Nipah Virus Assays and Animal Models for Vaccine Development

Landscape Analysis, January 2021

Author: Albert Price, IAVI

Secondary authors/reviewers: Donata Sizemore, IAVI Thomas Hassell, IAVI Raúl Gómez Román, CEPI Johan Holst, CEPI Paul Kristiansen, CEPI TABLE OF CONTENTS

I. Introduction 4

II. Background 5

1. Epidemiology 5

2. NiV Clinical Features and Pathogenesis in Humans 9

3. Diagnosis and Treatment 10

4. NiV Molecular Biology and Structure 11

5. Vaccine Development 13

III. Standardization of Assays and Animal Models 18

IV. NiV Serological Assays 21

1. Detection of antigen – specific serum IgG 21

2. Detection of serum neutralizing antibodies 22

V. NiV Animal Models 27

1. Syrian Golden Hamster 29

2. Ferret 32

3. African Green Monkey (AGM) 34

VI. Conclusions 38

VII. References 40

VIII. Statement of Support 46

1. Article H.20. Publication And Publicity 46

2 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 LIST OF FIGURES

Figure 1. Geographic distribution of Pteropid fruit bats and Henipavirus outbreaks (Enchery and Horvat, 2017) 5

Figure 2. NiV structure and organization of the 18.2 kB ssRNA (-) genome (Sun et al., 2018). 11

Figure 3. The Henipavirus infection and replication cycle (Aguilar and Lee, 2011). 12

LIST OF TABLES

Table 1. NiV Outbreaks by Year and Location* 7

Table 2. Differences in Clinical and Epidemiological Characteristics Between NiV Malaysia and Bangladesh Outbreaks 8

Table 3. NiV Viral-vector Vaccine Candidates Tested in Animals 15

Table 4. NiV Submit Vaccine Candidates Tested in Animals 16

Table 5. NiV vaccine candidates supported by CEPI 17

Table 6. Benefits and Potential Challenges of Implementing Biological Standards for NiV Vaccine Development 20

Table 7. Pros and Cons of NiV Serological Assays 24

Table 8. Serological Assays Used in NiV Pre-Clinical Vaccine Studies 25

Table 9. Serological Assays Used in Other NiV Research Studies 26

Table 10. Summary of clinical signs and pathology in the NiV hamster, ferret and AGM challenge models 28

Table 11. NiV Hamster Model Challenge Studies 30

Table 12. NiV Hamster Model Challenge Studies, Continued 31

Table 13. NiV Ferret Model Challenge Studies 33

Table 14. NiV African Green Monkey Model Challenge Studies 35

Table 15. NiV African Green Monkey Model Challenge Studies, Continued 36

Table 16. Pros and Cons of the Major NiV Animal Challenge Models 37

3 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 I. INTRODUCTION Nipah is an emerging, zoonotic viral disease that causes severe neurologic and respiratory symptoms and has an overall case fatality rate of 59%.

Although relatively rare and currently targeted for development To this end, CEPI is focusing on currently confined to sporadic of prophylactic vaccines as an developing biological standards, outbreaks in southern and urgent priority2 and is actively validating assays and supporting southeast Asia (including Malaysia, supporting efforts toward a the development and refinement of Singapore, Bangladesh and India), protective NiV vaccine. animal models for three emerging the extreme virulence, lack of a diseases in its vaccine development vaccine or effective therapeutic Development of new vaccines portfolio: Nipah, MERS-CoV options, broad species tropism against any disease is most and Lassa. The purpose of this and wide geographical distribution efficient when there is Landscape Analysis, supported by of the Nipah virus’ (NiV) primary standardization of key R&D tools, NIH/NIAID/DMID and prepared for animal reservoir (Pteropid fruit particularly analytical methods, CEPI, is to analyze the current state bats) led the World Health reagents and animal models, of NiV assays and animal models Organization (WHO) to label NiV so that experimental results currently in use within the context a “Priority Pathogen” for the from different investigators and of NiV biology, epidemiology, development of effective medical developers can be directly and and vaccine development. This countermeasures (MCMs), and in confidently compared. CEPI has document will serve both as an 2017 developed a Target Product identified a set of research and internal resource at CEPI to guide Profile (TPP) for a NiV vaccine.1 development activities needed to scientific discussions and as an The Coalition for Epidemic accelerate vaccine development external resource to inform the Preparedness Innovations (CEPI) by promoting standardization, Nipah scientific community. has selected NiV as one of seven transparency and comparability emerging infectious diseases between vaccine candidates.

1 https://www.who.int/blueprint/priority-diseases/key-action/Nipah_virus_vaccineTPP.pdf 2 https://cepi.net/research_dev/priority-diseases/

4 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 II. BACKGROUND 1. Epidemiology

Fruit bats (‘Flying Foxes’) of the concern over the potential spread Caledonia, and Papua New Guinea family Pteropodidae, particularly of NiV, is illustrated in Figure 1. (Sun et al., 2018). More recently, those of the genus Pteropus, are the a six-year study of Pteropus medius primary animal reservoir for NiV Pteropid bats of the genus Eidolon bats in Bangladesh indicates and are asymptomatically infected range over most of the African that Nipah virus may be more by the virus. Even experimental continent and have been found widespread than previously infections with very high doses to be seropositive for NiV, and thought: bats throughout the of NiV cause only sub-clinical thus are potentially an additional country, and not just those in what infection in fruit bats and viremia reservoir (Enchery and Horvat, is referred to as the “Nipah belt”, has not been reported, although 2017). A field survey found had similar patterns of Nipah virus the animals do seroconvert against that 9% to 25% of fruit bats in infection throughout the year.3 the virus and virus shedding has Malaysia, Cambodia, Thailand been observed, albeit rarely and and Bangladesh were seropositive only in urine (Geisbert et al., 2012; for NiV (Sharma et al., 2019) and Middleton et al., 2007). The broad NiV-seropositive bats have also geographical distribution of these been found in China, Vietnam, animals, one of the factors driving Indonesia, Madagascar, New

Figure 1. Geographic distribution of Pteropid fruit bats and Henipavirus outbreaks (Enchery and Horvat, 2017)

3 Epstein et al. PNAS 2020: https://doi.org/10.1073/pnas.2000429117

5 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 A number of domesticated animals and Tan, 2014). In the 2014 – pigs, dogs, cats and horses – can NiV outbreak in the Philippines be infected by NiV (Geisbert et al., (Malaysia strain), the majority 2012), but the virulence and rate of of cases were acquired by eating infection is variable. Mortality in contaminated horse meat or suckling pigs is high (40%) and 1-6 participating in slaughtering month-old pigs show respiratory horses, and the rest were likely due and neurological symptoms, but to human to human transmission. mortality is less than 5%. Adult 65% of the cases presented with pigs show less serious respiratory an acute encephalitic syndrome signs and mortality is rare (McLean and the overall case fatality rate and Graham, 2019). During NiV was 53% (Ching et al., 2015). outbreaks in Malaysia many dogs on pig farms were found to be The NiV outbreaks in Bangladesh NiV-seropositive, but only 2 had and India beginning in 2001 active disease (Hooper et al., 2001). showed a distinct pattern of Horses can be infected by NiV transmission and symptomology. (Hooper et al., 2001), most likely Humans were infected by drinking from eating fruit contaminated by raw date palm sap contaminated bats, and were intermediate hosts with bat saliva or urine, and there in an outbreak in the Philippines was no intermediate animal host. in 2014. During that outbreak There was a higher incidence investigations found disease among of respiratory illness (69% and horses including neurological a higher fatality rate (75%; see signs and 10 deaths. Four (4) cats Table 1). Patients infected with the and a dog were also likely infected Bangladesh strain (NiV-B) had by eating horse meat and died higher NiV RNA levels in the blood (Ching et al., 2015). However, no and more virus in oral secretions published reports of cats or dogs (Hossain et al., 2008). Finally, serving as intermediate hosts for there was evidence of human-to- transmission of NiV have been human transmission, primarily found. to healthcare workers or family caregivers, in the Indian and The locations and human fatality Bangladeshi outbreaks (Chadha et rates of all NiV outbreaks to date al., 2006). The mechanism(s) of are summarized in Table 1. human to human transmission has The first reported NiV outbreaks not been conclusively established, occurred in Malaysia and Singapore but exposure to bodily fluids in 1998-99. In those outbreaks (saliva, cough, vomit, blood) the majority of human cases were elevates the risk of transmission due to contact with infected pigs compared to physical contact that had acquired NiV by eating alone or being near the patient fruit contaminated by bat saliva, (Kumar et al., 2019). Experiments urine, or feces. Humans (mostly in non-human primates have pig farmers and slaughterhouse demonstrated infection via small workers) acquired NiV from and medium particle size aerosols infected pig urine or respiratory (Cong et al., 2017; Hammoud secretions. A majority of cases et al., 2018). Comparisons in were characterized by an acute transmission rates between the encephalitic syndrome. The case Malaysia and Bangladesh outbreaks fatality rate was 40% (see Table have not been found in the 1) and, at the time, there was published literature. inconclusive evidence of human- to-human transmission (Ahmad

6 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 1. NiV Outbreaks by Year and Location*

Number of Number of Case Month/Year Country Location Cases Deaths Fatality (%)

Sep 1998 – Apr 1999 Malaysia Perak, Selangor, Negeri Sebilan 265 105 40

Mar 1999 Singapore Singapore 11 1 9

Jan – Feb 2001 India Siliguri 66 45 68

Apr – May 2001 Meherpur 13 9 69

Jan 2003 Naogaon 12 8 67

Jan 2004 Rajbari 31 23 74

Apr 2004 Faridpur 36 27 75 Bangladesh Jan – Mar 2005 Tangail 12 11 92

Jan – Feb 2007 Thakurgaon 7 3 43

Mar 2007 Kushtia, Pabna, Tatore 8 5 63

Apr 2007 Naogaon 3 1 33

Apr 2007 India Nadia 5 5 100

Feb 2008 Manikgon 4 4 100

Apr 2008 Rajbari, Faridpur 7 5 71

Jan 2009 Gaibandha, Rangpur, Nilphamari 3 0 0

Jan 2009 Rajbari 1 1 100

Feb – Mar 2010 Faridpur, Rajbari, Gopalganj, Madaripur 16 14 87.5

Bangladesh Lalmohirhat, Dinajpur, Comilla, Nilphamari , Jan – Feb 2011 44 40 91 Rangpur

Feb 2012 Joypurhat, Rajshahi, Tatore, Rajbari, Gopalganj 12 10 83

Gaibandha, Natore, Rajshahi,Naogaon, Rajbari, Jan – Feb 2013 24 21 87.5 Pabna, Jhenaidah, Mymensingh

Manikganj, Magura, Faridpur, Rangpur, Feb 2014 Shaariatpur, Kushtia, Rajshahi, Tatore, 18 9 50 Dinajpur, Chapai Nawabganj, Naogaon

Mar – May 2014 Philippines Tinalon, Midtungok 17 9 53

Nilphamari, Pnchoghor, Faridpur, Magura, Feb 2015 Bangladesh 9 6 67 Naugaon, Rajbari

May 2018 India Kozhikode, Malappuram 19 17 89

Overall Total 643 379 58.9

Malaysia/Singapore/Philippines Total 293 115 39.2

Bangladesh/India Total 350 264 75.4

*Adapted from (Thakur and Bailey, 2019) and (Sharma et al., 2019)

7 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 A comparison of key clinical and the Bangladesh outbreaks. and convulsions) were seen in epidemiological characteristics This, coupled with the higher level the Bangladesh outbreaks at rates of NiV outbreaks in Malaysia and of NiV-B RNA in oral secretions comparable to the Malaysia cases Bangladesh is compiled in Table could be linked to the higher level (Ang et al., 2018; Hossain et al., 2. Three clinical characteristics of human to human transmission, 2008). This phenomenon is largely between the outbreaks stand out. which is likely via oral/respiratory unexplained but could reflect The first is the shorter time from secretions or bodily fluids (Ahmad involvement of different areas of disease onset to death (7 days vs. and Tan, 2014). Finally, in the the central nervous system (CNS) 16 days) and higher case fatality Malaysia outbreak there was a due to differences in virus tropism, rate (74% vs. 38%; Table 1) in high incidence of segmented differences in the route of infection the Bangladesh vs. the Malaysia myoclonus (muscle jerking), or slower disease progression outbreaks (Ahmad and Tan, 2014; which was not reported in the allowing infection of different Ang et al., 2018; Hossain et al., Bangladesh outbreaks, although neural tissues. 2008; Lo and Rota, 2008). The other indications of encephalitis or second is the higher incidence neurological involvement (such as of respiratory involvement in altered mental status, hyporeflexia

Table 2. Differences in Clinical and Epidemiological Characteristics Between NiV Malaysia and Bangladesh Outbreaks

Characteristic Malaysia-Singapore Bangladesh-India

• Bat to human via consumption of • Bat to pig → pig to human Transmission contaminated date palm juice and fruits. • Rare human to human • Human to human

Fever 95% 100%

Headache 75% 73%

Vomiting 32% 58%

Diarrhea 18% 29%

Respiratory involvement 14-29% 62-69%

• Segmental myoclonus 32-54% • Segmental myoclonus not reported

• Hyporeflexia 60.5% • Hyporeflexia 65% Encephalitis/neurological involvement • Convulsion 23% • Convulsion 23%

• Altered mental status 72% • Altered mental status 100%

Disseminated small, high-signal-intensity Confluent high-signal brain lesions (limited MRI lesions MRIs were performed)

Relapsed and late-onset encephalitis ~5-10% 4 out of 22 patients (18%) in a follow-up study

Persistent neurological deficits ~20% ~30%

Incubation Period Mean = 10 days 6-11 days

Average (mean) time from disease onset to 16 days 7 days death

Sources: (Ang et al., 2018; Hossain et al., 2008); (Ahmad and Tan, 2014; Chong et al., 2002; Lo and Rota, 2008)

8 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Further clinical commonalities • Ferrets infected with NiV-B AGMs appear to reproduce the NiV and differences in Nipah showed comparable disease strain differences in virulence and infections across countries were progression, and histopathology pathology seen in humans more discussed during the NIAID co- of the lungs and CNS compared faithfully than hamster and ferrets, sponsored Nipah@20 Conference to ferrets infected with NiV-M. and the results suggest that the in Singapore, December 20204. However, NiV-B infected ferrets distinct clinical characteristics and The cause of the differences in shed more virus in oral secretions epidemiology seen in the Malaysia disease between the Malaysia/ than NiV-M infected animals and Bangladesh outbreaks is at Singapore and Bangladesh/India (Clayton et al 2012). Increased least partly genetic. However, outbreaks remains uncertain and human shedding of NiV-B was geographic differences in virus is complicated by the possibility of seen in the Bangladesh outbreaks transmission (including route of differing diagnostic methods and (Hossain et al., 2008). infection and dose), population case definitions. Experiments in health and the quality of animal models have demonstrated • African Green Monkeys (AGM) subsequent health care may also some differences in disease course Lethality of NiV-B was 100%, play a role. More information on and symptomology between the compared to 50% for monkeys the major animal challenge models Malaysia and Bangladesh strains, infected with an equivalent dose for NiV is presented in Section V. suggesting a genetic component: of NiV-M. NiV-B also causes more severe lung histopathology than • Hamsters infected with NiV-M NiV-M and has a shorter window showed accelerated virus for therapy with the monoclonal replication, pathology and death antibody m102.4 (Mire et al., compared to hamsters infected 2016). with equivalent doses of NiV-B (DeBuysscher et al., 2013). This finding is opposite to the human fatality rates in the Malaysia and Bangladesh outbreaks.

2. NiV Clinical Features and Pathogenesis in Humans

The initial clinical presentation of rapidly to an encephalitic syndrome encephalitis survivors suffer NiV infection (by either strain) is in approximately 60 percent long-term neurologic dysfunction non-specific and characterized by of patients. The time course of characterized by persistent flu-like symptoms including fever, disease progression from initial seizures, disabling fatigue and headache, dizziness, myalgia, symptoms to the encephalitic behavioral abnormalities (Mazzola and loose stools (Banerjee et al., syndrome has not been reported and Kelly-Cirino, 2019; Sejvar 2019). Mild or asymptomatic in detail. Neurological symptoms et al., 2007). In addition, some infections have also been reported include meningismus (central patients with initially mild, non- in various outbreaks, but the nervous system inflammation) and encephalitic disease develop a late- overall incidence is relatively low seizures in approximately one- onset or recurrent neurological and appears to be strain dependent, third of patients. A deterioration disease (Ramphul et al., 2018). with the Malaysia strain causing in consciousness, coma and death Some patients also present with less severe illness, a lower case typically occur within an average of severe respiratory symptoms, fatality rate and higher prevalence 7 days of disease onset (Bangladesh and respiratory involvement of asymptomatic infections than outbreaks) or an average of 16 has been more common in the the Bangladesh strain (Kumar et days (Malaysia outbreak) (Ang Bangladesh outbreaks compared al., 2019). The incubation period et al., 2018; Hossain et al., 2008; to the Malaysia outbreaks (see ranges from 7 to 40 days and Rahman and Chakraborty, 2012). Epidemiology). from onset the disease progresses Approximately 20-30% of NiV

4 Conference Proceedings: https://cepi.net/wp-content/uploads/2020/06/2019-CEPI-Duke-WHO-NIAID-Nipah-Conference_FINAL.pdf.

9 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Although the route of infection throughout the body, and the virus by severe vasculitis and syncytia in humans has not been may then enter the blood stream formation, resulting in endothelial conclusively determined, work with and disseminate throughout the damage due to vasculitis-induced experimental animal challenge host, infecting the brain, spleen thrombosis and the presence of models has shown that inhalation and kidneys (Escaffre et al., 2013). viral inclusion bodies. Necrotic of NiV virus particles is sufficient Experiments in hamsters suggest plaques are found in both grey and to initiate infection (Cong et al., that entry to the central nervous white matter of the CNS (Escaffre 2017; Hammoud et al., 2018). In system (CNS) may occur either et al., 2013). Lessons learned from humans, early infection appears through olfactory neurons or via pathology and disease course in to occur in lung epithelial cells, the choroid plexus and cerebral humans were discussed in the and in later stages moves to lung blood vessels (Baseler et al., 2016; Transmission/Case Management endothelial cells. Vasculitis in Munster et al., 2012). Infection of Session of the Nipah@20 small blood vessels may be present the human CNS is characterized Conference.

3. Diagnosis and Treatment

Laboratory diagnosis of NiV was not randomized and the In a lethal challenge study in infection can be performed using treated patients may have received African Green Monkeys (AGMs), a variety of nucleic acid-based or better overall care, thus making the monkeys could be successfully serological assays. The currently outcome uncertain (Banerjee et al., treated up to 5 days post-infection preferred methods for detecting 2019). Subsequent animal challenge with the NiV Malaysia strain active NiV infection are PCR- studies in hamsters showed that (NiV-M). Although half of the based tests such as conventional ribavirin delayed, but did not treated monkeys developed overt reverse transcriptase (RT) PCR, prevent, death after NiV infection clinical signs (fever, respiratory nested RT-PCR and real-time PCR (Freiberg et al., 2010; Georges- and neurological), all the animals (qPCR). PCR-based tests usually Courbot et al., 2006). A similar fully recovered (Geisbert et al., target the conserved N, M or P viral result was obtained after infection 2014). The window for successful genes. ELISA assays detecting IgM of African Green Monkeys with the treatment with m102.4 is only 3 against NiV antigens are typically closely related Hendra Virus (HeV; days post-infection when AGMs the first-line serological tests (Rockx et al., 2010)). are challenged with the NiV for NiV infection (Mazzola and Bangladesh strain (NiV-B) (Mire Kelly-Cirino, 2019). Progress and The broad-spectrum antiviral et al., 2016). These results suggest challenges in diagnostics, including drug Remdesivir (GS-5734) was that m102.4 may have utility as a presentation on the WHO Nipah recently shown to protect African a post-exposure prophylactic diagnostics Target Product Profile, Green Monkeys when administered or therapeutic in humans. The were featured in Session 4 of the 24 hours post-inoculation with m102.4 antibody has also been Nipah@20 Conference. a lethal dose of NiV (Bangladesh administered on an emergency strain) (Lo et al., 2019). Treated basis as post-exposure prophylaxis Treatment of NiV infection animals developed mild respiratory to a handful of humans in cases of consists primarily of supportive symptoms, reduced appetite and high risk of exposure to NiV or HeV care including maintaining fluids, showed local virus replication but (Broder et al., 2013). In all cases the anticonvulsants, treatment of no viremia. All the Remdesivir- patients did not become ill, but it secondary infection and mechanical treated animals recovered fully, is impossible to know if illness was ventilation. (Ang et al., 2018). while all the control-treated prevented by the antibody. No effective therapeutics for NiV animals succumbed to the infection are currently approved infection. for use in humans. The antiviral drug ribavirin was administered to A human monoclonal antibody, 140 patients during the 1998-99 m102.4, targeting the Ephrin-B2/ NiV Malaysia outbreak, resulting B3 binding site on the NiV and HeV in a 36% reduction in mortality G glycoproteins (see NiV Molecular compared to 52 untreated control Biology and Structure), has been patients (Chong et al., 2001). tested in NiV animal models for However, the treatment allocation prophylactic and therapeutic use.

10 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 4. NiV Molecular Biology and Structure

NiV is an enveloped, negative- host cell plasma membrane. Three glycoprotein mediates target cell sense, single-stranded RNA virus non-structural proteins, W, V, attachment via the cell surface of the family Paramyxoviridae, a and C, are produced by alternative receptors Ephrin-B2 and -B3. group which also includes measles, initiation or RNA editing within the The tissue tropism of Henipavirus mumps, parainfluenza viruses P gene open reading frame (Wang infection is determined by the and Sendai Virus. NiV shares the et al., 2001). These gene products tissue distribution of these genus Henipavirus with a handful inhibit host cell antiviral responses receptors. Ephrin-B2 is expressed of other recently identified viruses, such as Type 1 interferon signaling in neurons, endothelial cells, including Hendra Virus (HeV) and and are major determinants of smooth muscle surrounding Cedar Virus (Sharma et al., 2019). A viral pathogenicity (Mathieu et arteries, placental tissue and schematic of the NiV viral structure al., 2012b; Satterfield et al., 2015; spleen. High levels of Ephrin-B2 and genome organization is shown Yoneda et al., 2010). The viral mRNA have also been detected in Figure 2 below (Sun et al., 2018). envelope is studded with two in cardiomyocytes and bronchial The 18.2 kb NiV genome encodes transmembrane glycoproteins, epithelial cells. Ephrin-B3 is six structural proteins and three the trimeric F glycoprotein and expressed in the CNS and in lymph non-structural proteins. NiV RNA the tetrameric (dimer of dimers) nodes (Xu et al., 2012). Ephrin-B2/ is associated with nucleoprotein G glycoprotein (Aguilar and Lee, B3 expression levels in target (N) and phosphoprotein (P) to form 2011). The F and G glycoproteins tissues also impact the rate of the virus ribonucleocapsid (RNP). are the major targets of NiV virus replication (Sauerhering The NiV genome encodes its own neutralizing antibody responses in et al., 2016), but have not been RNA-dependent RNA polymerase animals and humans (Satterfield et extensively characterized. This (L) which, together with N and P, al., 2016b). is an important area for future forms the catalytic subunit of the investigation to understand replicase complex that enables The Henipavirus infection and differences in NiV disease virus replication. The matrix replication cycle is depicted pathogenesis in humans and (M) protein is required for virion schematically in Figure 3 (Aguilar animal challenge models (see assembly and budding from the and Lee, 2011). The viral G Section V.)

Figure 2. NiV structure and organization of the 18.2 kB ssRNA (-) genome (Sun et al., 2018).

11 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Following attachment, the G which is then re-exported with G 91.8% similarity at the nucleotide glycoprotein activates the F glycoprotein for assembly into the level (Rockx et al., 2012). The glycoprotein, which then mediates budding viral envelope. Assembly nucleotide changes are not fusion of the viral envelope and budding of new viral particles distributed uniformly; in most with the host cell membrane. from the plasma membrane is cases homologies are higher in the After cell entry the viral genome mediated primarily by the M coding regions than in non-coding [vRNA(-)] serves as a template for (matrix) protein. (Aguilar and regions. Nucleotide homologies transcription of mRNAs by the Lee, 2011). The interaction of cell range from 92.0% to 98.5% in the viral RNA polymerase (NiV L gene surface-displayed NiV F and G with open reading frames. While the 5’ product) which are then translated Ephrin B2/3 also mediates syncytia untranslated region of the N gene into proteins, the vRNA(-) is also formation by infected cells (Rockx is 100% conserved between the two a template for cRNA(+), which is et al., 2012). major strains, homologies in the 5’ then a template for production of and 3’ untranslated regions of all vRNA(-) genomes for packaging Two genetically distinct NiV the other viral genes range from into new viral particles. Precursor F strains, Malaysia (NiV-M) and 75.5% to 91.4% (Harcourt et al., glycoprotein (Fo) is exported to the Bangladesh (NiV-B), have been 2005). plasma membrane, endocytosed identified. The two strains share and proteolytically matured to F1/2, 92% amino acid homology and

Figure 3. The Henipavirus infection and replication cycle (Aguilar and Lee, 2011).

12 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 5. Vaccine Development

A number of factors suggest that little attention. One challenge study Due to safety issues associated development of a safe, efficacious conducted in pigs suggested that with production (i.e., BSL-4 human prophylactic vaccine cellular immune responses may containment) and administration against NiV is scientifically be important for achieving full of a live-attenuated or inactivated feasible. Natural infection by other protection, but the mechanism was NiV vaccine, and the need to paramyxoviruses, such as measles not defined, and the conclusions elicit neutralizing antibodies, and mumps, results in long-term are complicated by the fact that, most attempts at NiV vaccine immunity and vaccines for those unlike with other animal hosts, NiV development have focused on diseases have been successfully infects a range of porcine immune recombinant viral vectors and developed. A vaccine protecting cells (Pickering et al., 2016). More adjuvanted protein subunit horses against the closely related work is needed to elucidate the role vaccines. In all cases the target Hendra Virus (HeV; Equivac®) of cellular immune responses in antigen(s) have been the F and/or G has been approved for use in protection against NiV infection. glycoproteins (see Tables 3 and 4). Australia (Tan et al., 2018). Passive immunization experiments in a NiV The WHO developed a Target The NiV vaccines described below animal challenge model (hamsters) Product Profile (TPP) for a human are all research-stage candidates using immune sera and monoclonal NiV vaccine, including preferred focused on demonstrating antibodies have demonstrated as well as critical or minimal immunogenicity and protection that neutralizing antibodies product characteristics.1 Key against lethal NiV challenge. confer protection against NiV vaccine performance attributes No safety issues associated with challenge (Guillaume et al., 2004; recommended in the TPP are: vaccination or subsequent virus Guillaume et al., 2006). Finally, as challenge (due to antibody- discussed below, multiple modes • Intended use: For reactive use in dependent disease enhancement; of active vaccination have resulted outbreak settings ADE) were reported. However, in protection from lethal NiV more in-depth safety studies challenge in animal models. • Efficacy : ≥ 90% efficacy in will necessarily be performed preventing disease (preferred); on any NiV vaccine candidates There is broad consensus that ≥70% (minimal); rapid onset prior to advancing into human neutralizing antibodies confer of protection, less than 2 weeks clinical testing. protection against NiV infection after the first dose (preferred); and all vaccine development efforts protection ≤ 2 weeks after the last to date have focused on their dose (minimal). elicitation (Broder et al., 2012; Prescott et al., 2012; Satterfield et • Dose Regimen: Single-dose al., 2016b). However, a correlate of primary series (preferred); no protection based on neutralizing more than 2 doses, with some antibody titer has not been defined. protection after the first dose Neutralizing antibody titers in (minimal). animal vaccine challenge studies where protection was conferred • Durability of Protection: ≥ 1 are reported in Tables 3 and year (preferred); ≥ 6 months 4. However, since virtually all (minimal). animals were protected in these studies a threshold of protection • Product Stability and Storage: cannot be defined. The role of Shelf life of 5 years at 2-8oC cell-mediated immune responses (preferred); shelf life of at (CMI) in either natural immunity least 12 months at -20oC and or vaccine-induced protection demonstrated stability of ≥ 1 against NiV has received relatively month at 2-8oC (minimal).

13 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Viral Vector Candidates

A number of viral vector platforms Other recombinant viral vector expressing the NiV F or G vaccine platforms expressing NiV glycoproteins have been tested F or G have been tested, including: as vaccine candidates. The most vaccinia virus (Guillaume et widely used vector platform to date al., 2004), canarypox (ALVAC) has been the Vesicular Stomatitis (Weingartl et al., 2006), Measles Virus (VSV). Three types of VSV virus (Yoneda et al., 2013), vectors have been employed: Venezuelan Equine Encephalitis 1) replication-incompetent VSV Virus (VEEV) (Defang et al., pseudotypes expressing NiV 2010), Rabies virus (Keshwara F or G (Lo et al., 2014); 2) VSV et al., 2019), Newcastle disease virions expressing NiV F or G virus (Kong et al., 2012), Adeno that can undergo a single round Associated Virus (AAV) (Ploquin of replication (Mire et al., 2019); et al., 2013) and chimpanzee and 3) replication-competent adenovirus (ChAd; (van Doremalen recombinant viruses in which et al., 2019)). All these candidates the VSV-G protein is replaced by conferred full protection against the Ebola glycoprotein (ZEBOV) lethal challenge and/or elicited and also co-expressing NiV F high titers of neutralizing or G (DeBuysscher et al., 2014; antibodies. However, only the DeBuysscher et al., 2016; Prescott AAV and chimpanzee adenovirus et al., 2015). All three VSV-vaccine (ChAd) vectored vaccines types, whether expressing NiV F reported protection after a single or G antigens and administered vaccination. A summary of NiV viral singly, or co-administered, elicited vector vaccine candidates tested in neutralizing antibodies and fully animals is shown in Table 3. protected immunized animals from clinical disease in at least one of the 3 major NiV lethal challenge models (hamsters, ferrets or non-human primates; see Section V). Additionally, all three VSV vaccine types conferred protection after a single dose (see Table 3). Additional details on the animal challenge models and their use in vaccine studies are given in Section V.

14 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 3. NiV Viral-vector Vaccine Candidates Tested in Animals

NiV Neutralization Animal(s) Vaccination Route/Regimen/Challenge Reference Vaccine Description Titers (Pre- immunized (Strain3) challenge)2

Guillaume et al Vaccinia virus (VV) vector SC/2 vaccinations 1 month apart/ challenge 3 Hamsters ~ 1:10 – 1:25 (2004) expressing NiV G or F months after last vaccination (M)

Weingartl et al Canarypox vector (ALVAC) IM/2 vaccinations 14 days apart/ challenge on day Pigs 1:200 – 1:1280 (2006) expressing NiV G or F 28 post 2nd vaccination (NiV strain not specified)

Venezuelan Equine Defang et al Footpad inoculation/ 3 vaccinations on week 0, 5 Encephalitis Virus (VEEV) Mice ~ 1:215 – 1:217 * (2011) and 18/ no challenge expressing NiV G or F

Newcastle Disease Virus Kong et al (NDV) vector expressing Pigs IM/2 vaccinations 4 weeks apart/ no challenge ~ 1:27 – 1:212* (2012) NiV F or G

Single-cycle replication (Mire et al., IM/one vaccination/ challenge on day 20 post- VSV-∆G vector expressing Ferrets ~1:40 – 1:160 2013) vaccination (M). NiV G or F

Adeno-Associated Virus (Ploquin et al., IM/one vaccination/ challenge at 5 weeks post- (AAV) vector expressing Hamsters < 1:10 to 1:160 2013) vaccination (M). NiV G

Hamster: IP/ 2 vaccinations 21 days apart/ Measles virus vaccine Yoneda et al Hamsters, challenge 7 days post 2nd Vaccination AGM. SC/2 Hamster: Not reported vector expressing NiV G (2013) AGM* vaccinations 28 days apart/ challenge 2 weeks glycoprotein AGM: 1:1600 – 1:3200 post 2nd vaccination (NiV strain not specified).

Replication-competent DeBuysscher et IP/ one vaccination/ challenge on day 28 post- VSV vector expressing NiV Hamsters 1:80 - ≥ 1:640 al (2014) vaccination (M). G or F

Replication-defective IM/ one vaccination/ challenge at day 32 post- Lo et al (2014) VSV-∆G vector expressing Hamsters ~ 5 x103 – 1 x 104 vaccination (M) NiV G or F.

(Guillaume- Canarypox vector (ALVAC) Vasselin et al., Ponies (horses) IM/2 vaccinations 21 days apart/ no challenge ~ 1:2128* expressing HeV G or F 2016)

Prescott et al Live-attenuated VSV IM/one vaccination/ challenge on day 29 post- AGM1 1:80 – 1:160 (2015) vector expressing NiV G vaccination (M).

DeBuysscher et Live attenuated VSV IP/one vaccination/ challenge one day post- Hamsters Not reported al 2016 vector expressing NiV G vaccination (100% survival) (M).

Live-attenuated Rabies Keshwara et al ~1:10 to 1:600 Virus vaccine vector Mice IM/2 vaccinations 28 days apart/ no challenge (2019) (RABV) expressing NiV G. (no challenge)

Single-cycle replication IM/one vaccination/ challenge on day 28 post- Mire et al 2019 VSV-∆G vector expressing AGM1 1:160 – 1:640 vaccination (B). NiV G or F

(van Chimpanzee adenovirus IM/ one or two vaccinations (28 days apart)/ Doremalen et (ChAd) vector expressing Hamsters challenge 70 days post-prime or 42 days post- ~1:40 - ~1:100 al., 2019) NiV G boost (M and B).

1=African Green Monkey 2~ Indicates titer values estimated from data presented graphically 3 Challenge strain M= Malaysia; B=Bangladesh; *endpoint neutralization titers determined by 2-fold serial dilution and expressed as exponentials of 2. IM = intramuscular IP = intraperitoneal, SC = sub-cutaneous

15 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Subunit Vaccine Candidates

The most widely studied NiV does not efficiently generate HeV comprised of an enveloped virus- subunit vaccine candidates have cross-neutralizing antibodies like particle (VLP) created by co- utilized a purified, recombinant G (Ploquin et al., 2013). Various expression of the NiV M (matrix), glycoprotein from Hendra Virus adjuvant formulations have been F and G glycoproteins and (HeV) in which the transmembrane tested, including aluminum + CpG adjuvanted in either aluminum domain has been removed to allow (Bossart et al., 2012; McEachern hydroxide (Alhydrogel®), soluble G protein expression (sG). et al., 2008), CpG alone (Pallister monophosphoryl lipid A (MPLA), The high sequence conservation et al., 2013), and Quil A/DEAE- or CpG has also been tested and between the NiV and HeV G dextran/Montanide (Mungall et al., was 100% protective in the hamster glycoproteins (83% amino acid 2006). All formulations were 100% challenge model (Walpita et al., homology; (Wang et al., 2001) efficacious, eliciting neutralizing 2017). A summary of NiV subunit allows for the elicitation of antibodies and protecting all vaccine candidates tested in potent NiV cross-neutralizing vaccinated animals against lethal animals is shown in Table 4. antibodies (Sun et al., 2018), NiV challenge with no signs of although vaccination with NiV G clinical disease. A vaccine candidate

Table 4. NiV Submit Vaccine Candidates Tested in Animals

NiV Neutralization Animal(s) Vaccination Route/Regimen/Challenge Reference Vaccine Description Titers (Pre- immunized (Strain3) challenge)2

sGNiV or sGHeV Mungall et al adjuvanted with SC/ 3 vaccinations 2 weeks apart/ challenge 15 Cats 1:2,560 –1:20,480 (2006) Montanide/QuilA/DEAE- weeks after the first vaccination (M). dextran

Recombinant soluble HeV McEachern et IM/ 2 vaccinations 21 days apart/ challenge on G glycoprotein adjuvanted Cats 1:32 – 1:512 al (2008) day 42 post 1st vaccination (M). with CpG + AlhydrogelTM

Virus-like particles Walpita et al SC/ 3 vaccinations on days 0, 15 and 29/ no (VLPs) comprising NiV M, Mice 1:5 - >1:80 (2011) challenge G and F

Recombinant soluble HeV (Bossart et al., IM/ 2 vaccinations 21 days apart/ challenge 21 G glycoprotein adjuvanted AGM1 1:67 – 1:379 2012) days post 2nd vaccination (M). with CpG + AlhydrogelTM

Recombinant soluble HeV (Pallister et al., SC/ 2 vaccinations 20 days apart/ challenge 20 G glycoprotein adjuvanted Ferrets 1:16 – 1:128 2013) days or 14 months post 2nd vaccination (B) with CpG

Recombinant soluble Pickering et al HeV G glycoprotein in IM/ 2 vaccinations 21 days apart/ challenge 35 Pigs ~ 1:25- - 1:450 (2016) a proprietary adjuvant days post 1st vaccination (Strain not specified) (Zoetis, Inc.)

Single dose trial: IM/ one dose/ challenge on day 3-Dose Trial: ~ 1:200 Virus-like particles Walpita et al 28 post vaccination (M). – 1:2500 (VLPs) containing NiV M, Hamsters (2017) F and G. 3 dose trial: 3 doses on days 0, 21 and 42/ 1-Dose Trial: ~ 1:10 – challenge on day 58 (M). 1:200

1=African Green Monkey 2~ Indicates titer values estimated from data presented graphically 3 Challenge strain M= Malaysia; B=Bangladesh; *endpoint neutralization titers determined by 2-fold serial dilution and expressed as exponentials of 2. IM = intramuscular IP = intraperitoneal, SC = sub-cutaneous

16 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 More recently, novel antigen challenge with the Malaysian design options have been evaluated strain of Nipah virus. Authors using a structure-based design.5 noted immune responses were A stabilized prefusion F (pre-F), suboptimal. It is conceivable multimeric G constructs, and that the protection would have chimeric proteins containing both been superior under a two-dose pre-F and G were developed as regimen. protein subunit candidate vaccines. The proteins were evaluated In addition, some of the structure- for antigenicity and structural based designs described earlier integrity using kinetic binding for immunogen development5 assays, electron microscopy, and are being evaluated in the mRNA other biophysical properties. platform in collaboration with Immunogenicity of the vaccine Moderna, and clinical evaluation antigens was evaluated in mice is planned. using aluminum hydroxide as adjuvant. NiV Vaccine Candidates Supported by CEPI mRNA Vaccine Candidates As of August 2019, CEPI has four NiV vaccine candidates in its The US CDC has published proof- vaccine development portfolio, of-concept pre-clinical data on a three viral-vector platform Hendra virus glycoprotein mRNA candidates and one candidate vaccine in liquid nanoparticles.6 comprising an adjuvanted A single dose of the vaccine recombinant protein antigen protected up to 70% of hamsters (Table 5). against a lethal, intraperitoneal

Table 5. NiV vaccine candidates supported by CEPI

Developer Vaccine Platform Development Stage

University of Tokyo Recombinant Viral Vector Pre-clinical

Profectus Biosciences/Emergent Biosolutions/ Recombinant Protein Phase 1 (USA) PATH

Janssen Vaccines & University of Oxford Recombinant Viral Vector Pre-clinical

Replication-competent rVSV vector Public Health Vaccine, LLC Pre-clinical expressing NiV-G

Source: https://cepi.net/research_dev/our-portfolio/

5 https://www.frontiersin.org/articles/10.3389/fimmu.2020.00842/full 6 https://academic.oup.com/jid/article/221/Supplement_4/S493/5637464

17 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 III. STANDARDIZATION OF ASSAYS AND ANIMAL MODELS

Assays and animal models to quantify or characterize immune responses elicited by vaccination are, by their nature, inherently variable.

The reasons for this include the Immune Serum Reference and commercial manufacturing molecular complexity of the Standards (https://www.who.int/biologicals/ samples (serum or other biological vaccines/en/). Recent examples samples), the need to produce One of the most important tools include HPV 16 (Ferguson et al., reagents in complex biological for standardization of serological 2011), Typhoid Fever (Rijpkema et systems such as cell culture or in assays is immune reference al., 2018), Respiratory Syncytial vivo, variability in composition and serum. Even when similar assay Virus (McDonald et al., 2018) and stability of these reagents, and the formats are used for detection Zika (Source: WHO/BS/2018.2345). need to test immune responses in of antigen binding antibodies or vivo. Nevertheless, modern vaccine virus neutralizing antibodies, Three key factors determine development requires vaccines the resulting data can be highly the fitness of material for use and samples from vaccinated variable between laboratories due as a biological standard. First, humans and animals be tested to differences in assay methods the material must have similar with the highest possible precision. and reagents. For example, a composition and in vitro behavior The task is further complicated 10-laboratory collaborative study to the human sera test articles. by the collaborative and global assessing the precision of assays Second, the standard should be nature of modern vaccine for detection of serum antibodies commutable, meaning it should development. Multiple research against Human Papillomavirus 16 work for a wide range of serological laboratories, vaccine developers, (HPV 16) revealed inter-laboratory assays and vaccine platforms non-governmental organizations, variations in anti-HPV titer of being tested. Finally, a blinded and regulatory agencies are often up to 25-fold for the same test multi-laboratory collaborative involved in the development sample (Ferguson et al., 2006). A study must demonstrate the process and vaccine candidates similar, 15-laboratory collaborative utility of the standard for reducing utilizing different platform study evaluating assays for serum intra-laboratory assay variability. technologies are often evaluated for antibodies against H5N1 influenza (Source: CEPI 2nd Standards and the same disease indication. Thus, showed inter-laboratory variations Assays Workshop; June 2019). standardization of methods and in titer of 10 to 35-fold, depending reagents is important to facilitate on the sample and type of assay Serum reference standards for a development of new vaccines such (Stephenson et al., 2009). The new vaccine are often established as for NiV. The goal is to enable purpose of establishing immune in a staged manner as the “like versus like” comparisons reference standards is to provide development process progresses. of data generated by different a common, external control to This mitigates the risk of producing laboratories and derived from improve the comparability of exhaustively characterized many assay types. Recognizing assay data between laboratories. materials which might not be the value of phase-appropriate With the standard in place, test required if vaccine development standardization early in the vaccine results are reported relative does not progress. For R&D and development process, CEPI is to the activity of the reference early clinical trials a working promoting assay, reagent, and standard. In the studies cited standard or interim standard may animal model standardization to above, use of a common reference be established by a collaborative accelerate development of vaccines standard significantly reduced study involving a relatively limited for NiV and other priority diseases intra-laboratory assay variability. number of laboratories, and in its portfolio. Reference standards have been relatively low volumes may be developed for many vaccine sufficient in the earlier stages. indications, both in development

18 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 A number of different sources of (Source: CEPI 2nd Standards common reagents (reference immune sera may be considered. and Assays Workshop; June sera and antigens) provided by For example, a collaborative 2019). A single, large lot of an regulatory agencies and using a study for establishment of an antibody standard is preferred single, validated assay method interim standard for antibodies to avoid potential variability to test and release new seasonal to Ebola virus (EBOV) tested between multiple lots and the vaccine formulations. In general, plasma samples from patients who need for subsequent bridging standards may improve inter- recovered from Ebola infection studies. Once suitable standard laboratory test performance. (convalescent sera), anti-EBOV sera candidates are available for However, the need, feasibility IgG preparations from trans- evaluation, a collaborative study is and level of standardization is chromosomal (Tc) cows immunized performed to evaluate serological typically considered on a case- with experimental vaccines and assays performed by a number of by-case basis according to the plasma from vaccinated volunteers participating laboratories. A broad stage of vaccine development, participating in an EBOV vaccine panel of test samples from different the types of assays in use and the trial (Wilkinson et al., 2017). An sources (e.g., sera from naturally potential of standards to facilitate interim standard for NiV will infected humans, animals infected development and licensure. probably be generated from non- in the laboratory, and vaccinated Standardized reference sera are human primates infected with a humans or animals) is assayed and relatively easy to implement since sub-lethal dose of NiV and which the intra-laboratory variability in they ideally should be commutable generate high titers of neutralizing assay results is assessed. Finally, across many assay types and are antibodies (Dhondt and Horvat, the test sample absolute values broadly recognized for improving 2013). Obtaining convalescent sera (for example, geometric mean both intra- and inter-laboratory from NiV survivors is also being titers) are expressed relative to the assay consistency. In contrast, considered (Source: CEPI 2nd activity of the candidate standard standardized assay formats are Standards and Assays Workshop; and the ability of the standard more challenging to implement, June 2019). However, given the to improve intra-laboratory especially for newer vaccines sporadic nature of NiV outbreaks comparability of test results is and those in development, since and relatively small number of assessed. Once the standard has vaccine antigens may differ cases (and available survivors), been chosen a full storage stability between candidates and there is obtaining sufficient quantities of program is conducted to ensure the less consensus on the ideal assay convalescent sera for long-term quality of the material over time. format. With these considerations use may be challenging. Therefore, Trending of assay performance in mind, the benefits and potential in the case of emerging infections over time is also performed. The challenges of standardizing such as NiV one of the alternative International Standard itself is various assay and animal model approaches described above may not intended for routine assay use. components to accelerate need to be employed. A working reference standard is NiV vaccine development are established for routine use and summarized in Table 6. Establishment of an interim a bridging study is conducted to standard usually precedes calibrate the working standard to establishment of an International the International Standard (Source: Standard (IS) , often called CEPI 2nd Standards and Assays an International Reference Workshop; June, 2019). Preparation (IRP) under the endorsement of the WHO Standardization of Other Biological Expert Committee on Biological Assay Reagents and Methods Standardization. This is a more formal process, taking up to 36 Many aspects of biological months and involving a larger assays for vaccine testing may and more in-depth collaborative be standardized to improve the study, often involving more comparability of intra-laboratory than 25 laboratories and a wide data. Common reagents (reference geographical distribution. This sera, antigens, virus stocks) may is the main difference from a be produced and standardized working or interim standard. assay methods established and Regulatory agencies generally validated. For example, potency expect an established International testing for release of subunit Standard to be used in pivotal seasonal influenza vaccines clinical trials for vaccine approval, is performed under a high unless specifically justified degree of standardization using

19 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 6. Benefits and Potential Challenges of Implementing Biological Standards for NiV Vaccine Development

Standard Benefits Potential challenge(s)

Finding human NiV convalescent donors may Long track record and high level of acceptance be challenging (low numbers). Generating for improving intra-laboratory assay Immune Standard Reference Sera NiV convalescent animal sera by sub-lethal comparability. Inter-laboratory performance infection requires BSL-4 containment and may also be improved. can be considered an interim mitigation

Choice of genotype/strain and ensuring Promote standardization of serum antibody reactivity to diverse serum isolates; storage Common stocks of ELISA coating antigen detection; relatively easy to produce, test, stability; heterogeneity in post-translational store and distribute. modifications; biochemical differences between strains.

Current use of pseudovirus assays by major NiV research groups is rare. Often Common pseudovirus(es) for assaying Promote use of an assay method which can be overestimate titer compared to wild-type neutralizing antibodies performed in low-level biocontainment NiV-based assays, so will require extensive characterization compared to traditional NiV neutralization assays to gain acceptance.

Production, testing, storage, stability and Common NiV virus stocks for neutralization Promote standardization of animal challenge distribution of live NiV and requirement for assays and animal challenge models experiments and NiV neutralization assays BSL-4; mutations during passaging of ssRNA virus.

The large number of potential variables in challenge model performance (ie., challenge Standards for performance of animal Promote standardization of challenge strain/stock, route, dose, animal species etc.) challenge models experiments may complicate agreement on and acceptance of performance standards.

20 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 IV. NIV SEROLOGICAL ASSAYS

Robust serological assays for quantifying and characterizing humoral immune responses in humans and animals are critical for vaccine development.

A number of methods have been assays for NiV, as well as newer experiments is compiled in Table developed for NiV serology, and the assays in earlier stages of use and 8 and Table 9 to illustrate the refinement and standardization acceptance. An analysis of the pros prevalence of use, variables in of such methods will be essential and cons of different serological assay performance, how reagents for facilitating development of assays for NiV vaccine development and methods have changed over safe and effective human vaccines is presented in Table 7. The usage time and opportunities for assay against NiV. This section describes of assays in a large number of NiV standardization. the commonly used serological vaccine studies and other research 1. Detection of antigen – specific serum IgG

Detection of antigen – specific The earliest ELISAs for detection the 17 ELISAs in Tables 8 and 9 serum IgG is essential to the of NiV antibodies in sera were use recombinant G or F for target vaccine development process to developed by the Centers for IgG capture. The sFNiV and sGNiV characterize the specificity and Disease Control (CDC; USA). have been produced in a variety of magnitude of the vaccine-induced Different ELISAs were developed recombinant expression systems humoral immune response. The for NiV-specific serum IgG and (E. coli, insect cells, mammalian most common assay method is IgM. These ELISAs were used cells) and are usually epitope the traditional “indirect” ELISA. for surveillance and diagnosis of tagged for ease of purification In this assay a target antigen is disease in humans and pigs, and (Eshaghi et al., 2005; Eshaghi et al., plated (adsorbed) onto a 96- well used detergent and radiation- 2004; Keshwara et al., 2019; Kurup microtiter plate. After blocking inactivated, NiV – infected Vero et al., 2015). The glycosylation and the plate to suppress non-specific cell lysates as the target antigen disulfide bonding in the NiV F and binding, dilutions of immune (Daniels et al., 2001). Several of G glycoproteins make eukaryotic or control sera are added to the NiV animal vaccination studies cells preferable for recombinant the wells. After washing away and research experiments detailed expression of these antigens. The unbound antibody, bound IgG is in Table 8 and Table 9 utilized many successful tests of vaccines usually detected by the addition inactivated crude NiV- infected targeting the G glycoprotein in NiV of a species-specific anti-IgG Vero extracts or gradient-purified animal challenge models make it secondary antibody conjugated NiV as the target antigen. However, likely that this antigen will be used to a chromogenic enzyme. While the use of NiV as an assay reagent in human vaccine candidates. serum IgG is measured to elucidate is obviously problematic since vaccine responses, as well as for the initial preparation requires surveillance and epidemiology, BSL-4 containment. ELISAs using measurement of NiV-specific NiV-infected crude extracts also serum IgM is usually performed suffered from non-specific binding for diagnosis of active infection (Daniels et al., 2001). The discovery (Mazzola and Kelly-Cirino, 2019). that NiV F and G glycoproteins are The advantages of the ELISA assay the major target of neutralizing format include its broad use and antibodies and their use in vaccine familiarity throughout biomedical formulations spurred the use science, relatively low-tech and of recombinant, soluble NiV F low-cost application and wide (sFNiV) and G (sGNiV) as the target availability of reagents. antigens in ELISA assays. Six of

21 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 A newer assay format that has been bead and determines the analyte ability to develop multiplexed used for detection of NiV antigen- being detected. The second laser assays for discrimination of specific serum IgG is the bead- detects the PE-derived signal, different antibody types and based liquid protein array system which is in direct proportion to specificities. However, the commonly known as Luminex® the amount of anti-NiV G bound utilization of this system for (Vignali, 2000). In this assay (Source: R&D Systems website). NiV vaccine development is (Bossart et al., 2007), purified One vaccine study in Table 8 limited by availability of the sGNiV protein is covalently coupled (Pallister et al., 2013) and two specialized instrumentation to fluorescent microspheres. After other research studies in Table 9 and relative unfamiliarity of binding to the analyte (NiV G- utilized this system for detection the technology compared with specific IgG) biotinylated Protein A of antigen-specific serum IgG. ELISA-based assays. Nevertheless, is added, followed by streptavidin- Luminex®-based assays are standardization of such an assay phycoerythrin (PE), a fluorescence capable of high sensitivity and would be similar to the ELISA and indicator that emits at a different specificity and the experimental would involve use of standard wavelength than the microspheres. manipulations are no more immune sera and common stocks The bead mixture is analyzed on complicated than running ELISA- of target antigen. a dual laser flow-based detection based assays. Another potential instrument. One laser detects the advantage of this platform is the

2. Detection of serum neutralizing antibodies

The quantitation of serum fixed with methanol. Plaques are in 50% of replicate wells (Crameri neutralizing antibodies is essential then detected by immune-assay et al., 2002). This assay, or variants for measuring vaccine potency and using rabbit antisera specific of it, was used in 11 of 24 NiV is often important for establishing for a NiV antigen. Serum titer is animal studies detailed in Tables 8 correlates of protection. As shown expressed as the reciprocal of the and 9. in Tables 8 and 9, most published serum dilution that reduces the NiV vaccine studies and other number of plaques to 75% of that The major drawback of both the research investigations have of control untreated virus. The PRNT and SNT assays is that they utilized traditional assays for virus PRNT assay was used in 10 of the use live NiV and the procedures neutralization based on inhibition 24 NiV animal studies described (or at least the first steps) must of NiV-mediated killing or in Tables 8 and 9. be performed in BSL-4 cytopathology in Vero cell cultures. containment (Table 7). This would One common assay format is the The other common traditional be a major hurdle to any attempts Plaque Reduction Neutralization assay for NiV neutralization is the to standardize assays of this type Test (PRNT), of which there are Serum Neutralization Test (SNT) since scaled-up production and a number of variations. In the (Daniels et al., 2001) In this assay, testing of a common stock of NiV original method developed for serum dilutions are incubated with under BSL-4 containment would NiV (Crameri et al., 2002) the test approximately 200 TCID50 NiV in be extremely challenging. sample (immune or control serum) 96-well microtiter plates prior to The need for BSL-4 containment is diluted and mixed with a NiV the addition of Vero cells. Cultures is also a significant hinderance to suspension. After an incubation are visually (microscopically) developers lacking such facilities. period the mixture is applied to a examined for the presence or Thus, other assay formats have confluent monolayer of Vero cells. absence of cytopathic effect after been pursued that do not require After an adsorption period the virus a three-day incubation. The high-level biocontainment, and mixture is removed and replaced neutralization titer is expressed that may thus facilitate NiV vaccine with fresh culture medium. After as the reciprocal of the highest development. incubating overnight, the cells are dilution that prevents virus growth

22 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Pseudotype viruses (pseudoviruses) Overall, these and other results have been constructed for use suggest that pseudovirus-based in surrogate NiV neutralization assay systems are a promising assays which can be performed alternative to classical NiV under BSL-2. A pseudovirus is an neutralization assays for use in enveloped virus expressing one vaccine development. However, or more foreign virus envelope acceptance of NiV pseudovirus- proteins that mediate cell based neutralization assays attachment and membrane fusion. for vaccine development and In most cases the pseudovirus licensure will require thorough also carries a reporter gene such characterization to understand as GFP or luciferase which serves the comparability in sensitivity as an indicator of infection. The and specificity with traditional most common parental viruses NiV neutralization assays used for pseudovirus construction (Table 7). The ability of a single are vesicular stomatitis virus reference standard to harmonize (VSV) and lentivirus (HIV); (Wang pseudovirus-based assays as well and Daniels, 2012). The first NiV as NiV-based assays will also play pseudovirus assay (Tamin et al., a major role in the acceptance 2009) utilized a VSV constructed pseudovirus-based assays. to display both the NiV F and G glycoproteins on its envelope and Another promising NiV surrogate carrying the luciferase gene as neutralization assay uses a the reporter. This assay showed variation of the Luminex® method generally good correlation of described for detection of serum titers with a classical PRNT assay, IgG binding. Like the binding although some serum samples with antibody assay, this Luminex® very low titers showed reduced assay (Bossart et al., 2007) uses sensitivity in the pseudovirus sGNiV covalently conjugated assay. Another assay system to fluorescent microspheres. developed by Kaku et al., (2009) Biotinylated Ephrin B2, the cellular also utilized VSV pseudotyped receptor for NiV G, is added and with NiV G and F but used green the binding interaction is detected fluorescent protein (GFP) as with streptavidin-phycoerythrin. the reporter gene (Kaku et al., Neutralizing antibodies targeting 2009). This assay showed good NiV G disrupt the sGNiV – Ephrin specificity and higher sensitivity B2 interaction and thus diminish compared with the NiV SNT the phycoerythrin fluorescence assay. A variation of this assay signal. The sensitivity and was developed (Kaku et al., 2012) specificity of this assay appears in which the GFP reporter gene to be comparable to the NiV SNT was replaced by secreted alkaline assay using control and immune phosphatase (SEAP), which can sera from a variety of sources. be assayed from the culture In addition to standard immune supernatant using an ordinary reference sera, standardization ELISA plate reader. This assay was of this assay format could benefit also generally more sensitive than from the availability of common the classical SNT assay. Three in stocks of sGNiV, purified Ephrin B2 vivo studies described in Tables 8 and a control anti-sGNiV mAb. and 9 used NiV pseudovirus-based neutralization assays.

23 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 7. Pros and Cons of NiV Serological Assays

Binding or Gaps in Reagent neutralizing Pros Cons Standardization* antibody assays

• Widely used method • Need for production of NiV coating • Production and characterization using standard laboratory antigen under BSL-4 increases cost and of common stocks of instrumentation and readily decreases accessibility of the method, inactivated, NiV-derived coating Indirect ELISA available reagents. antigen. • NiV reagent standardization would be (NiV coating antigen • Reactive to antibodies against overly complicated NiV F and G. • Purification of NiV is required to reduce non-specific assay background.

• Widely used method • Testing to ensure proper folding • Production and characterization using standard laboratory (conformation-specific mAbs) & post- of common stocks of purified, instrumentation and readily translational modifications (disulfide recombinant NiV G and F available reagents. bonding & glycosylation); storage glycoproteins. stability testing. Indirect ELISA • Biocontainment not required for (Recombinant protein antigen production. coating antigen) • Low non-specific binding compared to whole NiV coating antigen.

• Good accessibility for all laboratories.

• Capable of high sensitivity and • Rarely used by major NiV research labs. • Production and characterization precision. of common stocks of purified, • Lower familiarity with researchers and recombinant NiV G and F Luminex® (antibody • Potential for multiplexed developers compared to ELISA. glycoproteins. binding) format to simultaneously detect different antibody types and • Requires specialized instrumentation specificities. and reagents, therefore increased cost compared to ELISA.

• Wide use and acceptance by • Use of live NiV and requirement for • Production and characterization major NiV research labs. BSL-4 containment during production, of common stocks of live NiV testing, and storage increases cost, (Malaysia and Bangladesh Traditional NiV • Directly measures neutralization assays lowers accessibility of the method and strains) neutralization of the pathogen makes standardization complicated (PRNT, SNT) of interest (NiV) • Possibility of mutations in ssRNA genome during passaging.

• Performed under BSL-2. • A surrogate (indirect) method rarely • Construction, production and used by major NiV research labs; characterization of a common • Scale-up for production of extensive comparability testing with VSV-based pseudovirus standardized stocks cheaper & classical methods will be required to expressing the NiV F and G Pseudovirus-based less complicated than for NiV. neutralization assay gain acceptance. glycoproteins. • Some versions show higher • Variety of different pseudovirus designs sensitivity than classical NiV makes acceptance and standardization neutralization assays. more complicated

• Capable of high sensitivity and • No reports of use for any NiV vaccine or • Common stocks of purified, selectivity. animal challenge studies. recombinant NiV G and F glycoproteins. Luminex® • Cell-free method should offer • Need for specialized instrumentation. (serum neutralization) a high level of precision and • Common stocks of purified, ease of method qualification/ biotinylated Ephrin B2. validation

*In addition to standard reference sera used in all assays

24 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 8. Serological Assays Used in NiV Pre-Clinical Vaccine Studies

Serum Antibody Binding Serum Neutralizing Reference Vaccine Type Assay Antibody Assay

sGNiV or sGHeV adjuvanted wit None SNT* (Mungall et al., 2006) Montanide/QuilA/DEAE-dextran

Recombinant canarypox vector ELISA; plates coated with purified PRNT** (Weingartl et al., 2006) (ALVAC) expressing NiV G & F NiV

Recombinant Venezuelan Equine ELISA; plates coated with purified Pseudovirus (HIV-1) (Defang et al., 2010) Encephalitis Virus (VEEV) recombinant HeV/NiV G or F expressing NiV G or F proteins

Recombinant soluble HeV G ELISA; plates coated with purified SNT (McEachern et al., 2008) glycoprotein adjuvanted with CpG recombinant sGHeV or sGNiV + AlhydrogelTM

Immunofluorescence assay using PRNT Virus-like particles (VLPs) (Walpita et al., 2011) 293T cells transfected with NiV comprising NiV M, G and F M, G or F.

Recombinant Newcastle Disease ELISA; plates coated with purified Pseudovirus (VSV∆G-GFP) (Kong et al., 2012) Virus (NDV) expressing NiV G recombinant sGNiV or SFNiiV or F.

Recombinant, single-cycle Luminex® using recombinant PRNT (Mire et al., 2013) replication VSV-∆G vector soluble NiV F and G glycoproteins expressing NiV G or F

Recombinant soluble HeV G Luminex® using recombinant SNT (Pallister et al., 2013) glycoprotein adjuvanted with CpG soluble NiV G glycoprotein

Recombinant measles vaccine ELISA; plates coated with None (Yoneda et al., 2013) vector expressing NiV G recombinant sGNiV glycoprotein (AGM)

Recombinant, replication- ELISA; plates coated with purified SNT (DeBuysscher et al., 2014) competent VSV vector expressing NiV. NiV G or F

Recombinant, replication- None Pseudovirus (VSV∆G-GFP) (Lo et al., 2014) defective VSV-∆G vector expressing NiV G or F.

Recombinant canarypox vector None SNT (Guillaume-Vasselin et al., 2016) (ALVAC) expressing HeV G or F

Recombinant, live-attenuated ELISA; plates coated with purified SNT (Prescott et al., 2015) VSV vector expressing NiV G NiV (AGM)

Recombinant, live attenuated VSV ELISA; plates coated with purified None (DeBuysscher et al., 2016) vector expressing NIV G NiV

Recombinant soluble HeV G ELISA; plates coated with purified PRNT (Pickering et al., 2016) glycoprotein in a proprietary recombinant sGNiV. adjuvant (Zoetis, Inc.)

ELISA; Plates coated with NiV SNT with fluorescence reporter Virus-like particles (VLPs) clarified supernatant (for NiV (Walpita et al., 2017) containing NiV M, G and F IgM ELISA) and NiV-infected cell extract (for NiV IgG ELISA).

Recombinant, live-attenuated ELISA; Plates coated with SNT with fluorescence reporter (Keshwara et al., 2019) Rabies Virus vaccine vector recombinant sGNiV. (RABV) expressing NiV G.

Recombinant, single-cycle None SNT (Mire et al., 2019) replication VSV-∆G vector expressing NiV G or F

*Serum Neutralization Test; **Plaque Reduction Neutralization Test

25 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 9. Serological Assays Used in Other NiV Research Studies

Serum Antibody Binding Serum Neutralizing Reference Study Type Assay Antibody Assay

ELISA; plates coated with crude Development of hamster (Wong et al., 2003) extracts from NiV-infected Vero None challenge model cells

ELISA; plates coated with crude (Guillaume et al., 2004) Antibody prophylaxis (hamster) extract from NiV-infected Vero PRNT** cells

ELISA; plates coated with NiV clarified extract or recombinant (Guillaume et al., 2006) Antibody prophylaxis (hamster SNT* NiV N protein produced in insect cells

ELISA; plates coated with purified (de Wit et al., 2011) NiV transmission in hamsters SNT NiV

Impact of non-structural (Mathieu et al., 2012b) proteins on NiV virulence Purified NiV None (hamster)

Foodborne transmission of NiV in ELISA; plates coated with (de Wit et al., 2014) None hamsters purified, inactivated NiV.

Multiplexed microsphere assay Therapeutic mAb treatment of (Geisbert et al., 2014) (Luminex) using recombinant SNT NiV infection (AGM) soluble NiV F glycoprotein

Analysis of NiV AGM challenge (Johnston et al., 2015) None Pseudovirus (VSV∆G-RFP) model

Impact of non-structural (Satterfield et al., 2015) proteins on NiV disease course None PRNT (ferret)

Monoclonal antibody study (Borisevich et al., 2016) None PRNT (hamster)

Multiplexed microsphere assay (Mire et al., 2016) NiV strain differences in AGM (Luminex) using recombinant PRNT soluble NiV F glycoprotein

Impact of non-structural (Satterfield et al., 2016b) None PRNT proteins on NiV virulence (ferret)

Favipiravir prophylaxis study (Dawes et al., 2018) None PRNT (hamster)

(Mathieu et al., 2018) Peptide prophylaxis (AGM) None SNT

ELISA; plates coated with NiV (Lo et al., 2019) Remdesivir prophylaxis (AGM) SNT antigen

Immune response/pathogenesis (Schountz et al., 2019) None SNT study in hamsters

*Serum Neutralization Test; **Plaque Reduction Neutralization Test

26 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 V. NIV ANIMAL MODELS

Vaccine Licensure under the FDA “Animal Rule”

Well characterized, robust animal study endpoint is clearly related attributes of relevant animal challenge models are another to the desired benefit in humans, challenge models to support FDA critical component for development generally the enhancement of licensure of vaccines under the of any vaccine and, as with survival or prevention of major Animal Rule are summarized below assays, standardization of animal morbidity; and 4) The data or (Golding et al., 2018): models can accelerate vaccine information on the kinetics development by promoting like- and pharmacodynamics of the 1. Animal species should show key versus-like comparisons between product or other relevant data characteristics of the human laboratories. In the case of NiV or information, in animals and disease following exposure to the and other emerging diseases the humans, allows selection of an challenge pathogen (time from refinement and standardization of effective dose in humans. For exposure to onset of disease, animal models takes on additional vaccines, prediction of clinical time course/progression of importance regarding the pathway benefit is determined by bridging disease, clinical manifestations, to vaccine licensure. Since NiV from the human immunogenicity morbidity and lethality. outbreaks are sporadic and data to the animal model infect relatively small numbers, immunogenicity and efficacy 2. The challenge agent used in the performing controlled vaccine data (Source: Draft FDA Guidance animal study should be relevant efficacy studies in humans becomes for Industry; Animal Models – to the human disease. very challenging. A potential Essential Elements to Address alternative licensure pathway is the Efficacy Under the Animal Rule; 3. The immune marker(s) selected FDA “Animal Rule”, a mechanism January 2009). To date, one FDA should reflect the protective designed for disease indications vaccine approval has followed immune responses generated by for which efficacy studies would this pathway, a post-exposure humans. be infeasible or unethical. Under prophylaxis indication for an this mechanism Phase I/II safety anthrax vaccine (Beasley et al., 4. The vaccine dose and vaccination and immunogenicity testing 2016). The European Medicines schedule chosen for adequate is conducted in humans, but Agency (EMA) does not currently and well-controlled studies in efficacy is demonstrated in a have a detailed mechanism animals should elicit an immune well-established animal model(s) analogous to the FDA Animal Rule. response in animals reflective of that provides substantial evidence However, current EMA guidance that in humans. of effectiveness when all of the leaves open the possibility following four criteria are met: of using data from animal 5. Ideally, the immunological 1) There is a reasonably well- models to demonstrate vaccine assays should be species- understood pathophysiological efficacy (Source: EMEA/CHMP/ independent. mechanism of the toxicity of VWP/164653/05 Rev.1; 2018) the substance (pathogen) and 6. There should be a robust its prevention or substantial According to the FDA guidance, statistical plan. reduction by the product (vaccine); animal model(s) should be 2) The effect is demonstrated in highly refined and characterized more than one animal species with regard to understanding of expected to react with a response disease etiology, progression and predictive for humans, unless the pathology and their performance effect is demonstrated in a single (along with validated assays) animal species that represents a should be standardized as much sufficiently well-characterized as possible ( (Williamson and animal model for predicting the Westlake, 2019); WHO Nipah response in humans; 3) The animal R&D Roadmap Draft, 2018). Key

27 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 NiV Animal Challenge Models

NiV infects a wide range of wild (Dhondt et al., 2013). No reports of how the models have been and domesticated animals, NiV strains lab-adapted to mice or used. Key variables relevant to including: fruit bats, cats, dogs, any other animal species have been standardization, such as challenge swine, and horses (see Section found in the published literature. strain, dose, and route of infection II, Epidemiology). However, the The NiV animal challenge models are provided, along with other symptomology is often variable, that most accurately recapitulate available details such as survival and in some cases does not closely the symptomology and pathology time, clinical signs, and pathology. resemble the human disease. Fruit of the human disease are Syrian A summary comparison of clinical bats are asymptomatically infected Golden Hamsters, ferrets and signs and pathology of NiV (Geisbert et al., 2012). In the African Green Monkeys. The infection in the hamster, ferret and laboratory setting mice are largely following sections describe the AGM models is given in Table 10, resistant to NiV infection unless key characteristics and use of and Table 16 provides an analysis delivered intracranially (Mungall the models. For each model of the pros and cons of each model et al., 2006), administered to aged a table compiling published vis a vis its usefulness in vaccine mice (C57B6 or BALB/c), or given challenge experiments is provided development. to Type I interferon knockout to illustrate experimental (IFNAR KO) transgenic mice performance variables and

Table 10. Summary of clinical signs and pathology in the NiV hamster, ferret and AGM challenge models.

Clinical Signs Species Gross lesions Histology Virus found in: Respiratory Neurological

Labored breathing, Imbalance, muscle Vasculitis, meningitis, Lung, nasal epithelium, CNS, Edema, hemorrhages, Hamster serosanguineous twitching, tremor, encephalitis, endothelial heart, liver, spleen, kidney, congestion nasal exudates limb paralysis syncytia bladder, urine

Depression, Necrotizing alveolitis, Brain, lung, lymphoid Dyspnea, cough, tremors, Edema, hemorrhages, glomerular necrosis, organs, adrenal, kidney, Ferret serous nasal myoclonus (muscle enlarged lymph nodes vasculitis, endothelial testes, uterus, liver discharge twitching), hind syncytial, meningitis. pharyngeal and rectal swabs limb paresis

Pulmonary consolidation, Severe Dyspnea, congestion of lungs, Alveolar hemorrhages, Lung, lymph nodes, heart, African open-mouth enlarged lymph pulmonary edema and Muscle twitches, liver, spleen, kidney, Green breathing, nodes, congested inflammation, alveolitis, seizures adrenal gland, brain, urinary Monkey serosanguineous liver, inflammation fibrinoid necrosis, bladder, sex organs nasal discharge of gastrointestinal vasculitis, meningitis tract, congestion in the brain.

Adapted from (Dhondt and Horvat, 2013)

28 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 1. Syrian Golden Hamster

The Syrian Golden Hamster encephalitis and lesions in multiple had accelerated virus replication, (Mesocricetus auratus) is the organs, including the lungs pathology, and death compared most commonly used animal and brain, caused by vasculitis to the Bangladesh strain, which challenge model for NiV owing and thrombosis (Williamson had a more delayed disease to its small size and economical and Torres-Velez, 2010). Some progression. The accelerated housing. Tables 11 and 12 provide variances in NiV disease pathology disease course of the Malaysia a compilation of most of the have been reported in different strain in hamsters appears contrary NiV hamster challenge studies hamster studies. The NiV (Malaysia to what has been observed in found in the published literature, strain) infection in hamsters human outbreaks. However, the including challenge strain and reported by Wong et al., (2003) overall symptomology and viral available identifying information was dominated by neurological distribution of the two strains were on the challenge virus, route of signs. In contrast, (Rockx et al., comparable (DeBuysscher et al., administration, dose and notes 2011) reported that infection by 2013). on the disease course. Hamsters the same NiV strain, by the same develop clinical symptoms and route and at a similar dose caused Several key variables in pathologies which closely resemble a primarily respiratory disease. performance of the NiV hamster NiV disease in humans, including The cause of the discordance in model are apparent from Tables respiratory and neurological results in these studies is not clear 11 and 12. A wide range of doses is signs (Guillaume et al., 2006). and suggests that further studies reported, and two different routes Infection by the intranasal (IN) or are needed to fully understand of administration are used. Many intraperitoneal (IP) routes induces NiV infection in the hamster. This of the published studies provide similar symptoms and pathologies, will be particularly important for only limited information on the but the disease course may vary standardization of the model. To identity and origin of NiV challenge with dose and route (Rockx et our knowledge, a formal natural stocks. Most of the stocks appear al., 2011; Wong et al., 2003), with history study of the NiV hamster to be different isolates from the IP inoculation causing a more challenge model has not been same outbreaks: Malaysia 1999 and rapid disease course than the IN performed. However, several Bangladesh 2004 (Source: CEPI 2nd route. Respiratory signs in the publications have investigated Standards and Assays Workshop, hamster include labored breathing the course of NiV infection and June 2019). However, the identity and severe respiratory distress, pathology in hamsters in detail, of many individual isolates, how bloody nasal discharge, pulmonary including: virus strain, routes their activities compare to one infiltrates (fluid build-up), and of infection and dose, time to another, and how they have been hemorrhagic lesions of the lung death, and virus replication and stored, amplified, and tested (Rockx, 2014). Neurological signs pathology/histopathology in major is unclear. Establishment of include behavioral deterioration, organs and tissues (Baseler et al., common, well-characterized virus lethargy, imbalance, muscle 2015; DeBuysscher et al., 2013; challenge stocks is important for twitching, tremors and limb Rockx et al., 2011; Wong et al., standardization of this model. It paralysis (Dhondt and Horvat, 2003). is also unclear why the Malaysia 2013). Dose levels appear to play a strain has been the preferred role in NiV symptomology in the Both the Malaysia and Bangladesh challenge strain although the hamster. High doses, particularly NiV strains have been tested in Bangladesh strain causes most when delivered IN, induce a the hamster model, but most human outbreaks. Many of the primarily respiratory syndrome. studies have used Malaysia as same gaps are also true for the Lower doses also induce respiratory the challenge virus (see Tables 11 ferret and African Green Monkey symptoms, but neurological and 12). A direct comparison of Models, as discussed below. symptoms are also present (Rockx the two NiV strains in hamsters et al., 2011). Hamsters develop suggested that the Malaysia strain

29 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 11. NiV Hamster Model Challenge Studies

Challenge Route Reference NiV Strain (Source) Notes (Dose)

IN (up to 1 x 106 pfu) IP: 100% lethality by 8 days post-infection (Wong et al., 2003) Malaysia (patient cerebrospinal fluid) IP (up to 1 x 104 pfu) (p.i.) IN: 5 out of 6 lethality by day 15 p.i.

Vaccine efficacy study. 100% lethality by 8 (Guillaume et al., 2004) Malaysia (patient cerebrospinal fluid) IP (1 x 103 pfu) days p.i. (placebo).

Passive antibody protection study. Mean (Guillaume et al., 2006) Malaysia (patient cerebrospinal fluid) IP (750 pfu; 100 LD50) survival time for placebo was 7.5 days p.i.

(Georges-Courbot et Mean survival time for placebo Malaysia (patient cerebrospinal fluid) IP (35 and 350 LD50) al., 2006) was 7.3 ± 2.9 days p.i.

Mean survival time for placebo (Freiberg et al., 2010) Malaysia (CDC; passaged on Vero cells) IP (1 x 104 TCID50) was 9.2 ± 3.0 days p.i.

Malaysia (patient cerebrospinal fluid; (Yoneda et al., 2013) IP 10-1 to 104 pfu LD50 was approximately 10 pfu at day 8 passaged on Vero cells)

Malaysia (patient cerebrospinal fluid; (Yoneda et al., 2010) IP (100 to 105 pfu) LD50 at day 12 p.i. was 3.8 x 101 pfu passaged on Vero cells)

(Porotto et al., 2010) Not reported IP (1 x 103 LD50) Max survival time for placebo was 7 days

LD50 at day 5 p.i. was approximately 106 (de Wit et al., 2011) Malaysia (CDC) IN (103 to 107 TCID50 TCID50. Inter-animal transmission was demonstrated

IN or IP (102 or 105 LD50 <1 TCID50 for IN and 6 TCID50 for IP. (Rockx et al., 2011) Malaysia; GenBank AF017149 TCID50) Higher dose results in more lung pathology.

(Mathieu et al., 2012a) Malaysia (UMMC1; GenBank AY029767) IP (104 pfu) Death beginning at day 5 p.i.

(Mathieu et al., 2012b) Malaysia (UMMC1; GenBank AY029767) IP (102 and 103 pfu) Both doses 100% lethal by day 6 p.i.

30 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 12. NiV Hamster Model Challenge Studies, Continued

Challenge Route Reference NiV Strain (Source) Notes (Dose)

All animals developed neurological (Munster et al., 2012) Malaysia (CDC) IN (105 TCID50) symptoms between day 7 and day 12 p.i.

First analysis of NiV-B in an animal model. (DeBuysscher et al., Malaysia (1999 human CNS sample); IP (1 to 105 TCID50) NiV-M IP LD50 = 68 TCID50; NiV-B IP LD50 = 2013) Bangladesh (2004 human throat swab) IN (105 TCID50) 528 TCID50; Slower disease progression for NiV-B by IP or IN.

IP (105 TCID50; >103 Vaccine study; 100% of moribund animals (Lo et al., 2014) Malaysia LD50) euthanized by day 6 p.i.)

IN (107 TCID50); Oral gavage (107 TCID50); IN 100% lethal by day 14; Oral gavage 20% (de Wit et al., 2014) Bangladesh/200401066 (CDC) drinking palm sap lethal; Drinking 108 TCID50 was 40% lethal. (107 and 108)

(DeBuysscher et al., IP (6.8 x 104 TCID50; Malaysia (CDC) Vaccine efficacy study 2014) 103 LD50)

Malaysia (1999 human CNS sample); Histological comparisons at days 2 & 4 p.i.; (Baseler et al., 2015) Oronasal (107 TCID50) Bangladesh (2004 human throat swab) lethality not reported.

(Borisevich et al., 2016) Malaysia (CDC) IP (105 TCID50) 100% lethality by day 7 p.i.

Histopathology study of early infection; Malaysia (1999 human CNS sample); lethality not reported. NiV-B disseminates (Baseler et al., 2016) Bangladesh (2004 human throat swab). IN (5 x 106 TCID50) within the nasal cavity and lung more slowly Both from CDC. than NiV-M.

Favipiravir efficacy study. Moribund animals (Dawes et al., 2018) Malaysia (CDC) IP (104 pfu) euthanized at 5-6 days p.i.

31 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 2. Ferret

The domestic ferret (Mustela clinical symptoms within six days showing similar respiratory and putorius furo) is a small carnivore of infection, including fever, severe neurologic signs and reaching the which is susceptible to a range depression, myoclonus (muscle humane endpoint by days 7 – 10 of respiratory viruses including spasms), and hind-limb paresis post-infection. However, as with influenza, paramyxoviruses such (partial paralysis); (Clayton et the hamster challenge model, a as NiV and HeV, pneumoviruses al., 2012). Respiratory symptoms variety of Malaysia and Bangladesh such as Respiratory Syncytial Virus include dyspnea (labored strain isolates have been used and and human metapneumovirus, breathing), severe respiratory limited information is published coronaviruses, and morbillivirues distress, cough, and serous nasal regarding the identity and passage such as Canine Distemper Virus discharge. Pathology includes history of the challenge stocks. (CDV). Compared to the smaller subcutaneous edema of the head, Most NiV challenge studies in the Syrian Golden Hamster, ferrets hemorrhagic lymphadenopathy ferret have been performed with can be more easily monitored (swollen lymph nodes), petechial the Malaysia strain. Agreement on, for physiological signs such (spotted) hemorrhages of the and production of, a common, well as temperature, respiration, lung and kidney, and generalized characterized challenge stock(s) and balance, and have more vasculitis (Bossart et al., 2009). is critical for standardization of easily observable behavioral Vasculitis and pulmonary alveolitis the ferret model. Performance of characteristics (Enkirch and von are common and meningitis may IN and oronasal administration Messling, 2015). The larger snouts be present, although encephalitis methods should be reported in of ferrets are also reported to make was not observed (de Wit and more detail since the amount intranasal (IN) challenge easier Munster, 2015). Lesions frequently of challenge virus which is to perform than on hamsters contain syncytial cells (Williamson delivered to the upper versus (R. Gomez-Roman, personal and Torres-Velez, 2010). Ferrets lower respiratory tract could communication). infected with the Malaysia and be a significant variable. Bangladesh NiV strains show Table 13 provides a compilation of similar clinical signs and disease most of the NiV ferret challenge course, although Malaysia-infected studies found in the published animals show a more pronounced literature, including challenge hemorrhagic state, while the strain and available identifying Bangladesh strain induces more information on the challenge virus, oral shedding (Clayton et al., 2012; route of administration, dose and Leon et al., 2018). notes on the disease course and pathology. Several publications Intra-laboratory performance of have compiled detailed descriptions the NiV ferret challenge model of the NiV disease course in ferrets, appears to be somewhat more including dose range, symptoms, consistent compared to the viral loads, tissue tropism and hamster model (see Tables 11 and pathology/histopathology in major 12). Most challenge studies used organs ((Bossart et al., 2009; a similar dose, 5 x 103 pfu or 5 x Clayton et al., 2012) ; (Pallister 103 TCID50, delivered oronasally et al., 2013). NiV-infected ferrets or IN, although the dose could develop serious respiratory and be better standardized by using neurological disease. NiV (500 – 5 the same NiV titer assay. Disease x 104 TCID50) is usually introduced progression and symptomology via the oronasal or intranasal (IN) appear comparable between routes. Infected animals develop most of the studies, with animals

32 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 13. NiV Ferret Model Challenge Studies

Challenge Route Reference NiV Strain (Source) Notes (Dose)

Passive antibody prophylaxis study. Symptoms included: Oronasal; dose fever, severe depression, lack of appetite, diarrhea, sneezing, ranging; (5 x 101 to nasal discharge, tremors, hind limb paralysis. Euthanasia of 5 x 104 TCID50). 5 x high dose animals 8 – 10 days p.i. Histopathology included (Bossart et al., 2009) Malaysia (EUKK 19817) 103 TCID50 was given acute focal necrotizing alveolitis and pulmonary vasculitis, oronasally for the acute glomerular necrosis, focal necrosis of the spleen and mAb efficacy study severe diffuse subacute inflammation of the organs of the head and neck.

Malaysia (Malaysia/ Clinical signs of lower respiratory tract (nasal discharge, human/99 from sneezing) and neurologic system infection (tremors, cerebrospinal Oronasal (5 x 103 (Clayton et al., 2012) paralysis, stupor). Malaysia-infected animals had cutaneous fluid); Bangladesh TCID50) petechial hemorrhage, bleeding from oral, nasal and rectal (Bangladesh/2004/Rajbari/ mucosa. Euthanasia on humane grounds at 7 to 10 days p.i. R1)

Vaccine efficacy Study. Control animal pathology: ventral cervical subcutaneous hemorrhage; pulmonary & renal (Mire et al., 2013) Malaysia (1999011924) IN (5x103 pfu) lesions; hemorrhagic interstitial pneumonia; multifocal renal hemorrhage; diffuse reticulation of the liver. Control animals succumbed within 7-8 days p.i.

Vaccine efficacy study. The humane endpoint for euthanasia was defined as “rapidly progressive clinical illness of up to Bangladesh Oronasal (5 x 103 2 days’ duration including fever and depression, possible (Pallister et al., 2013) (Bangladesh/2004/Rajbari/ TCID50) confirm accompanied by increased respiratory rate or posterior R1) paresis or ataxia”, usually occurring within 10 days post- challenge.

Clinical signs: fever, moderate respiratory disease and severe neurological signs; depression, discharge (ocular, oral, Malaysia (UMCC1; GenBank (Satterfield et al., 2015) IN (5 x 103 pfu) nasal) sneezing, oral frothing, ataxia, tremors. All animals AY029767) infected with wt. virus reached humane endpoint and were euthanized on days 7-8 p.i.

All NiV-M wt. infected animals reached the humane (Satterfield et al., Malaysia (UMCC1; GenBank endpoint and were euthanized on days 7 to 8 p.i. Clinical IN (5 x 103 pfu) 2016a) AY029767) signs included fever, severe respiratory disease and mild to moderate neurological signs.

Malaysia (Malaysia/ Clinical signs from day 5 p.i.: fever, agitation, disorientation, human/99 from ataxia, facial edema, hunched posture, tachypnea/ cerebrospinal (Clayton et al., 2016) Oronasal (5 x 103 pfu) dyspnea, straining to defecate. Virus shedding from the fluid); Bangladesh respiratory tract. Assisted ferret-to-ferret transmission was (Bangladesh/2004/Rajbari/ demonstrated via oronasal fluids from infected animals. R1)

IN dose ranging with 101 to 105 TCID50. The Clinical signs included fever, lack of grooming, hunched LD50 was 22 TCID50 posture, ataxia, severe depression, labored breathing, Malaysia (1998 patient); for NiV-M and 32 subcutaneous edema of the neck and head, vomiting and (Leon et al., 2018) Bangladesh (2004 patient); TCID50 for NiV-B. neurological signs (tremor, paralysis, seizure). High dose both from CDC. 5 x 103 TCID50 was animals infected with NiV-M succumbed by Day 9 p.i. and used for challenge NiV-B infected animals by Day 10 p.i. experiments.

33 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 3. African Green Monkey (AGM)

The most relevant non-human consistent in the AGM than in the experiments have used the intra- primate model for NiV disease is hamster and ferret models. Some tracheal (IT) route, and some the African Green Monkey (AGM; animals show muscle twitches studies have included IN as well, Chlorocebus sabaeus). Tables 14 and and partial paralysis (Geisbert recent experiments have tested 15 provide a compilation of most of et al., 2014), histopathological aerosolized delivery (Cong et al., the published NiV AGM challenge signs of encephalitis and tremors 2017; Lo et al., 2014). This form of studies, including challenge (Johnston et al., 2015), but overt administration may best mimic strain and available identifying brain abnormalities are usually not human-to-human transmission. information on the challenge seen. One study identified lesions The question of the utility, general virus, route of administration, by brain imaging (Hammoud et al., feasibility and necessity of this dose and notes on disease course, 2018) while another did not (Cong route of administration may need clinical signs and pathology. et al., 2017). The different findings to be settled to fully standardize Several publications have compiled from these studies may be due performance of the NiV AGM detailed descriptions of NiV disease to differences in dose or route of model. course in AGMs, including dose administration. range, symptoms, viral loads, tissue tropism and pathology/ Infection with NiV Malaysia and histopathology in major organs Bangladesh strains has been (Geisbert et al., 2010; Johnston directly compared in the AGM et al., 2015; Mire et al., 2016). Of model, and in that study morbidity particular interest, Johnston et and mortality was accelerated al., (2015) conducted a detailed in the NiV Bangladesh-infected characterization study of NiV animals by approximately three infection in AGMs with the aim of days compared with the NiV supporting future therapeutic and Malaysia-infected animals. This vaccine development, although this acceleration of disease progression study only used the NiV Malaysia appears distinct from what is strain. seen in hamsters and ferrets, but consistent with human NiV Intra-tracheal (IT) inoculation disease. Otherwise, symptomology of AGMs with NiV is lethal, with between the two groups of AGMs death occurring in 9-12 days. was comparable (Mire et al., 2016). Higher challenge doses generally accelerate the disease course and Several different isolates of NiV reduce survival time (Geisbert et Malaysia have been used in AGM al., 2012). Animals initially show challenge studies, while the same signs of depression, lethargy, fever, isolate of NiV Bangladesh has been and loss of appetite. NiV causes an used (Tables 14 and 15). As with acute respiratory disease in AGMs, the hamster and ferret challenge characterized by labored breathing models, production of a common, and bloody nasal discharge. The well-characterized stock of virus lungs become enlarged with for AGM challenge studies would edema and areas of congestion help to standardize performance and hemorrhage (Geisbert of the model. There appears to et al., 2010). Histopathology be an overall trend toward lower includes alveolar hemorrhage in challenge doses since the first the lungs, prominent syncytia AGM challenge experiments formation in lung endothelial (Tables 14 and 15). However, cells, and generalized vasculitis. the route of administration is Neurological signs are less not entirely settled. While most

34 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 14. NiV African Green Monkey Model Challenge Studies

Challenge Route Reference NiV Strain (Source) Notes (Dose)

Clinical signs: Depression, lethargy, fever, loss of appetite, dyspnea (difficulty breathing), pleural effusions (by x-ray). 7 of 8 animals succumbed or were euthanized between days Intratracheal (IT) or 9 and 12 p.i. (Geisbert et al., 2010) Malaysia (1999) IT and oral (2.5 x103 to 1.3 x 106 pfu) Gross pathology: Thrombocytopenia, blood-tinged pleural fluid, enlarged lungs with multifocal areas of congestion and hemorrhage, hemorrhage on mucosal surface of the urinary bladder, frothy fluid exuding from the nose and mouth.

Clinical Signs: Severe depression, reduced mobility, loss of body weight and reduced food consumption day 5 p.i. Animals were seriously moribund by day 14 p.i.

IP or IN (1 x 106 or 1 x Gross pathology: Advanced lesions in many abdominal (Yoneda et al., 2013) Not reported 108 TCID50) organs and lungs, pulmonary congestion with edema, hemorrhage, necrosis and lymphocyte depletion in the spleen. Virus antigen was found in vascular epithelial cells of lung capillaries and small blood vessels. No apparent changes in the brain were seen.

Passive immunization efficacy study.Clinical Signs: Control animals had fever, depression and decreased activity, loss of appetite, dyspnea, thrombocytopenia and changes in coagulation factors. 3 of 4 control animals had (Geisbert et al., 2014) Malaysia 1998-99 (CDC) IT (5 x 105 pfu) neurologic signs (muscle twitches and partial paralysis). Control animals succumbed between 8 and 10 days p.i. Histopathology: NiV antigen was found in the lung, spleen and brainstem of control animals

Vaccine efficacy study.Clinical signs: Control animals had shallow and increased respiration, hunched posture (Prescott et al., 2015) Malaysia IT (1 x 105 pfu) and decreased appetite. 2 of 3 control animals recovered by 15 days post-challenge; the other control animal was euthanized at day 9 p.i.

Clinical signs: fever, lymphadenopathy (enlarged lymph nodes), bloody exudate in the nose and mouth, tremors, increased heart rate and labored respiration, decreased responsiveness and weight loss. (Johnston et al., 2015) Malaysia 1998-99 (CDC) IT (2.5 x 104 pfu) Histopathology: red, mottled edematous lungs, necrotic lesions in most tissues examined centered on small blood vessels (vasculitis) often with endothelial syncytia, and evidence of encephalitis is some animals/

35 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 15. NiV African Green Monkey Model Challenge Studies, Continued

Challenge Route Reference NiV Strain (Source) Notes (Dose)

Morbidity and mortality was accelerated for animals infected with NiV-B compared to NiV-M by approximately 3 days. The incidence of respiratory and neurologic symptoms was Malaysia 199912916; (Mire et al., 2016) IT + IN (5 x 105 pfu) comparable between the NiV-B and NiV-M infected groups, Bangladesh 200401066 as was overall symptomology. Animals in both groups showed primarily respiratory symptoms, and one in each group showed some neurological signs (tremors)

Animals challenged by either method showed similar symptomology. Clinical signs: Lethargy, cough, difficulty breathing, decreased fluid and food consumption. There were no overt signs of neurological disease or hemorrhage, IT or small-particle or bloody froth from the mouth/nose. There was no weight (Cong et al., 2017) Malaysia aerosol inhalation (1 x loss and only half of the animals had elevated temperature. 104 pfu) Survival time for all animals was approximately 8 days. IT administration induced a significant congestion and loss in lung volume, while small-particle aerosol confined infection to the lower respiratory tract. Imaging did not reveal significant lesions in the brain.

Peptide prophylaxis study. Clinical signs: Depression, loss of appetite, dyspnea, labored breathing, epistaxis Malaysia (UMMC1, GenBank (hemorrhage from nose or pharynx), paresis of hindlimbs, (Mathieu et al., 2018) IT (2 x 107 pfu) AY029767) uncoordinated motor movements and brain hemorrhage. Control animals succumbed to infection by day 13 post- challenge.

Clinical signs: lethargy, reduced appetite, cough. No animals Medium-large showed overt neurological symptoms. Average survival time Malaysia 1998 (GenBank particle aerosol was 12.5 days p.i. There were no clear differences between (Hammoud et al., 2018) AF212302) inhalation (102 or the dose groups. Gross pathology showed congestion and 103pfu) hemorrhage in the lungs, but no overt brain abnormalities. Imaging showed loss of lung volume and brain lesions.

Remdesivir therapeutic efficacy study.Clinical signs: Control (Lo et al., 2019) Bangladesh/200401066 IN (1 x 105 TCID50) animals developed severe respiratory symptoms and were euthanized on day 7 or 8 p.i.

Vaccine challenge study. Clinical signs: control animal showed loss of appetite, labored breathing & succumbed on day 8 p.i.

(Mire et al., 2019) Bangladesh/200401066 IT + IN (5 x 105 pfu) Clinical and gross pathological findings: lymphopenia (low lymphocyte count); serosanguinous oral & nasal discharge; severely enlarged lungs with severe congestion and hemorrhage; hemorrhage of the mucosal surface of the urinary bladder.

36 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Table 16. Pros and Cons of the Major NiV Animal Challenge Models

NiV Animal Pros Cons Challenge Model

• The difference in virulence of the NiV-M and NiV-B • Small size & low cost facilitate large experimental strains appears to be reversed in comparison with numbers. humans. Hamster • Multiple routes of administration (IN and IP). • Limited physiological and behavioral monitoring compared to larger animals.

• Limited immunological reagents.

• A common animal model for a number of respiratory • No obvious differences in virulence or disease course viruses. between NiV-M and NiV-B strains, as observed in Ferret • Larger size facilitates better physiological and humans. behavioral monitoring, and pathological examination • Limited immunological reagents. compared to the hamster.

• As a NHP, closest to human physiology and immune response. • High cost and potential ethical concerns limit • Aerosol challenge route may best mimic human-to- African Green Monkey experimental numbers. human transmission. (AGM) • Neurological symptoms are inconsistent compared to • The difference in virulence of the NiV-M and NiV-B the hamster and ferret models strains seen in humans appears to be recapitulated in the AGM.

Session 5 of the Nipah Virus International Conference reviewed the latest developments in pathogenesis and animal models, including experiments with aerosols.4

Standardization of virus challenge stocks The primary opportunity for 1. Strain Selection: 2. Standardized Growth Protocol standardizing all three of the • Strains should come from and Virus Infectivity Assays - NiV lethal challenge models clinical isolates with known To ensure the consistency of described above is development of lethal outcome. working virus stocks. a common, fully characterized, and controlled virus challenge stock • The passage history should be 3. Release Criteria - Testing for (or one each for the Malaysia and well documented, with a low potency, identity and purity Bangladesh strains). Best practices passage number through a for identification and development well-characterized cell line. 4. Characterization Criteria of viral challenge stocks for use – Including ratio of genomic in vaccine development under the • A panel of stocks should be equivalents and/or viral particle FDA “Animal Rule” have been developed representing the full count to PFU, viral particle count. advanced by the Filovirus Animal range of viruses relevant to the Deep sequencing, infectivity and Nonclinical Group (FANG), a disease. lethality testing combined US and UK government interagency group, to facilitate • Full genomic characterization, Source: The Filovirus Animal the testing of Filovirus vaccines. particle infectivity ratios Nonclinical Group (FANG) These recommendations provide and QC testing for sterility, a roadmap for standardizing virus mycoplasma, endotoxin and stocks for NiV and other emerging adventitious viruses. viral diseases, and include the following:

37 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 VI. CONCLUSIONS

Development of an effective prophylactic vaccine against NiV appears to be scientifically feasible, and the basic R&D components and knowledge are in place to enable successful development programs.

A large number of vaccine 2019). Nevertheless, it is important BSL-4 containment would create candidates using a range of to generate data on the protective serious technical and logistical platform technologies have been threshold for several reasons. challenges for vaccine developers tested and showed very good The first reason is for evaluating needing to perform the assays efficacy in a variety of lethal vaccine candidates in early on large numbers of samples, animal challenge models. There is development. What minimal titers often under cGMP (current Good broad consensus in the NiV field are needed for protection and what Manufacturing Practices) for lot that neutralizing antibodies are dose(s) of a particular candidate are release. Are research institutions the mechanism of protection and needed to elicit them, and in which with BSL-4 capability willing to will form the basis for defining animal model(s)? Which candidates collaborate with vaccine developers a correlate of protection once elicit minimal protective titers after in this way, and are they capable appropriate studies are performed. a single dose, as is the preference of processing large numbers of A variety of assay platforms exist in the WHO Target Product Profile? samples under the quality systems to quantitate NiV serum binding Answers to these questions are necessary for clinical development antibodies and virus neutralizing critical for vaccine candidate and licensure? Could a centralized antibodies. While standardization evaluation and advancement. The BSL-4 facility be created/funded of assays is in an early stage, second reason is to anticipate what to support all the NiV vaccine there is broad acceptance of the neutralizing antibody responses developers in this effort? pathways this process will follow. will be needed in formal animal Alternatively, surrogate assays for Finally, three animal challenge studies for vaccine licensure under NiV serum neutralizing antibodies models (hamsters, ferrets and the “Animal Rule”, in which have been developed which can be African Green Monkeys) appear to antibody responses in animal performed by vaccine developers faithfully replicate many aspects challenge models will be bridged without high-level biocontainment, of NiV infection and pathology back to those seen in human most notably those using in humans. Nevertheless, gaps, Phase I/II clinical studies. Finally, pseudoviruses expressing the NiV challenges and further questions knowing the correlate of protection F and G glycoproteins. However, remain which must be resolved to will make it possible to determine these assays have not been used accelerate NiV vaccine development the durability of protection after extensively by major NiV research and ensure success. Below are three vaccination in humans. laboratories, and even the most key, forward-looking topics and recent animal challenge and questions to be addressed: Choice of assay(s) for serum vaccine studies continue to use neutralizing antibodies the live NiV-based assays almost Defining a correlate of protection As discussed earlier, there are exclusively. At a minimum, Many types of vaccines tested well-established live NiV-based extensive testing will be required in NiV lethal animal challenge assays for serum neutralizing to characterize the sensitivity models elicited serum neutralizing antibodies (SNT, PRNT) that are and specificity of these assays in antibodies. However, none of the widely accepted and have been comparison with the live NiV- studies determined a correlate of used in the majority of published based assays and evaluate their protection based on neutralizing NiV animal challenge studies. In potential for validation. Who would antibody titer because in most theory, it should be possible to perform these studies? And what studies all the animals were standardize and validate these else needs to be done (and by protected. Encouragingly, assays, and use them for vaccine whom) to facilitate acceptance of anecdotal evidence from NiV development, human clinical these assays by NiV researchers researchers suggests that even studies and animal challenge and regulatory bodies? This is an very low neutralizing antibody studies for licensure under the area in which major stakeholders in titers may be protective in animal “Animal Rule”. However, the use NiV vaccine development (such as models (Source: CEPI 2nd Assays of live NiV and the requirement CEPI) have a key role to play. and Standards Workshop; June for performing these assays under

38 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 Characterization of animal challenge models challenge models for NiV infection which recommends early and – hamsters, ferrets and the frequent engagement when use FDA draft guidance on the use African Green Monkey – have all of the “Animal Rule” is being of animal challenge models for been characterized in multiple contemplated. This is another licensure under the “Animal Rule” publications, in some cases in a area in which leadership by emphasizes the requirement for high degree of detail. However, major stakeholders, such as CEPI, a high degree of characterization is the existing characterization is needed to facilitate vaccine of the animal model, such as sufficient to support use under development and licensure by identity and characterization of the “Animal Rule”? If not, what ensuring these issues are resolved the challenge agent, route and additional studies are needed to and the animal models are ready quantification of exposure and close the gaps, and who would for use when vaccine candidates the course and pathophysiology perform them? Ultimately, these are ready for clinical testing. of infection compared to humans. questions should be addressed The three well established animal in consultation with FDA,

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45 Nipah Virus Assays and Animal Models for Vaccine Development, Landscape Analysis, January 2021 VIII. STATEMENT OF SUPPORT

1. ARTICLE H.20. PUBLICATION AND PUBLICITY

In addition to the requirements set forth in HHSAR Clause 352.227-70, Publications and Publicity incorporated by reference in SECTION I of this contract, the Contractor shall acknowledge the support of the National Institutes of Health whenever publicizing the work under this contract in any media by including an acknowledgment substantially as follows:

"This project has been funded in whole or in part with Federal funds from the Division of Microbiology and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201800010l".

Acknowledgements Dan Stoughton, NIAID/DMID

Janet Lathey, NIAID/DMID

Larry Wolfraim, NIAID/DMID

Colleen Sico, NIAID/DMID

Eun-Chung Park, NIAID/DMID

Sara Woodson, NIAID/DMID

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