genome segments based on established nucleotide percentage cut-off values. The rotavirus classification working group maintains and evaluates this classification system.

Table 1: Rotavirus gene segments and properties of encoded proteins

Nucleotide % Segment Product identity cut- Genotype name Function off values 1 VP1 83 RNA-dependent RNA RNA-dependent RNA polymerase, ss-RNA polymerase binding, complex with VP3 2 VP2 84 Core protein RNA binding, required for replicase activity of VP1 3 VP3 81 Methyltransferase Guanylyltransferase, methytransferase, ss-RNA binding, complex with VP1 4 VP4 80 Protease-sensitive P type neutralization antigen, viral attachment, homotrimer, protease- enhanced infectivity, virulence, putative fusion region 5 NSP1 79 Interferon Antagonist Interferon antagonist, putative viral E3 ligase, RNA binding 6 VP6 85 Inner capsid Subgroup antigen, trimer, subgroup antigen, protection (intracellular neutralization?); required for transcription 7 NSP3 85 Translation enhancer Acidic dimer, binds 3’ end of viral mRNAs, cellular eIF4G, Hsp90, surrogate of PABP, inhibits host translation 8 NSP2 85 NTPase Basic, octamer, RNA binding, NTPase, NDP kinase, RTPase, helix destabilizing activities, forms viroplasms with VP1 and NSP5 9 VP7 80 Glycosylated G type neutralization antigen, glycoprotein, calcium-dependent trimer 10 NSP4 85 Enterotoxin RER transmembrane glycoprotein, viroporin, intracellular receptor for DLPs, role in morphogenesis of TLPs, interacts with viroplasms and autophagy pathway, modulates intracellular calcium and RNA replication, enterotoxin secreted from cells, virulence 11 NSP5 91 pHosphoprotein Basic, phosphoprotein, RNA binding, protein kinase, forms viroplasms with NSP2, interacts with VP2 and NSP6 NSP6 Interacts with NSP5, present in viroplasms and most virus strains, RNA binding

Source: Adapted from David M. Knipe and Peter Howley. Fields Virology, by permission of the publisher (Wolters Kluwer Health)

6 WHO immunological basis for immunization series - Module 21: Rotavirus 3. Classification methods for rotaviruses

Rotaviruses were earlier classified by a scheme of groups and subgroups, with multiple serotypes/genotypes within each group. On the basis of the electrophoretic mobility of the 11 dsRNA segments, as well as the serological reactivity and genetic variability of VP6, 10 different groups or species of rotavirus, RVA−RVJ, have been identified (Banyai et al., 2017; Matthijnssens et al., 2012; Mihalov-Kovacs et al., 2015). Of these, group A rotaviruses are the most common cause of infections in children worldwide and are the focus of this module. A typical electrophoretic pattern of the group A rotavirus genome comprises four high molecular weight dsRNA segments (1−4), two medium-sized segments (5 and 6), a distinct triplet of segments 7−9, and two smaller segments (10 and 11) (Estes and Cohen, 1989; Pedley et al., 1986) (Figure 3). Three major electropherotypes have been observed in group A rotaviruses, including the long, short and super-short patterns. Four different subgroups (SG) – SGI, SGII, SGI and II, and SG non I/non II – have been distinguished within group A on the basis of reactivity to two VP6 monoclonal antibodies (Greenberg et al., 1983; Hoshino et al., 1984; 2002; Mattion et al., 1989).

In 1985, a binary classification system based on the serological reactivity and genome sequence of VP7 and VP4 – similar to the classification approaches used for influenza viruses – was proposed for group A rotaviruses. Rotaviruses were classified into G serotypes (G for Glycoprotein VP7) and P serotypes (P for Protease-sensitive protein VP4) on the basis of serum neutralization assays using hyperimmune sera raised to prototype viruses and laboratory strains. However, with the increasing availability of sequencing methods and lack of widespread access to serological reagents, the serotype classification has largely been replaced by a classification of rotaviruses into G and P genotypes based on sequence analyses of VP7 and VP4 genes respectively (Matthijnssens et al., 2008). For VP7, the G serotype designations chiefly correlate with G genotype designations and rotavirus G types are described as G1, G2, G3 and so on. Such a correlation is much less evident for VP4, and a dual nomenclature has been adopted to denote the VP4 serotype and genotype classification. The P serotype is first indicated, when known, followed by the genotype number in square brackets. For example, the description P1A[8] suggests that the virus belongs to the 1A VP4 serotype and P[8] VP4 genotype. As of February 2018, 36 G genotypes and 51 P genotypes were described in the database maintained by the rotavirus classification working group (RCWG).1

1 See: https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg (accessed 14 July 2019).

7 In 2008, a comprehensive sequencing-based complete genome classification system was developed for group A rotaviruses whereby a specific genotype was assigned to each of the 11 dsRNA genome segments based on established nucleotide percent cut-off values (Table 1) (Matthijnssens et al., 2008). The abbreviations Gx-P[x]-Ix-Rx-Cx-Mx-Ax- Nx-Tx-Ex-Hx (where x is an Arabic numeral, starting from 1) is used to describe the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 genes, respectively. The RCWG maintains and evaluates this classification system, with updates maintained on the website (Matthijnssens et al., 2011). The group recommends a uniform nomenclature for the description of individual strains, including the following details: RV group/species of origin/country of identification/common name/year of identification/G-and P-type. For instance, the description RVA/Human-tc/USA/ Wa/1974/G1P[8] refers to a tissue culture adapted group A rotavirus isolated from the United States of America in 1974. The common name for this virus isolate is Wa, and it belongs to the G1 VP7 genotype and P[8] VP4 genotype.

Figure 3: Electrophoretic mobility of rotavirus dsRNA segments

Polyacrylamide gel electrophoretic patterns of 11 dsRNA segments of rotavirus strains SA11 (G3P[2]), Wa (G1P[8]), KUN (G2P[4]), AU19 (G1P[6]) and 69M (G8P[10]) are visualized by silver staining. SA11 and Wa represent long patterns, KUN represents a short pattern, and AU19 and 69M represent strains with super-short pattern. Source: Figure adapted from Nakagomi et al., by permission of the publisher (American Society for Microbiology)

8 WHO immunological basis for immunization series - Module 21: Rotavirus 4. Rotavirus epidemiology

4.1 Burden of disease

Rotavirus has often been described as a “democratic” virus because of the ability to cause infections in almost every child worldwide by the age of 5 years (Parashar et al., 1998). Improvements in hygiene and sanitation alone are insufficient to decrease the incidence of disease. Indeed, data from hospital-based sentinel surveillance networks from 2000 to 2008 revealed that the prevalence of rotavirus in children hospitalized with gastroenteritis was approximately 40% in both developing and developed countries (Crawford et al., 2017). However, more than 90% of mortality attributed to rotavirus occurs in low-income countries. A systematic review and meta-analysis conducted before the introduction of global rotavirus vaccination programmes determined that in 2008, rotavirus gastroenteritis resulted in 453 000 deaths globally in children younger than 5 years (Tate et al., 2012). Five countries – Democratic Republic of the Congo, Ethiopia, India, Nigeria and Pakistan – accounted for more than 50% of all deaths attributable to rotavirus. A more recent study estimated that, following the introduction of vaccines, the number of rotavirus deaths in children declined to 215 000 (Figure 4) (Tate et al., 2016). Ten countries – Afghanistan, Angola, Chad, Democratic Republic of the Congo, Ethiopia, India, Kenya, Niger, Nigeria and Pakistan – accounted for 65% of rotavirus-associated deaths in 2013.

4.2 Rotavirus strain distribution

The epidemiology of rotaviruses is complex; human strains that include more than 70 individual G and P genotype combinations have been reported from over 100 countries worldwide, and spatiotemporal fluctuations in genotype prevalence occur in different populations (Banyai et al., 2012; Patel et al., 2013b). Cross-species transmission of rotavirus has been documented particularly in low-income countries, with detection of human strains that share genetic and antigenic homology with animal rotavirus strains. Despite this diversity, comprehensive reviews of strain surveillance studies prior to vaccine introduction showed that six rotavirus genotypes, G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8] were the predominant cause of rotavirus hospitalizations worldwide (Banyai et al., 2012) . In addition to these six, certain genotypes (e.g. P[6] strains in parts of Africa) are known to be important regionally (Todd et al., 2010).

Surveillance networks continue to monitor rotavirus strains even after the introduction of rotavirus vaccines. These studies allow for the assessment of potential changes in rotavirus epidemiology following vaccine introduction and can track the evolution of escape mutants based on selective pressure due to the vaccines. Genotyping data from 46 967 samples from 81 countries between 2007 and 2012 showed that the

9 six globally dominant strains accounted for more than 70% of all global genotypes (Doro et al., 2014). Regional differences in strain distribution and spatiotemporal changes were observed in countries both with and without rotavirus vaccination programmes. Reports of increased prevalence of G2P[4] rotaviruses in parts of Australia, Europe and South America, where the monovalent G1P[8] vaccine was used, have raised questions about heterotypic immunity to the vaccine and vaccine-induced selection pressure on G2P[4] viruses (Carvalho-Costa et al., 2009; Kirkwood et al., 2011; Zeller et al., 2010; ). In Australia, a state-based competitive tender process resulted in the use of RotaTeq in some states and Rotarix in others, providing an opportunity to assess the impact of each vaccine on genotypic diversity post-vaccine introduction. Increased prevalence of the G12P[8] genotype was reported in states using RotaTeq while the dominance of equine-like G3P[8] and G2P[4] was reported in states and territories using Rotarix (Roczo-Farkas et al., 2018). While these data suggest a role for different immunological pressures from each vaccine driving the diversity of rotavirus strains, the absence of selective pressure has been reported in other countries. Higher prevalence of G2P[4] was reported simultaneously in other countries with low rates of monovalent vaccine use and where the pentavalent rotavirus vaccine was used in the national immunization programme. Although less common, vaccine virus-derived genes have also been detected in virulent reassortant wild-type strains, emphasizing the need for continuing strain surveillance studies even after the introduction of rotavirus vaccines (Boom et al., 2012; Bucardo et al., 2012; Donato et al., 2012; Hemming and Vesikari, 2012; Rose et al., 201). Whole genome characterization studies will be important for tracking the evolution of emerging rotaviruses and for determining whether vaccine introduction induces selective pressure on circulating strains resulting in antigenic changes to escape immunity (Patton, 2012).

4.3 Seasonality of rotavirus disease

The seasonality of rotavirus disease varies widely across the world. Typically, rotavirus infections are seen during the winter months in temperate regions of the world, while infections occur throughout the year in warmer tropical countries (Cook et al., 1990). A number of studies have attempted to determine the link between rotavirus seasonality and conditions such as temperature, rainfall and humidity; however, the results remain inconclusive (D’Souza et al., 2008; Hashizume et al., 2008; Levy et al., 2009; Patel et al., 2013b; Purohit et al., 1998). Interestingly, a recent study found that, rather than the geographical region, a country’s income level was the strongest predictor of seasonality (Patel et al., 2013b). Higher birth rates and higher transmission rates were suggested as possible factors facilitating the year-round circulation of rotavirus in some regions (Pitzer et al., 2009; Pitzer et al., 2011). Other factors that affect seasonality of rotaviruses remain to be ascertained.

Changes in the timing and seasonality of rotavirus disease have been described in some regions since the introduction of rotavirus vaccines. Delayed start of the rotavirus epidemic season and blunting of peaks have been described (Enweronu-Laryea et al., 2014; Leshem et al., 2015; Muhsen et al., 2015; Richardson et al., 2010). In the United States of America, the highly regular annual epidemic peak has been replaced by biannual peaks (Aliabadi et al., 2015). Mathematical models which predicted biannual epidemics at high vaccine coverage have been used to gain insight into the population dynamics underlying these changes (Pitzer et al., 2012). One hypothesis is that, because of moderate vaccine coverage rates, there is an accumulation of unvaccinated susceptible children over successive rotavirus seasons and this leads to larger epidemics every alternate year.

10 WHO immunological basis for immunization series - Module 21: Rotavirus 4.4 Age at infection

While rotavirus infects almost every child by the age of 5 years, differences in median age at primary infection are observed between developing and developed countries. Neonatal rotavirus infections are predominantly asymptomatic (Cicirello et al., 1994; Dunn et al., 1993; Sukumaran et al., 1992), although the association of neonatal infections with gastrointestinal symptoms has been documented in some settings (Cameron et al., 1978; Iturriza Gomara et al., 2004; Ramani et al., 2008; Rochi et al., 1981; Sharma et al., 2002). Overall, the median age of primary infection is younger in developing countries, with a majority of infections occurring before the age of 1 year (Bresee et al., 1999). By contrast, the vast majority of infections in children in developed countries occur after 1 year of age. Among other factors, differences in seasonality resulting in year- round exposure to the virus in developing countries are thought to contribute to these differences in age at primary infection (Bresee et al., 1999). Cohort studies of natural infection in children show that reinfections are common although, in most settings, the severity of disease decreases with each repeat infection (Bishop et al., 1983; Fischer et al., 2002; Gladstone et al., 2011; Velazquez et al., 1996). This suggests that protection from natural infection may be short-lived. Rotavirus can also cause infections in older children and adults (Anandan et al., 2014; Anderson and Weber, 2004). This is particularly well documented in parents of children with rotavirus diarrhoea and has also been described in immunocompromised persons and older individuals. However, rotavirus infections in these age groups are largely asymptomatic or are mild-to-moderate in severity. Changes in age demographics have been documented following the introduction of rotavirus vaccines. For example, rotavirus infection was most common in children over 5 years of age before vaccine introduction in Finland; following vaccine introduction, rotavirus infections are now most common in unvaccinated children aged 6−16 years of age and individuals over 70 years of age (Markkula et al., 2017).

Figure 4: Trends in the number of rotavirus deaths by region (2000–2013)

Source: Tate et al., 2016, reprint by permission by the publisher (Oxford University Press)

11 5. Rotavirus replication

Much of our understanding of the rotavirus replication cycle comes from infections in monkey kidney epithelial cells or polarized, transformed intestinal epithelial cell cultures. In vivo studies demonstrate that rotavirus replication occurs primarily in differentiated enterocytes at the tip of the small intestinal epithelium (Estes and Greenberg, 2013). Replication in enteroendocrine cells has also been documented in both mice and in vitro cultures (Hagbom et al., 2011; Saxena et al., 2015). Virus entry is thought to occur predominantly through the apical membrane in polarized intestinal epithelial cells, although basolateral entry has been documented for some human rotaviruses (Cevallos Porta et al., 2016; Realpe et al., 2010). The role of basolateral entry in virus pathogenesis has not been fully elucidated but may be important given that extraintestinal spread of rotavirus, antigenaemia and viraemia are well documented (Blutt et al., 2007).

Rotavirus attachment and entry into cells is a complex, multi-step process that begins with the interaction of the outer capsid protein VP4 with cellular receptors (Figure 5, step 1). Attachment to host cells is mediated by the interaction of the VP8* domain of VP4 with cellular glycans in a virus genotype-dependent manner (Ramani et al., 2016a). For decades, sialic acid was considered the key mediator of interactions with VP8*; animal rotaviruses were thought to bind terminal sialic acid residues on cellular surfaces while human rotaviruses were thought predominantly to bind internal sialic acid residues (Ciarlet et al., 2002; Delorme et al., 2001; Dormitzer et al., 2002a; Dormitzer et al., 2002b; Fukudome et al., 1989; Haselhorst et al., 2009; Yolken et al., 1987; Yu et al., 2011; Yu et al., 2012). Recent studies resulted in a paradigm shift in our understanding of rotavirus−glycan interactions. The VP8* of many human rotavirus strains have been shown to bind nonsialylated glycoconjugates called histo-blood group antigens (HBGAs) and these interactions are genotype-dependent (Bohm et al., 2015; Hu et al., 2012a; Hu et al., 2015; Huang et al., 2012; Liu et al., 2013; Liu et al., 2012; Ramani et al., 2013; Liu et al., 2016). HBGAs are blood group glycan determinants that are present on epithelial cell surfaces and in mucosal secretions, with an expression profile that is genetically determined. The binding of human rotavirus VP8* to HBGA appears to be critical for virus pathogenesis, interspecies transmission, differences in epidemiology, and susceptibility and immune response to live-attenuated rotavirus vaccines (Gunaydin et al., 2016; Imbert-Marcille et al., 2014; Kazi et al., 2017; Lee et al., 2018; Payne et al., 2015; Van Trang et al., 2014; Zhang et al., 2016). In addition to sialic acid and HBGAs, other receptors/coreceptors for different rotavirus strains have been identified – including integrins, heat shock protein 70 (HSP70), junctional adhesion molecule A (JAMA) and tight junction proteins ZO-1 and occludin (Graham et al., 2003; Guerrero et al., 2002; Guerrero et al., 2000; Torres-Flores et al., 2015; Zarate et al., 2003; Zarate et al., 2000; Zarate et al., 2004). Many of these receptors are associated

12 WHO immunological basis for immunization series - Module 21: Rotavirus with lipid rafts and interact with the VP5* domain of VP4 or with VP7 to facilitate virus entry. The virus is then internalized into cells by either clathrin-dependent or clathrin- and caveolin-independent endocytic pathways, depending on the virus strain (Diaz-Salinas et al., 2013; Diaz-Salinas et al., 2014; Gutierrez et al., 2010) (Figure 5, step 1). Following entry, the removal of the outer capsid proteins occurs due to the low Ca2+ concentrations in endosomes to release the transcriptionally active double-layered particles (DLPs) into the cytoplasm (Figure 5, step 2).

Within the cytoplasm, DLPs produce capped, nonpolyadenylated, (+) single-stranded RNA (ssRNA) transcripts that function both as a template for complementary negative strand synthesis and for translation of viral proteins (Jayaram et al., 2004; Silvestri et al., 2004). Viral RNA and newly synthesized proteins are packaged into newly synthesized DLPs in specialized cytoplasmic inclusion bodies called viroplasms (Figure 5, step 3). Viroplasm formation requires the presence of at least two nonstructural proteins NSP2 and NSP5, and viroplasms are associated with lipid droplets (Cheung et al., 2010; Crawford and Desselberger, 2016). Nonstructural protein NSP4 then serves as an intracellular receptor for the budding of DLPs into the endoplasmic reticulum (ER) (Au et al., 1989; Lopez et al., 2005) (Figure 5, step 4). In the ER, transiently enveloped rotavirus particles are formed (Figure 5, step 5). However, the envelope is lost when the outer capsid proteins VP7 and VP4 are added to the particles to form TLPs (Cuadras et al., 2006) (Figure 5, step 6). Progeny virions have been shown to be released from nonpolarized cells through cell lysis or by a Golgi-independent nonclassical vesicular transport mechanism in polarized epithelial cells (Jourdan et al., 1997; McNulty et al., 1976) (Figure 5, step 7).

13 Figure 5: Overview of rotavirus replication cycle

Rotavirus entry involves attachment to cellular receptors (gangliosides such as GM1 and GD1a or histo-blood group antigens) in a virus VP8* genotype-dependent manner and internalization by clathrin-dependent or clathrin- and caveolin-independent endocytic pathways (step 1). Other proposed receptors/coreceptors for rotavirus cell entry include integrins, heat shock protein and junctional proteins such as junctional adhesion molecule A. The removal of the viral outer capsid proteins following entry results in the release of the transcriptionally active double-layered particles into the cytoplasm (step 2). Viral mRNA serves as a template for new RNA synthesis and is translated into proteins within specialized virus replication factories called viroplasm (step 3). The newly formed double-layered particles bud into the endoplasmic reticulum (ER) using NSP4 as an intracellular receptor (step 4). In the ER, the outer capsid proteins VP4 and VP7 are added to the double-layered particles and t he virus acquires a transient envelope (step 5). The envelope is subsequently lost (step 6) and the mature progeny virions are released from cells through cell lysis or by a Golgi-independent nonclassical vesicular transport mechanism (step 7).

Source: Adapted from David M. Knipe and Peter Howley. Fields Virology, by permission of the publisher (Wolters Kluwer Health).

14 WHO immunological basis for immunization series - Module 21: Rotavirus 6. Rotavirus pathogenesis

6.1 Clinical presentation

As noted above, the spectrum of clinical presentations following rotavirus infections range from subclinical, asymptomatic infections and mild diarrhoea to severe diarrhoea and vomiting resulting in dehydration (Parashar et al., 2013). The incubation period for rotavirus diarrhoea is short and is estimated to be less than 48 hours. Rotavirus disease in children presenting to emergency rooms and those requiring hospitalization is often characterized by watery diarrhoea, vomiting and fever that can result in electrolyte imbalance, shock and, in some cases, death (Uhnoo et al., 1986). Vomiting is a predominant feature of rotavirus gastroenteritis. The onset of vomiting and diarrhoea is abrupt, with vomiting preceding the other symptoms in many cases. Moderate fever (temperature > 39°C) and malaise are also associated with rotavirus disease.

Rotavirus-associated diarrhoea is nonbloody and noninflammatory, with almost no apparent change in inflammatory markers such as faecal calprotectin and serum C reactive protein levels (Chen et al., 2012; Wiegering et al., 2011). While rotavirus-induced gastroenteritis is generally clinically indistinguishable from diarrhoeal diseases caused by other enteric viruses, such as norovirus and astrovirus, the disease is more severe than diarrhoea caused by other agents. Specifically, the proportion of diarrhoea episodes caused by rotavirus increases from patients in the community requiring home care only (5−10%), or outpatient care (15−20%) to those requiring hospitalization (30−50%) (Tate et al., 2016). In addition, seasonality is a predictor, at least in temperate climates where rotavirus infections are highly likely to be the causative agent in children with gastrointestinal symptoms during the winter (Cook et al., 1990; Patel et al., 2013b). Replacement of fluids and electrolytes is the main treatment for rotavirus gastroenteritis (King et al., 2003). In immunocompetent infants, symptoms typically resolve within 3−7 days. Rotaviruses are also an important cause of nosocomial infections (Chandran et al., 2006). Chronic diarrhoea and prolonged virus shedding have been documented in immunocompromised individuals – particularly those with severe combined immunodeficiency (SCID) and in transplant patients (Adeyi et al., 2010; Patel et al., 2012b; Peigue-Lafeuille et al., 1991; Stelzmueller et al., 2007). While infections are usually subclinical in adults, severe illness can occur in older persons (Anderson and Weber, 2004; Echeverria et al., 1983).

Apart from diarrhoea and vomiting, the association of rotavirus with necrotizing enterocolitis (NEC), intestinal perforation and pneumatosis intestinalis in neonates has also been described (Capitanio and Greenberg, 1991; de Villiers and Driessen, 2012; Keller et al., 1991; Sharma et al., 2004; Sizmaz et al., 2012; Stabell et al., 2013). Among other gastrointestinal presentations, the relationship between rotavirus

15 and intussusception is particularly important in the context of rotavirus vaccines. Intussusception is a condition in which the intestine folds in on itself and may lead to bowel obstruction. While many cases of intussusception self-resolve, others can be fatal in the absence of surgical intervention (Bines et al., 2004). In 1998, the first rotavirus vaccine, a rhesus-human reassortant strain (Rotashield) was licensed in the USA and was recommended for routine immunization of children (Murphy et al., 2001; Patel et al., 2009). Ultimately, the vaccine was withdrawn because post-licensure studies found an increased risk of intussusception following vaccination. Vaccination with Rotashield was estimated to result in one excess case of intussusception per 10 000 infants (Peter et al., 2002). Assessment for the risk of intussusception, therefore, became a key component of subsequent rotavirus vaccine trials. Two other rotavirus vaccines, RotaTeq and Rotarix that were in various stages of clinical testing when Rotashield was withdrawn were tested in 60−70 000 infants to assess the risk of intussusception prior to licensure. Although the large clinical trials identified no risk of intussusception, the much larger post-licensure studies identified a low risk of intussusception with both rotavirus vaccines. Post-marketing surveillance conducted following vaccine introduction in several high- and middle-income countries (Australia, Brazil, Mexico, Singapore, United Kingdom and USA) showed approximately 1−6 excess cases of intussusception for every 100 000 vaccinated infants (Carlin et al., 2013; Haber et al., 2013; Weintraub et al., 2014; Yih et al., 2014; Yung et al., 2015). This risk is primarily seen during the week following the first dose of vaccine. However, the global health agencies strongly support rotavirus vaccination in infants since the benefits of vaccination far outweigh the risk of intussusception (Yen et al., 2016). Furthermore, a recent study found that the risk of intussusception after rotavirus vaccine administration was not higher than the background risk of intussusception in seven low-income countries in sub-Saharan Africa (Tate et al., 2018). A number of factors are hypothesized as playing a role in the reduced risk of intussusception in low-income countries compared to high-income countries, including younger age at vaccination and poor vaccine uptake in these settings compounded by the administration of oral poliovirus vaccine.

6.2 Extraintestinal infections

While gastroenteritis is the primary clinical manifestation of rotavirus infection, systemic infections and extraintestinal presentations have also been described. The virus has been detected in several nonintestinal tissues in animal models. Rotavirus RNA has been detected in the cerebrospinal fluid of children presenting with seizures and encephalitis; however, causality has remained unclear. A more direct evidence for the association between rotavirus with neurological manifestations has come from the observation of a reduction in the number of seizure hospitalizations in vaccinated children following the introduction of rotavirus vaccines (Payne et al., 2014; Pringle et al., 2018).

Rotavirus antigenaemia (detection of viral antigens in serum) and viraemia (detection of infectious virus in serum) have been reported in children in many countries (Blutt et al., 2007; Chiappini et al., 2005; Fischer et al., 2005; Hemming et al., 2014; Moon et al., 2012; Ramani et al., 2010a). However, direct evidence supporting the role of antigenaemia or viraemia in extraintestinal disease is lacking. In one study, children with rotavirus antigenaemia had more severe gastrointestinal symptoms than those without antigenaemia; however, there were no differences in extraintestinal presentations between the two groups (Ramani et al., 2010a). A transient increase

16 WHO immunological basis for immunization series - Module 21: Rotavirus in serum transaminases has been observed in children with rotavirus gastroenteritis (Akelma et al., 2013; Grimwood et al., 1988; Kovacs et al., 1986; Teitelbaum and Daghistani, 2007). Rotavirus has also been associated with a number of other extraintestinal diseases such as type I diabetes, otitis media, Kawasaki syndrome and respiratory illnesses (Hadziselimovic et al., 1984; Honeyman et al., 2000; Matsuno et al., 1983; Zheng et al., 1991). It remains to be ascertained whether the association with these diseases is merely temporal or if rotavirus plays an etiological role.

6.3 Pathophysiology and mechanisms of disease

Much of the description of pathological changes following rotavirus infections comes from studies in animals (Estes and Greenberg, 2013). Almost all studies confirm that most changes are restricted to the small intestine, although the severity of findings varies between animal species and age at infection. Studies in calves and piglets have revealed that rotavirus infection caused the villus epithelium to change from columnar to cuboidal, resulting in shortening and stunting of the villi (Mebus et al., 1974; Pearson & McNulty, 1979). The cells at the villus tips became denuded (Figure 6), while in the underlying lamina propria, the numbers of reticulum-like cells increased and mononuclear cell infiltration was observed. Early studies of biopsies from children also demonstrated varying degrees of histological changes, ranging from no changes or slight lesions, including enterocyte vacuolization and loss, to significant changes such as villous atrophy and blunting, crypt hyperplasia and increased infiltration of inflammatory cells in the lamina propria (Davidson and Barnes, 1979; Holmes et al., 1975). Studies in piglets and mice also revealed that disease symptoms can occur prior to the development of histological changes, indicating that the histopathology alone does not explain clinical disease (Osborne et al., 1991; Ramig, 2004; Ward et al., 1996). The mechanisms of enterocyte damage are not fully understood. Proposed mechanisms include disruption of the microvillus cytoskeletal network, virus-mediated apoptosis and disruption of normal cellular homeostasis pathways (Brunet et al., 2000a; Brunet et al., 2000b; Bhowmick et al., 2012; Boshuizen et al., 2003; Chaibi et al., 2005; del Castillo et al., 1991; Hyser et al., 2010; Michelangeli et al., 1991).

Rotavirus-induced diarrhoea is multifactorial. The two main proposed mechanisms are: 1) osmotic diarrhoea due to malabsorption, secondary to enterocyte damage or decreased epithelial absorptive function, and 2) secretory diarrhoea due to the activity of the virus-encoded enterotoxin NSP4 and activation of the enteric nervous system (ENS) (Ball et al., 1996; Hagbom et al., 2011; Lundgren et al., 2000; Osborne et al., 1991). Apart from the loss of absorptive cells in the villi and the proliferation of secretory crypt cells, rotavirus infection and NSP4 mediate the inhibition of the Na+ solute cotransport systems involved in fluid reabsorption (Halaihel et al., 2000). However, the impact of reduced absorptive capacity on clinical disease is unclear as a result of the success of oral rehydration solutions (ORS) in rapidly correcting electrolyte and water loss in children with severe diarrhoea. This suggests that rotavirus infection in the small intestine may be patchy, and sufficient numbers of intact enterocytes are present to facilitate ORS uptake (Crawford et al., 2017). NSP4 is a viral enterotoxin and calcium agonist and was first shown to induce diarrhoea in mice (Ball et al., 1996). NSP4 increases cytoplasmic calcium levels resulting in the activation of calcium- dependent chloride channels that leads to excessive secretion of chloride ions and water, thereby resulting in secretory diarrhoea (Dong et al., 1997; Hyser et al., 2010; Pham et al., 2017). Further, NSP4 may cause disruption of tight junctions between enterocytes and may alter paracellular permeability (Dickman et al., 2000;

17 Tafazoli et al., 2001). Virus infection and NSP4 also activate the ENS through secretion of 5-hydroxytryptamine (5-HT) (Bialowas et al., 2016; Hagbom et al., 2011). The release of 5-HT is calcium-dependent and can activate enteric nerves in the gut leading to altered intestinal motility. The release of 5-HT can also activate vagal nerves associated with nausea and vomiting. In mice, opioid receptor agonists and drugs that inhibit parasympathetic nerves attenuate rotavirus diarrhoea, supporting the role of the ENS in rotavirus disease (Istrate et al., 2014).

Figure 6: Histological changes following rotavirus infection

The top panel shows the normal histological appearance of ileum from an 8-day old gnotobiotic pig. Normal mature vacuolate absorptive cells cover the villi (haematoxylin and eosin [H&E)] stain). The lower panel shows the ileum from an 8-day old gnotobiotic pig after oral inoculation with virulent human rotavirus (Wa strain). Severe villous atrophy and early crypt hyperplasia are evident in the H&E stain. Source: Reproduced by permission of Dr LA Ward, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH

18 WHO immunological basis for immunization series - Module 21: Rotavirus 7. Rotavirus immunity

7.1 Methods for detection of rotavirus

Rotavirus can be detected in stool samples from children with gastroenteritis by a number of methods, including electron microscopy (EM), polyacrylamide gel electrophoresis (PAGE), enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and isolation of virus by cell culture (Desselberger, 2014; Herring et al., 1982; Morinet et al., 1984; Xu et al., 1990). Historically, the first diagnosis of rotavirus was made by EM, including the use of hyper-immune sera to increase sensitivity of detection by immuno-electron microscopy. PAGE was also widely used, with the electrophoretic migration pattern of the 11 dsRNA segments being used to characterize virus strains. Currently, children presenting with gastroenteritis are not tested routinely for rotavirus in most clinical settings because treatment focuses on rehydration and the diagnosis does not alter treatment strategies (Parashar et al., 2013). When laboratory diagnosis for rotavirus is requested by the child’s consulting physician, viral antigen is usually detected by commercially-available ELISA or immunochromatography assays (Crawford et al., 2017). In surveillance studies and vaccine trials, ELISAs are complemented with RT-PCR or RT-qPCR arrays for detection and characterization of rotavirus genotype and viral load, respectively. In children with rotavirus gastroenteritis, the virus can be detected by ELISA for about a week following infection. However, by using more sensitive RT-PCR assays, the virus can be detected for longer periods (Ramani et al., 2010b; Richardson et al., 1998). The correlation between viral load and severity of disease has been documented in children but not in neonates (Kang et al., 2004; Ramani et al., 2010b).

7.2 Rotavirus vaccines

Early epidemiological studies showed that, while repeat infections with rotavirus were common, subsequent infections were often less severe than the primary infection (Bishop et al., 1983; Fischer et al., 2002; Gladstone et al., 2011; Velazquez et al., 1996). This observation provided a rationale for the development of rotavirus vaccines, with a target of reducing the morbidity and mortality associated with severe rotavirus gastroenteritis. Several live-attenuated candidate vaccines have been tested in clinical trials and licensed. Efforts are ongoing to improve responses to current vaccines and to develop next-generation vaccines. Live-attenuated candidate and licensed vaccines can be broadly categorized as animal rotavirus strains, animal-human reassortant strains, and attenuated human rotavirus strains. In addition, nonreplicating vaccines are also undergoing clinical trials.

19 7.2.1 Animal rotavirus vaccines Initial efforts for rotavirus vaccine development focused on a “Jennerian” approach based on the natural attenuation of animal rotaviruses in humans. In this approach, animal rotaviruses were further attenuated through multiple passages in cell culture (Table 2). The cell culture-adapted bovine rotavirus strain RIT4237 was the first rotavirus vaccine tested in humans. The original bovine isolate, Nebraska Calf Diarrhoea Virus (NCDV), was highly attenuated with over 150 cell culture passages, primarily for the purpose of preventing disease in calves (Delem et al., 1984). In the first efficacy studies in Finland, a single dose of RIT4237 induced 50% protection against any gastroenteritis and 88% protection against severe disease in children (Vesikari et al., 1984). Subsequent studies in Gambia and Peru showed lower efficacy of only 33% against gastroenteritis in Gambia, although the severity of disease was not measured (Hanlon et al., 1987; Lanata et al., 1989). No protection was conferred by the vaccine in a study in Rwanda and in a Native American population (De Mol et al., 1986; Santosham et al., 1991). The development of this vaccine was discontinued primarily because of lower protection in developing countries although this finding is now documented with all licensed rotavirus vaccines and in studies of natural history in children. Other key observations with RIT4237 also remain broadly applicable to several other rotavirus vaccines, including greater efficacy against severe rotavirus gastroenteritis than mild- to-moderate disease and interference by oral poliovirus vaccine (OPV) (Vesikari, 2016).

The next vaccines to be developed were the bovine Wistar Calf 3 (WC3) vaccine and a rhesus rotavirus vaccine (RRV). WC3 is a G6P[5] strain isolated from a calf with diarrhoea in Pennsylvania (Clark et al., 1986). Passaged about 20 times, this vaccine showed 87% efficacy in clinical trials in the USA against relatively severe rotavirus gastroenteritis (clinical severity score of > 8) and only 31% protection in a trial conducted in the Central African Republic (Georges-Courbot et al., 1991; Treanor et al., 1995). The strain was not developed further as a monovalent vaccine. However, the WC3 strain forms the backbone for the bovine-human reassortant vaccine currently licensed as RotaTeq. RRV was originally isolated from a 3-month-old rhesus monkey with acute diarrhoea and developed as a vaccine candidate after passage in monkey kidney and fetal lung cell lines (Stuker et al., 1980). This vaccine showed varying efficacy in developed countries (12−90%) but was found to be highly efficacious in studies in Venezuela (90%) (Christy et al., 1988; Perez-Schael et al., 1990; Vesikari et al., 1990). However, the vaccine was also highly reactogenic, causing fever in humans (Vesikari et al., 1986). Although this candidate vaccine was not itself continued for development, it formed the backbone for RRV-TV, a tetravalent reassortant rotavirus vaccine that was the first rotavirus vaccine to be licensed.

The only animal rotavirus vaccine to be licensed for use in humans is the Lanzou lamb rotavirus vaccine (LLR). The virus was isolated from a lamb with diarrhoea in 1985 and attenuated through serial passages in cell culture (Bai et al., 1994). The vaccine was developed at the Lanzhou Institute for Vaccines, China, and has been available since 2000. As of the end of 2014, a total of 60 million doses of LLR had been distributed to children in China (Zhen et al., 2015). A 35% protection rate was estimated in one effectiveness study, while a one-dose vaccine effectiveness of 44% against laboratory- confirmed rotavirus gastroenteritis and 77% against rotavirus hospitalizations was reported in another study (Fu et al., 2012; Fu et al., 2010; Zhen et al., 2015). There are no formal head-to-head comparisons of the LLR vaccine with other licensed rotavirus vaccines.

20 WHO immunological basis for immunization series - Module 21: Rotavirus Table 2: Animal rotavirus vaccine candidates

Vaccine Species Virus strain Status Key finding

RIT4237 Bovine G6P[5] Not continued First rotavirus vaccine in trials for humans. Varied efficacy between developed and developing countries WC3 Bovine G6P[7] Not continued Low efficacy in clinical trials. Backbone for RotaTeq vaccine RRV Rhesus G3P[5] Not continued Efficacious but highly reactogenic. Backbone for RRV- TV reassortant vaccine LLR Lamb G10P[12] Licensed in No formal efficacy comparisons with other rotavirus China vaccines

7.2.2 Animal-human reassortant rotavirus vaccines Animal human reassortant rotavirus vaccines follow a modified Jennerian approach; they contain an animal strain backbone and incorporate one or more human VP7 or VP4 genes, with the goal of developing multivalent, attenuated vaccines that induce protection against severe illness caused by human rotavirus infections. The rhesus–human reassortant tetravalent vaccine RotaShield (RRV-TV, manufactured by Wyeth Laboratories) was the first reassortant vaccine licensed for human use. RRV-TV contains the G3 RRV virus and reassortants with the VP7 genes of G1, G2, or G4 human rotavirus serotypes on the rhesus G3P[5] backbone (Midthun et al., 1985; Midthun et al., 1986). Similar to RRV, RRV-TV was reactogenic but was more efficacious than the parental vaccine. Three doses of the vaccine were tested in clinical trials and high efficacy of 70−90% against severe gastroenteritis was demonstrated in Finland, USA and Venezuela (Joensuu et al., 1997; Perez-Schael et al., 1997; Rennels et al., 1996). RotaShield was licensed for use in the USA in 1998–99; however, the vaccine was withdrawn from the market by the manufacturer less than a year after licensure because of an association with intussusception (Murphy et al., 2001; Patel et al., 2009). Vaccination with Rotashield resulted in one excess case of intussusception per 10 000 and most cases occurred within the first week after the first dose of the vaccine, coinciding with the peak virus replication and febrile reactions. The majority of cases of intussusception occurred in infants between 3 and 9 months of age who were part of a vaccination catch-up programme. It is now known that this age risk window is similar for naturally-occurring cases of intussusception, placing an emphasis on age at vaccination for rotavirus vaccines. Rotashield was withdrawn before the vaccine was launched in Europe or tested in developing countries, and post-licensure studies have identified a low risk of intussusception even with the other rotavirus vaccines. Since RotaShield was disproportionately associated with cases of intussusception in infants receiving their first dose at ≥ 90 days of age, RRV-TV has been further evaluated recently in Ghana using a modified dose schedule. The first dose was given during the neonatal period and the second before 60 days of age. The vaccine showed 63% efficacy in this population (Armah et al., 2013).

21 Bovine-human reassortant vaccines have also been licensed for use. RotaTeq (also called RV5, manufactured by Merck & Co. Inc,) is a pentavalent bovine-human reassortant rotavirus and contains four human G-type reassortants (G1–G4) and the P[8] P-type reassortant on the G6P[5] WC3 bovine rotavirus backbone (Clark et al., 1996). The vaccine is given in a three-dose schedule with each dose having a titre of approximately 106 plaque forming units (PFU). The titre was determined in a small efficacy study in Finland that showed that a dose that was one log higher did not substantially increase efficacy and was likely to be more expensive to produce, while a dose that was one log lower was less efficacious (Vesikari et al., 2006a). In a large clinical trial of nearly 70 000 infants for the assessment of safety and efficacy of RotaTeq in 11 countries, efficacy against any G1–G4 rotavirus gastroenteritis in the first rotavirus season following vaccination was 74% (95 % confidence interval [CI], 67−80%) and efficacy against severe gastroenteritis was 98% (95% CI, 88−100%) (Vesikari et al., 2006b). Shedding of infectious virus after the first dose of vaccination was found in about 9% by ELISA, although shedding could be measured by RT-PCR in 50% of the recipients (Markkula et al., 2015). No significant differences in the incidence of fever and irritability were detected between the vaccine and placebo groups. The efficacy of RotaTeq was, however, lower in developing countries. In a study conducted in Ghana, Kenya and Mali, the efficacy against severe gastroenteritis was 39% (95% CI, 19−55%) (Armah et al., 2010). Similarly in Bangladesh and Viet Nam, the efficacy against severe gastroenteritis was 48% (95% CI, 2−66%) (Zaman et al., 2010). Post-licensure effectiveness analyses continue to reflect these numbers. On the basis of a systematic review of post-licensure data from 2006 to 2016 in 20 studies from countries with low mortality, the median vaccine efficacy of RotaTeq was estimated to be 90% (range, 63–100%) (Jonesteller et al., 2017). For high-mortality countries, the median efficacy was 45% (range, 43–92%) based on seven studies (Figure 7A). Overall vaccine efficacy point estimates were higher among children under 12 months of age than among those of 12 months and older. It has been shown that the G1 and P[8] reassortants included in RotaTeq may reassort with each other to form vaccine- derived G1P[8] viruses (Donato et al., 2012; Hemming and Vesikari, 2012). These viruses may be more virulent than the original single-vaccine viruses, resulting in a low rate of diarrhoea after vaccination. These strains may also be transmissible, particularly to immunocompromised persons, resulting in prolonged shedding (Payne et al., 2010; Patel et al., 2010).

A second set of bovine-human reassortant vaccine candidates are based on the bovine United Kingdom (UK) strain as the parental backbone (Hoshino et al., 1997). Originally developed by Wyeth, the technology for production of bovine(UK)-human rotavirus reassortants has been licensed by the United States National Institutes of Health (NIH) to manufacturers in China, Brazil and India, (Kirkwood et al., 2017). A bovine (UK) reassortant pentavalent vaccine (BRV-PV, also called RotaSIIL), containing G1, G2, G3, G4 and G9 genotypes developed by the Serum Institute of India, was found to be safe and well-tolerated in adults, children and infants. A phase IIb study showed IgA seroconversion rates of 56.7% and 60% after the second and third doses of the vaccine respectively (Zade et al., 2014). A phase III clinical efficacy study showed that BRV-PV reduced severe rotavirus diarrhoea by 39.5% and very severe rotavirus gastroenteritis by 55% (Kulkarni et al., 2017). A heat-stable form of BRV-PV was tested in a clinical trial among children in Niger (Isanaka et al., 2017). Three doses of the heat-stable BRV-PV were safe and had an efficacy of 67% against severe rotavirus gastroenteritis. RotaSIIL received WHO prequalification for global use in 2018.

22 WHO immunological basis for immunization series - Module 21: Rotavirus A tetravalent vaccine (BRV-TV) containing G1, G2, G3 and G4 was produced by Shantha Biologicals of India using the same bovine UK backbone and G1−G4 human reassortant viruses (Dhingra et al., 2014). This vaccine was tested in a clinical trial in Indian infants and did not meet the immunogenicity non-inferiority primary endpoint when compared to the RotaTeq vaccine (Saluja et al., 2017). The anti-rotavirus IgA seroconversion rate was 47% (95% CI, 43−52%) for BRV-TV compared to 61% (95% CI, 57−66%) for RotaTeq. Phase I studies are ongoing for a hexavalent vaccine containing G1−G4, G8 and G9 produced by Wuhan Institute of Biological Products, China and a pentavalent vaccine (G1−G4, G9) produced by the Institute Butantan in Brazil (Kirkwood et al., 2017).

7.2.3 Human rotavirus vaccines The other rotavirus strains developed for use as live vaccine candidates have been derived from infected humans. As humans are the natural hosts for these viruses, it has been suggested that – even after attenuation with passage in cell culture – the immune responses stimulated by human strains may be greater and more consistent than the responses elicited after vaccination with animal strains. Human rotavirus vaccine Rotarix (also called RV1, manufactured by GlaxoSmithKline Biologicals) is currently the most widely used rotavirus vaccine worldwide. Rotarix belongs to the G1P[8] genotype and was derived from a human strain (89-12) isolated from a child with rotavirus gastroenteritis in Cincinnati, USA, in 1989 (Bernstein and Ward, 2006). The strain was chosen because children with infection from the virus appeared to be fully protected against subsequent severe gastroenteritis from homotypic rotavirus strains (Bernstein et al., 1991). The vaccine is recommended in a two-dose schedule with about 105 PFU/dose as the titre in the final formulation (Bernstein et al., 1998). Rotarix replicated efficiently in the human gut and shedding was observed in 38−60% of vaccine recipients after the first dose (Vesikari et al., 2004). In a large study of over 60 000 infants in Finland and Latin America, 85% vaccine efficacy was seen against severe rotavirus disease and 42% against all diarrhoea hospitalizations (Ruiz-Palacios et al., 2006). In five European countries, Rotarix was shown to prevent 90% of the severe rotavirus gastroenteritis episodes and 72% of all-cause diarrhoea hospitalizations, indicating the potential for significant impact in different populations (Vesikari et al., 2007). However, subsequent trials of Rotarix in developing countries have demonstrated lower efficacy similar to that of other rotavirus vaccines. In South Africa, the vaccine provided 72% protection against severe rotavirus disease; however, in a more impoverished low-income country (Malawi), the efficacy of the vaccine was only 49% against severe rotavirus disease (Madhi et al., 2010). As with RotaTeq, post-licensure effectiveness analysis showed that the median vaccine efficacy was 84% (range, 19–97%) for 13 studies from low-mortality countries; 75% (range, −2−94%) for eight studies from medium-mortality countries; and 57% (range, 18–69%) for nine studies from high-mortality countries (Jonesteller et al., 2017) (Figure 7B).

23 Another human rotavirus G1P[8] strain is licensed for use in Viet Nam. This vaccine, designated Rotavin-M1, is manufactured by the Center for Research and Production of Vaccines and Biologicals (PolyVac). The G1P[8] strain was chosen as a vaccine candidate from among three rotavirus strains belonging to the G1P[8], G1P[4] and G4P[6] genotypes isolated from Vietnamese infants with diarrhoea and adapted to cell culture (Luan le et al., 2009). After primary assessment of safety in adults, the vaccine was evaluated in Phase II immunogenicity studies in infants, with Rotarix as a comparator (Dang et al., 2012). No significant differences in mild adverse reactions or IgA seroconversion were observed between the vaccines. The vaccine was licensed for use locally in Viet Nam on the basis of results from a Phase IIb trial with two doses of the vaccine whereby 92% seroconversion was observed after the second dose (Kirkwood et al., 2017).

Rotavirus vaccines have also been derived from strains associated with asymptomatic neonatal infections. Several early studies demonstrated protection from subsequent rotavirus gastroenteritis in infants that were asymptomatically infected during the neonatal period (Bhan et al., 1993; Bishop et al., 1983). One such neonatal virus-based vaccine is Rotavac (also called 116E) manufactured by Bharat Biotech of India. The 116E strain is a naturally-occurring bovine-human reassortant virus belonging to the G9P[11] genotype and was isolated from a maternity unit in Delhi, India (Das et al., 1993). Epidemiological studies showed that infants infected with this strain were protected against subsequent rotavirus disease (Bhan et al., 1993). The strain was developed as a vaccine candidate through an international collaboration between Bharat Biotech, the United States Centers for Disease Control and Prevention (USCDC) and the NIH (Bhan et al., 2014). A Phase III clinical efficacy study conducted in 6800 Indian infants demonstrated that the vaccine was safe and efficacious, with a vaccine efficacy of 54% against severe rotavirus gastroenteritis in the first year of life and 49% in the second year (Bhandari et al., 2014a; Bhandari et al., 2014b). The efficacy results with Rotavac were comparable to data on Rotarix and RotaTeq from other developing countries. In 2016, the vaccine was included in the Universal Immunization Programme of the Indian government. Rotavac has recently received WHO prequalification for global use (PATH, 2017).

Another asymptomatic neonatal rotavirus strain-based vaccine that is currently in phase III clinical trials is RV3-BB. This vaccine is developed from a G3P[6] neonatal isolate (RV3) from Australia that was shown to confer protection up to 3 years of age against subsequent rotavirus disease (Bishop et al., 1983). First developed by the Murdoch Children’s Research Institute of Melbourne, Australia, this vaccine candidate belongs to the P[6] VP4 genotype that is currently not a component of any other rotavirus vaccine and is prevalent in Africa and parts of Asia (Banyai et al., 2012). Initial studies with RV3 demonstrated poor immunogenicity (Barnes et al., 2002) and a new formulation was developed (designated as RV3-BB). The vaccine was found to be safe and immunogenic in adults, children and infants in a Phase I study in Australia (Danchin et al., 2013). Phase IIa studies were carried out in an infant schedule (6, 10 and 14 weeks of age) or a newborn schedule (birth, 6 and 10 weeks of age) (Bines et al., 2015). In these studies, the primary endpoint was vaccine take, as measured by serum IgA or vaccine shedding in stool. By these definitions, vaccine takes of 93% and 90% were observed for the infant and newborn dosing schedules respectively. Vaccine efficacy against severe rotavirus gastroenteritis was 75% in the newborn vaccine group and 51% in the infant vaccine group (Bines et al., 2018). The technology for the development of this vaccine has been transferred to PT Biofarma in Indonesia which is developing it as a vaccine for Indonesia.

24 WHO immunological basis for immunization series - Module 21: Rotavirus Vaccine efficacy data from selected clinical trials of licensed vaccines or those in development are summarized in Table 3.

Figure 7: Median vaccine effectiveness based on child mortality stratification for (A) RotaTeq (RV5) and (B) Rotarix (RV1)

Source: Jonesteller et al. 2017. Reprint by permission of the publisher (Oxford University Press)

25 Reference (Rennels et al., 1996) (Joensuu et al., 1997) (Perez-Schael et al., 1997) (Kawamura et al., 2011) (Linhares et al., 2008) (Madhi et al., 2010; Cunliffe et al., (Madhi et al., 2010; Cunliffe 2012; Schwartz et al., 2017) (Phua et al., 2009) (Ruiz-Palacios et al., 2006) (Vesikari et al., 2007) (Vesikari NA NA NA NA NA NA (95% CI) Year 2 % Year 90 (79−95) 79 (67−87) 86 (76−92) 18 (−59−56) 40 (−204−87) NA NA NA NA NA (95% CI) Year 1 % Year 95 (63−99) 83 (67−92) 49 (19−68) 77 (56−88) 61 (44−73) 96 (90−99) (95% CI) 80 (56−91) 91 (82−96) 88 (61−96) 92 (62−99) 81 (71−87) 49 (19−68) 77 (56−88) 61 (44−73) 85 (72−92) 90 (85−94) Efficacy % 96 (85−100) Country USA Finland Venezuela Japan Latin America Malawi South Africa Pooled Hong Kong, Singapore, Taiwan, China Latin America and Finland Europe strain Type of vaccine Type Rhesus-human reassortant with human G1, G2, G4 on rhesus G3P[3] RRV backbone Human G1P[8] Licence status Withdrawn by manufacturer Multiple countries, WHO prequalified Manufacturer Table 3: Vaccine efficacy data from selected clinical trials of licensed rotavirus vaccines or those in development 3: Vaccine Table Wyeth-Ayerst GlaxoSmithKline Vaccine RotaShield, RRV-TV Rotarix, RV1 NA: not available.

26 WHO immunological basis for immunization series - Module 21: Rotavirus Reference (Armah et al., 2010) (Zaman et al., 2010) (Vesikari et al., 2006b) (Vesikari (Bhandari et al., 2014a; Bhandari et al., 2014b) –55 (95% CI) Year 2 % Year 46 (1−71) 88 (49−99) 49 (17−68) (–1753−82) 29 (–65−71) 19 (–23−47) 20 (–16−44) 39 (–18−70) 65 (–48−94) (95% CI) Year 1 % Year 51 (13−73 83 (26−98) 65 (36−82) 64 (40−79) 46 (–1−72) 54 (35−57) 1 (–432−82) 72 (–45−97) 98 (88−100) No data No data (95% CI) 64 (8−91) 64 (-6−90) 56 (28−73) 39 (19−55) 43 (10−64) 48 (22−66) 56 (37−70) Efficacy % 18 (-23−45) 98 (88−100) Country Ghana Mali Kenya Pooled Bangladesh Viet Nam Viet Belgium, Costa Rica, Finland, Germany, Guatemala, Italy, Mexico, Puerto Rico, Sweden, China, Taiwan, USA Pooled India China Nam Viet strain Type of vaccine Type Bovine-human reassortant with human G1, G2, G3, G4 and P[8] on bovine G6P[5] WC3 backbone Naturally- occurring bovine-human reassortant neonatal G9P[11] Lamb G10P[12] Human G1P[8] Licence status Multiple countries, WHO prequalified Licensed for use in India, WHO prequalified Licensed for use in China Licensed for use in Nam Viet Manufacturer Merck Bharat Biotech Lanzhou Institute of Biological Products POLYVAC Vaccine RotaTeq, RotaTeq, RV5 Rotavac, 116E Lanzhou Lamb Rotavirus, LLR Rotavin-M1 NA: not available.

27 Reference (Armah et al., 2013) (Isanaka et al., 2017) (Kulkarni et al., 2017) (Bines et al., 2018) NA (95% CI) Year 2 % Year (95% CI) Year 1 % Year 61 (30−78) (95% CI) 63 (30−78) 67 (50−78) 63 (34−80) 36 (12−54) Efficacy % Country Ghana Niger Indonesia India strain Type of vaccine Type Rhesus-human reassortant with human G1, G2, G4 and rhesus G3P[3] RRV Bovine-human reassortant with human G1, G2, G3 and G4 bovine UK G6P[5] backbone Human neonatal G3P[6] Licence status Phase III Licensed for use in India, WHO prequalified Phase II Manufacturer International Medica Foundation, USA Serum Institute of India Bio Farma, PT Indonesia Vaccine RotaShield, RRV-TV Rotasiil, BRV-PV RV3-BB NA: not available.

28 WHO immunological basis for immunization series - Module 21: Rotavirus 7.2.4 Nonreplicating rotavirus vaccines While live-attenuated, oral rotavirus vaccines have been effective in preventing severe rotavirus gastroenteritis, poor efficacy of oral rotavirus vaccines continues to be seen in developing countries. In addition, although it is very evident that the benefits of vaccination far outweigh the risk of intussusception, the small risk of intussusception seen with oral rotavirus vaccines remains a concern. In this context, alternative nonreplicating vaccines are being developed and evaluated (Kirkwood et al., 2017). These include inactivated rotavirus vaccines, virus-like particle (VLP) vaccines and viral proteins as vaccines (Bertolotti-Ciarlet et al., 2003; Johansen et al., 2003; Moon et al., 2013a; Ward and McNeal, 2010).

Inactivated rotavirus vaccines are being developed by the USCDC. The CDC-9 strain was developed as a candidate vaccine; the strain grows to high titres (108 FFU/ml) in Vero cells and was found to produce predominantly triple-layered virus with robust stability (Jiang et al., 2013). Intramuscular immunization with a low dose of thermally inactivated CDC-9 formulated with aluminum hydroxide (AlOH) was found to be immunogenic in mice and gnotobiotic pigs (Jiang et al., 2008; Wang et al., 2010). The monovalent strain induced cross-reactive neutralizing antibodies against homotypic and heterotypic human strains, but not against bovine or bovine-human reassortant strains (Jiang et al., 2013). Three doses of the vaccine were more effective than two doses. Administration of the vaccine using a microneedle skin patch resulted in significant dose-sparing and induced higher IgG and neutralizing antibody responses compared to the intramuscular route (Moon et al., 2013a).

Another alternative to live rotavirus vaccines are virus-like particles (VLPs). Co-infection of insect cells with recombinant baculoviruses expressing the rotavirus structural proteins results in the self-assembly of VLPs, including 2/6 particles (formed by co-expression of VP2 and VP6) and 2/6/7-, or 2/4/6/7-VLPs (co-expression of VP2, VP6 and VP7, with or without VP4, respectively) (Crawford et al., 1994). The immunogenicity and/or protective efficacy of VLPs has been assessed in a number of animal models, including mice, rabbits, cows and pigs (Crawford et al., 1999; Ciarlet et al., 1998; Fernandez et al., 1996; Kim et al., 2002; Yuan et al., 2001). Intramuscular and intranasal administration of VLPs provided higher levels of protection against virus challenge in mice when compared to the oral route (Bertolotti-Ciarlet et al., 2003). Rotavirus VLPs have been used for immunizing cows to produce anti-rotavirus antibodies in the colostrum which in turn confers protection against scours in newborn calves. This product has recently received the approval of the United States Department of Agriculture (USDA) (Immucell, 2017). As of the date of publication, neither CDC-9 nor the VLP vaccines had been tested in humans.

The most advanced nonreplicating vaccine candidate is a recombinant VP8* parenteral rotavirus vaccine. The vaccine, developed at the NIH, contains the VP8* protein of P[4], P[6] and/or P[8] human rotaviruses expressed in E. coli. The monovalent P2- VP8*-P[8] vaccine was found to be safe and immunogenic in a Phase I trial in healthy American adults (Fix et al., 2015). The P2-VP8*-P[8] vaccine was also well tolerated and immunogenic in a dose-escalation trial in South African infants (Groome et al., 2017). Neutralizing antibody responses were induced in 40% of vaccinated infants. Further, a reduction in the shedding of Rotarix vaccine was observed when the live, oral vaccine was administered one month after the third dose of the P2-VP8*-P[8] vaccine. This suggests a potential effect of the parenteral vaccine at the mucosal surface

29 and the possibility of utilizing oral vaccine shedding as a surrogate marker for protection. A Phase I/II trial of a trivalent P2-VP8* vaccine – including the P[4], P[6] and P[8] genotypes – has been completed in South Africa.

7.2.5 Mechanisms of protection Immune correlates of protection in humans, both in cases of natural infection with rotavirus and of vaccination are not completely understood because of the complexities associated with the collection of samples for studying the induction and persistence of protective immunity in a disease that affects infants and young children. Furthermore, new studies show that susceptibility to rotavirus may also be modulated by non-immunological factors such as genetically controlled differences in HBGA receptor expression (Gunaydin et al., 2016; Imbert-Marcille et al., 2014; Payne et al., 2015; Van Trang et al., 2014; Zhang et al., 2016). Initially, immune responses to rotavirus were studied in detail in animal models, including neonatal and adult mice and gnotobiotic pigs (Desselberger and Huppertz, 2011). However, the broad applicability of these studies to infections with human rotavirus strains in children is unclear for a number of reasons, including reduced susceptibility of animals to infections with heterologous human rotavirus strains and the near-complete resistance to symptomatic disease following primary infection (Estes and Greenberg, 2013). This is particularly important because the natural history of rotavirus infection and disease studies in birth cohorts have shown that, while primary infections provide protection from subsequent severe disease, rotavirus immunity is not sterilizing. Further, population-specific differences in the level and number of infections needed to acquire protection have been observed and may be more difficult to recapitulate in animal models. Indeed, in a birth cohort in Mexico children, two infections conferred almost complete protection against moderate-to-severe rotavirus gastroenteritis while, in children in Vellore, India, three or more infections conferred around 80% protection (Gladstone et al., 2011; Velazquez et al., 1996). These differences are reflected in vaccine performance with more than 80% protection in Mexico and Latin American countries and with only 50% efficacy seen in Africa and Asia.

Antibody responses detected in serum samples are the most widely described immune response following rotavirus infections. Transplacentally-acquired antibodies have been demonstrated to confer protection early in infancy. In a study in Danish children, pre-existing levels of anti-rotavirus IgA, but not IgG, correlated with less severe clinical symptoms (Hjelt et al., 1987). By contrast, a study in rural Bangladesh found that children with rotavirus diarrhoea had significantly lower serum anti-rotavirus IgG titres than healthy controls (Clemens et al., 1992). Some studies suggest a requirement for critical levels of antibodies to achieve clinical protection. Longitudinal follow-up of infants in a day-care centre in the USA showed that both IgA titres of over 200 or IgG titres of over 800 correlate with protection (O’Ryan et al., 1994). The levels of antibodies required to confer protection is also likely to vary between populations; an anti-rotavirus IgA titre of over 800 provided about 80% protection against rotavirus, while IgG titres over 6400 correlated with 50% protection in the Mexican birth cohort (Velazquez et al., 2000). However, these cut-offs were not applicable in an Indian birth cohort (Premkumar et al., 2014). Recently, data from the birth cohorts in India and Mexico were reanalyzed using similar methods in order to better understand naturally-acquired protection (Lewnard et al., 2017). The risk for primary, secondary and subsequent infections to cause gastroenteritis decreased per log-month of age by 28%, 69% and 64% respectively in Mexico, and by 10%, 51% % and 67% respectively

30 WHO immunological basis for immunization series - Module 21: Rotavirus in Vellore, India. Elevated serum IgA and IgG antibodies were associated with partial protection against rotavirus infection. Further, the associations between age and reduced risk for disease persisted after controlling for antibody levels. A correlation between the levels of pre-existing homotypic neutralizing antibodies in serum and resistance to disease has also been observed. In a study in Japanese children, neutralizing antibody titres greater than 1:128 appeared to be protective against homotypic gastroenteritis caused by a G3 rotavirus, although heterotypic responses against G1 and G4 strains were also induced (Chiba et al., 1986). By contrast, a study in Bangladesh found that the correlation of protection with neutralizing antibody titres was not virus serotype- specific (Ward et al., 1992). Antibodies to NSP4 have also been evaluated as a correlate of protection. In a study evaluating serological response to NSP4 in samples from children with acute gastroenteritis, a significantly higher number of children who did not develop rotavirus-associated diarrhoea had NSP4 antibodies in their acute-phase serum samples compared to those with rotavirus diarrhoea. The role of NSP4 antibodies in vaccine response has not been examined extensively (Vizzi et al., 2005). The critical role of antibodies in protection against rotavirus recurrent infection can be seen in children with primary severe combined immunodeficiency (SCID), who develop chronic rotavirus infection and disease. Since children with SCID are also susceptible to chronic rotavirus vaccine shedding, it is recommended that children with SCID should not receive rotavirus vaccines (Morillo-Gutierrez et al., 2015; Patel et al., 2010).

In addition to serum, antibody responses in other biological specimens such as stool, saliva, duodenal fluid and breast milk have been examined. Correlations between faecal anti-rotavirus IgA and protection from rotavirus gastroenteritis has been described in children in Australia and the USA (Coulson et al., 1992; Matson et al., 1993). Increases in the levels of secretory immunoglobulin in duodenal fluids were reported in the first 10 days of infection in Danish children and correlated with the levels detected in serum, suggesting that the intestinal mucosal immune system could be a source of serum secretory immunoglobulin (Hjelt et al., 1985b). Rotavirus-specific IgA and neutralizing antibodies have also been detected in breast milk (Hjelt et al., 1985a; Jayashree et al., 1988; Santos et al., 2013). In a study in India, IgA titres in breast milk from mothers of infants infected with rotavirus early in life were significantly lower than the titres in breast milk from the mothers of uninfected infants (Jayashree et al., 1988). No differences in neutralizing antibody titres were seen between the breast milk samples from mothers of rotavirus infected and uninfected infants in Mexico (Brussow et al., 1993). Non-antibody components of breast milk, such as lactoferrin and lactadherin, have been shown to neutralize rotavirus infectivity, although results vary between studies (Moon et al., 2013b; Moon et al., 2010; Superti et al., 1997).

Studies of T-cell responses to rotavirus in humans are limited. Healthy and rotavirus- infected adults have relatively low frequencies of CD4+ and CD8+ rotavirus-specific T cells and these cells secrete only interferon gamma (IFNγ), suggesting limited functionality (Jaimes et al., 2002; Mesa et al., 2010). Rotavirus-specific CD4+ T cells in adults have a terminal effector memory cellular phenotype, suggesting that they do not provide long-term immunity; this may explain the absence of sterilizing immunity and the re-occurrence of mild infections seen in adults (Parra et al., 2014b). Very low levels of rotavirus-specific CD8+ T cells can be detected in children with rotavirus disease (Mesa et al., 2010). These cells are below the detection limit in most healthy children. In children with rotavirus gastroenteritis, these responses are either undetectable or low. While virus-specific CD4+ T cells expressing mucosal homing receptors α4β7 and CCR9 have been detected (Parra et al., 2014a), little is known about intestinal T-cell

31 immunity in children or adults. Memory B cells in adult intestinal tissue that produced broadly cross-reactive and cross-neutralizing anti‑VP5* antibodies were described in one study; the role of these responses in protective immunity in children remains to be studied (Nair et al., 2017). The innate immune response to rotavirus has been examined primarily in animal models and little is known about cytokine responses in children. A few studies have demonstrated elevated levels of certain cytokines – such as IL-6, IL-8 and TGF-β – in serum or plasma from children with rotavirus diarrhoea (Chen et al., 2014; Jiang et al., 2003). The role of these cytokines in disease pathogenesis and clearance of infection remains to be elucidated.

The induction of serum antibodies after vaccination appears to play a role in protection. IgA, IgG and neutralizing antibody responses have been evaluated but correlates of protection are not clear (Clarke and Desselberger, 2015). Consequently, despite the evaluation of several immune effector molecules for protection against rotavirus infection and disease, anti-rotavirus IgA is the most widely used immunological marker of rotavirus vaccine take. A systematic review evaluating serum anti-rotavirus IgA as a correlate of vaccine efficacy showed that the level and duration of protection were lower in settings with lower IgA response (Patel et al., 2013a). For both Rotarix and RotaTeq, vaccine efficacy during the first two years of life was only 44% (CI, 30–55%) in countries with IgA geometric mean titres less than 90, while the efficacy in countries with IgA titres greater than 90 was about 85% (CI, 82–88%). The response measured in IgA assays is primarily against the inner capsid protein VP6. Single-chain antibodies against VP6 have been reported to possess neutralizing activity and to confer protection against diarrhoea in mice, while non-neutralizing antibodies against VP6 have also been found to be protective in mice, possibly acting by transcytosis through intestinal epithelial cells (Garaicoechea et al., 2008; Burns et al., 1996). Intracellularly acting anti-VP6 antibodies have also been hypothesized as blocking viral infection, potentially by stabilizing the virion and preventing the release of viral RNA upon infection (Feng et al., 2002). Neutralizing antibodies against VP7 and VP4 are induced following vaccination but the levels of these antibodies are often low and responses are inconsistent (Vesikari, 2012). Research aimed at understanding differences in homotypic and heterotypic protective immune responses conferred by Rotarix and RotaTeq is hindered by the lack of a clear correlate of vaccine-induced protection. However, the comparable efficacy of both vaccines suggests that immune responses are heterotypic and can protect against virus strains that are not included in the vaccine. A systematic review and meta-analysis of data from high-income and middle-income countries found vaccine effectiveness of over 70% against homotypic and heterotypic strains with both Rotarix and RotaTeq vaccines in high-income countries (Leshem et al., 2014). While the effectiveness rates were lower in middle-income countries, there were no significant differences between responses to homotypic and heterotypic strains. In one-year follow-up studies with Rotarix in middle-income and low-income settings in Malawi and South Africa, the vaccine efficacy was 64% against G1 strains and 60% against non-G1 strains (Steele et al., 2012). Similarly, pooled vaccine efficacy against severe rotavirus gastroenteritis caused by single human rotavirus VP7 or VP4 strains included in RotaTeq or not included in RotaTeq were 57% and 60% respectively in one-year follow-up studies in low-income and middle-income countries in Africa and Asia. These data indicate a limited role for strain diversity in rotavirus vaccine response (Velasquez et al., 2014). Interestingly, homotypic VP7-specific immune responses induced by the vaccines may be strong enough to protect against a subsequent dose of the homologous vaccine virus itself. This has been demonstrated by the lack of a booster effect to the monovalent RRV and Rotarix vaccines (Rennels et al., 1996; Vesikari et al.,

32 WHO immunological basis for immunization series - Module 21: Rotavirus 2004). Vaccine take as measured by virus shedding with the second dose of Rotarix was seen only if the first dose did not take. Giving three or more doses of the vaccine did not significantly improve the take rate in developing countries (Kompithra et al., 2014). While there are limited data on the interchangeability of different rotavirus vaccines, a randomized, multicentre, open-label study in children in the USA showed that immune responses to mixed vaccine schedules with Rotarix and RotaTeq were non-inferior when compared to the single vaccine reference groups (Libster et al., 2016). These new data suggest that interchangeability of rotavirus vaccines is feasible, although further evaluation may be needed with the different vaccines and in other settings.

Studies with all licensed rotavirus vaccines demonstrate the lack of interference of rotavirus vaccine co-administration on immune response to other routine infant vaccines (Chandola et al., 2017; Dennehy et al., 2008; Desai et al., 2018; Vesikari et al., 2010). However, a number of studies have documented diminished immunity to oral rotavirus vaccines when co-administered with OPV (Ciarlet et al., 2008; Emperador et al., 2016; Ramani et al., 2016b; Steele et al., 2010; Zaman et al., 2009). This may be due to the reduced replication of rotavirus in the presence of OPV, although the effect is greatest only after the first dose of OPV and appears to be overcome with subsequent rotavirus vaccine doses (Parker et al., 2018a; Patel et al., 2012a; Wang et al., 2012). Interestingly enterovirus carriage, specifically non-polio enteroviruses have also been associated with diminished rotavirus vaccine response (Taniuchi et al., 2016). Indeed, the role of non-immunological host components in rotavirus vaccine response is being increasingly recognized. Of particular importance is the role of genetically- determined HBGA expression in susceptibility to disease with specific rotavirus strains and susceptibility to vaccines (Ramani et al., 2016a). Recent data suggest that infants who lack a functional fucosytranferase 2 (FUT2) gene (also called non-secretors) are resistant to diarrhoea with P[8] rotavirus strains (Imbert-Marcille et al., 2014; Nordgren et al., 2014; Van Trang et al., 2014; Payne et al., 2015; Gunaydin et al., 2016; Yang et al., 2017). Increased susceptibility of Lewis HBGA-negative children to P[6] rotavirus diarrhoea has also been reported (Lee et al., 2018; Nordgren et al., 2014). Since the P[8] genotype is a component of both Rotarix and RotaTeq vaccines, these new findings raise questions about the susceptibility of non-secretor infants to these vaccines. Indeed, reduced immunogenicity in non-secretors has been documented in a study in Pakistan (Kazi et al., 2017). The Lewis A phenotype was identified as a restrictive factor for seroconversion to Rotarix and RotaTeq in Nicaragua (Bucardo et al., 2018). Only one study in Bangladesh has examined the role of HBGA in rotavirus vaccine efficacy (Lee et al., 2018). Rotarix was found to protect both secretors and non-secretors similarly, although the natural resistance to rotavirus disease among unvaccinated non-secretors had an impact on efficacy estimates. Two studies in Nicaragua and Zambia examined the effects of the breast-milk innate immune molecules lactoferrin, lactadherin and Tenascin-C on rotavirus vaccine seroresponse (Becker-Dreps et al., 2017a; Mwila-Kazimbaya et al., 2017). Higher levels of lactadherin correlated with lower immunity in Zambia while no associations were detected in Nicaragua. The composition of the intestinal microbiome prior to vaccination has also been associated with diminished vaccine efficacy in Ghana and Pakistan, although the mechanisms by which the microbiome influences vaccine response are unclear (Harris et al., 2017a; Harris et al., 2017b). However, no correlations of vaccine response with pre-vaccination microbiota composition was observed in a study from southern India (Parker et al., 2018b). While co-infections with other enteric pathogens have been hypothesized to play a role in diminished rotavirus vaccine response in low- and middle-income countries, direct evidence is currently limited (Mokomane et al., 2018;

33 Taniuchi et al., 2016). Biomarkers of environmental enteropathy have also been associated with reduced vaccine response in some settings (Becker-Dreps et al., 2017b; Naylor et al., 2015). While studies on protective immune responses continue to be made, the absence to date of a reliable immunological correlate of protection remains a challenge and is possibly the single largest barrier to the improvement of current rotavirus vaccines and the development of next-generation vaccines.

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60 WHO immunological basis for immunization series - Module 21: Rotavirus Department of Immunization, Vaccines and Biologicals

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