Safety, tolerability, pharmacokinetics, and immunogenicity of a human monoclonal antibody targeting the G glycoprotein of henipaviruses in healthy adults: a first-in-human, randomised, controlled, phase 1 study

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Safety, tolerability, pharmacokinetics, and immunogenicity of a human monoclonal antibody targeting the G glycoprotein of henipaviruses in healthy adults: a first-in-human, randomised, controlled, phase 1 study Elliott Geoffrey Playford, Trent Munro, Stephen M Mahler, Suzanne Elliott, Michael Gerometta, Kym L Hoger, Martina L Jones, Paul Griffin, Kathleen D Lynch, Heidi Carroll, Debra El Saadi, Margaret E Gilmour, Benjamin Hughes, Karen Hughes, Edwin Huang, Christopher de Bakker, Reuben Klein, Mark G Scher, Ina L Smith, Lin-Fa Wang, Stephen B Lambert, Dimiter S Dimitrov, Peter P Gray, Christopher C Broder

Summary

Background The monoclonal antibody m102.4 is a potent, fully human antibody that neutralises Hendra and Nipah viruses in vitro and in vivo. We aimed to investigate the safety, tolerability, pharmacokinetics, and immunogenicity of m102.4 in healthy adults. Methods In this double-blind, placebo-controlled, single-centre, dose-escalation, phase 1 trial of m102.4, we randomly assigned healthy adults aged 18–50 years with a body-mass index of 18∙0–35∙0 kg/m² to one of five cohorts. A sentinel pair for each cohort was randomly assigned to either m102.4 or placebo. The remaining participants in each cohort were randomly assigned (5:1) to receive m102.4 or placebo. Cohorts 1–4 received a single intravenous infusion of m102.4 at doses of 1 mg/kg (cohort 1), 3 mg/kg (cohort 2), 10 mg/kg (cohort 3), and 20 mg/kg (cohort 4), and were monitored for 113 days. Cohort 5 received two infusions of 20 mg/kg 72 h apart and were monitored for 123 days. The primary outcomes were safety and tolerability. Secondary outcomes were pharmacokinetics and immunogenicity. Analyses were completed according to protocol. The study was registered on the Australian New Zealand Clinical Trials Registry, ACTRN12615000395538. Findings Between March 27, 2015, and June 16, 2016, 40 (52%) of 77 healthy screened adults were enrolled in the study. Eight participants were assigned to each cohort (six received m102.4 and two received placebo). 86 treatmentemergent adverse events were reported, with similar rates between placebo and treatment groups. The most common treatment-related event was headache (12 [40%] of 30 participants in the combined m102.4 group, and three [30%] of ten participants in the pooled placebo group). No deaths or severe adverse events leading to study discontinuation occurred. Pharmacokinetics based on those receiving m102.4 (n=30) were linear, with a median half-life of 663∙3 h (range 474∙3–735∙1) for cohort 1, 466∙3 h (382∙8–522∙3) for cohort 2, 397·0 h (333∙9–491∙8) for cohort 3, and 466∙7 h (351∙0–889∙6) for cohort 4. The elimination kinetics of those receiving repeated dosing (cohort 5) were similar to those of single-dose recipients (median elimination half-time 472·0 [385∙6–592∙0]). Anti-m102.4 antibodies were not detected at any time-point during the study. Interpretation Single and repeated dosing of m102.4 were well tolerated and safe, displayed linear pharmacokinetics, and showed no evidence of an immunogenic response. This study will inform future dosing regimens for m102.4 to achieve prolonged exposure for systemic efficacy to prevent and treat henipavirus infections. Funding Queensland Department of Health, the National Health and Medical Research Council, and the National Hendra Virus Research Program. Copyright © 2020 Elsevier Ltd. All rights reserved.

Introduction Nipah virus and Hendra virus are enveloped, singlestranded, negative-sense RNA viruses and the prototype members of the genus Henipavirus in the family Paramyxoviridae.1 Bats of the genus Pteropus, from which all bat isolates of Hendra virus and Nipah virus have been derived, appear to be the major natural reservoir hosts for henipaviruses.2,3 Hendra virus and Nipah virus are two emerging zoonotic, transboundary viruses with host tropism that is uniquely broad, capable of infecting at least

18 animal species across six orders of mammals, including the viruses’ natural bat hosts.4,5 Hendra virus and Nipah virus are highly pathogenic and cause widespread infection with severe disease in multiple organ systems in at least 11 mammalian species. Fatality rates in these mammalian species range from 40% to 100%.6 In humans, Hendra virus and Nipah virus infections result in a systemic and often fatal respiratory or neurological disease, or both.7,8 Both Hendra and Nipah viruses can also cause a relapsing of encephalitis after recovery from

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Lancet Infect Dis 2020 Published Online January 29, 2020 https://doi.org/10.1016/ S1473-3099(19)30634-6 See Online/Comment https://doi.org/10.1016/ S1473-3099(19)30687-5 Infection Management Services, Princess Alexandra Hospital, Brisbane, QLD, Australia (E G Playford PhD); School of Medicine (E G Playford), Australian Institute for Bioengineering and Nanotechnology (T Munro PhD, S M Mahler PhD, M Gerometta PhD, K L Hoger BSc, M L Jones PhD, B Hughes BEng, K Hughes BSc, E Huang PhD, C de Bakker PGBSc, P P Gray PhD), ARC Training Centre for Biopharmaceutical Innovation (S M Mahler, M L Jones, P P Gray), Child Health Research Centre, Faculty of Medicine (K D Lynch, S B Lambert), University of Queensland Brisbane, QLD, Australia; Q-Pharm, Clive Berghofer Cancer Research Centre, Herston, QLD, Australia (S Elliott PhD, P Griffin MBBS, M E Gilmour RN); Clinical Tropical Medicine Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD, Australia (P Griffin); Communicable Disease Branch, Prevention Division, Queensland Health, Brisbane, QLD, Australia (K D Lynch MPHTM, H Carroll MBBS, D El Saadi MAPH, S B Lambert PhD); Health and Biosecurity Business Unit, CSIRO Australian Animal Health Laboratory, Geelong, VIC, Australia (R Klein BSc, I L Smith PhD, L-F Wang PhD); Henry M Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA (M G Scher PhD);

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Programme in Emerging Infectious Diseases, Duke-National University Medical School, Singapore (L-F Wang); Center for Antibody Therapeutics, University of Pittsburgh Medical School, Pittsburgh, PA, USA (D S Dimitrov PhD); and Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD, USA (C C Broder PhD) Correspondence to: Dr Elliott Geoffrey Playford PhD, Infection Management Services, Princess Alexandra Hospital, Woolloongabba QLD 4102, Australia [email protected]. gov.au

Research in context Evidence before this study We searched PubMed from database inception to May 31, 2019, for studies using the terms (“Hendra” OR “Nipah”) AND (“monoclonal antibody” OR “monoclonal antibodies”) without language or article-type restrictions. We found no articles describing clinical trials of monoclonal antibodies designed for human prophylaxis. The human monoclonal antibody m102.4 binds to the same or similar region of the G glycoprotein of Hendra and Nipah viruses as the ephrin receptors do. The m102.4 monoclonal antibody showed protection from infection and was effective when administered after experimental challenge with Hendra or Nipah viruses, or both, in ferret and non-human primate models. The m102.4 has been available since 2010 for compassionate therapy following high-risk Hendra virus exposures and has been used on 14 occasions (13 in Queensland, Australia, and one in the USA). The safety and tolerability of m102.4 has not been systematically assessed.

an acute infection, which appears to result from a recru­ descence of virus replication in the CNS.9 Hendra virus was first recognised in 1994, during an outbreak of fatal cases of a severe respiratory disease in horses and humans, which occurred in the Brisbane suburb of Hendra (QLD, Australia).10 14 horses and their trainer succumbed to infection and seven additional horses and a stablehand had non-fatal infections. To date, Hendra virus has appeared in eastern Australia on 61 occasions, causing fatalities in four of seven human cases, death or euthanasia of more than 100 horses, and euthanasia of two dogs which were Hendra virus antibody-positive.4,11 A large outbreak of encephalitis in 1998 among pig farmers in Peninsular Malaysia (Malaysia) led to the identification of Nipah virus, a Hendra-like virus isolated from the cerebrospinal fluid of two patients.12 Outbreaks of human cases of Nipah virus infection have been recorded almost annually since 2001 in Bangladesh. Between 1998 and 2018, 637 cases of human Nipah virus infection with 373 fatalities were reported in south and southeast Asia. The mode of Nipah virus transmission was direct human contact with infected pigs, con­sumption of raw date palm sap that might have been contaminated by fruit bats, or human-to-human trans­mission.13 Corpse-to-human trans­ mission of Nipah virus has also been documented in a case-series report of sporadic cases in Bangladesh in 2010.14 At least two strains of Nipah virus have been identified in genetic analysis: one associated with the 1998 Malaysian outbreak, and another that has caused repeated out­breaks in Bangladesh and northeastern India.15 Human isolates of Nipah virus show considerable genetic heterogeneity, particularly those from the Bangladesh outbreaks. This feature of Nipah virus, in combination with transmission modes that include human-to-human, raises concerns of a pandemic potential.16 2

Added value of this study This is the first study to assess the safety, tolerability, pharmacokinetics, and immunogenicity of m102.4 in healthy adults using various single dosing regimens and a repeat dosing regimen. The four single and one repeat dosing regimens of the m102.4 were generally safe and well tolerated, and had a half-life of 16∙5–27∙6 days, with no observed immunogenicity associated with the m102.4. Implications of all the available evidence Our findings support the use of the m102.4 in humans following high-risk henipavirus exposure. Two doses of 20 mg/kg separated by 72 h were as well tolerated as a single dose of 20 mg/kg was. Further research is needed to better quantify the risk of infection following henipavirus exposure and understand the outcomes of exposed humans receiving m102.4.

No approved vaccines or therapeutics exist to prevent or treat disease in humans. Nipah and Hendra viral infections are listed among the WHO priority diseases for which a research and development blueprint of action to prevent epidemics has been developed.17 Both Nipah and Hendra viruses are also emerging viruses selected for the human vaccine development initiative funded by the Coalition for Epidemic Preparedness Innovations.18 Hendra and Nipah virus particles possess surface pro­ jections composed of the viral transmembrane-anchored fusion (F) and attachment (G) glycoproteins, which are the mediators of virus attachment and host cell infection. The glycoproteins are also the major antigenic targets of virusneutralising antibodies. The G glycoproteins of both Hendra and Nipah virus bind to the host cell membrane proteins ephrin-B2 and ephrin-B3, which are highly conserved across known susceptible hosts.4 Human antibody fragments reactive to the G glycoproteins of both Hendra and Nipah virus and capable of neutralising the viruses have previously been isolated using recombinant antibody technology.19 One monoclonal antibody, m102, possessed strong cross-reactive and neutralising activity against Hendra virus and Nipah virus, and was sub­ sequently affinity matured to m102.4, formatted as an IgG1 monoclonal antibody, and produced in a CHO-K1 cell line.20 The m102.4 epitope localises to the ephrin receptor binding site on the G glycoprotein and binds in a similar way to the ephrin molecule.21 In a post-exposure Nipah virus challenge experiment in a ferret model, a single dose of m102.4 administered by intravenous infusion 10 h after a lethal virus challenge was shown to provide complete protection.22 m102.4 was also shown to be effective against Nipah and Hendra virus infections in non-human primates in studies designed to reflect a potential realworld scenario of infection exposure,15,23,24 high­lighting clear potential for future approved human use.

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In 2010, the m102.4 cell line was provided to the Queensland Government (Queensland Health, Brisbane, QLD, Australia) to produce m102.4 for compassionate emer­­ gency use in the event of high-risk exposure to Hendra virus. Following an unpre­cedented number of equine Hendra virus notifications (22 horses on 18 properties) in Queensland and New South Wales (Australia) in 2011,11 planning com­menced, in 2012, for this first phase 1 clinical trial of m102.4 in human participants.25 We aimed to assess the safety, tolerability, immunogenicity, and pharma­ cokinetics of single and multiple intravenous administrations of m102.4 in healthy participants.

Methods

Study design and participants We did a single-centre, randomised, double blind, placebo-controlled, dose-escalation, phase 1 study to assess the safety, tolerability, pharmacokinetics, and immunogenicity of m102.4 in healthy adults at Q-Pharm (Brisbane, QLD, Australia). Healthy adults aged 18–50 years with a body-mass index of 18∙0–35∙0 kg/m², a weight of 50–100 kg, and normal blood haematology values, coagulation profiles, clinical chemistry, and urinalysis results were eligible for inclusion. Exclusion criteria were: close, unprotected exposure to horses with an unexplained illness within the previous 4 weeks; known exposure to potentially Hendra virusinfected horses; positive tests for HIV or hepatitis B or C viruses; treatment with an investigational drug or biologic agent within the previous 60 days; use of prescription drugs within the previous 14 days or five half-lives before monoclonal antibody administration. Sexually active men with a partner who was capable of becoming pregnant and women who were capable of becoming pregnant in the absence of an effective method of birth control or were pregnant or breastfeeding were also excluded. Additional details of inclusion and exclusion criteria are outlined in the trial protocol (appendix p 43). Participants were recruited via external advertising or web-based media, and were mainly from the Brisbane area. Participants provided written informed consent before any study-specific tests or procedures were done. Ethics approval was obtained from the study site’s Human Research Ethics Committee at the QIMR Berghofer Medical Research Institute before participant enrolment com­menced. The study was done under the Therapeutic Goods Administration Clinical Trial Notification Scheme and in accordance with the Declaration of Helsinki, Good Clinical Practice guidelines, and the National Health and Medical Research statement on ethical conduct in human research.

Randomisation and masking Participants were given a sequential screening number at their screening visit. Eligible participants were assigned to one of five cohorts, each with eight

participants. In each cohort, two individuals in a sentinel pair were randomly assigned in a double-blinded manner to receive either active treat­ment or placebo (0∙9% saline solution) in a 1:1 ratio. The remaining participants in each cohort received either m102.4 or placebo in a 5:1 ratio, following a review of safety data by the investigator and the medical monitor. Randomisation misation was done with a computer-generated rando­ schedule and in accordance with the randomisation plan prepared and maintained by Clinical Network Services (Brisbane, QLD, Australia), the contract research organisation. Because this was a single site trial with eight participants allocated to five cohorts, no block randomisation was done. To maintain the masking of the study, the rando­ misation plan was available only to the unmasked pharmacist. Treatment allocation was double blinded, and m102.4 and placebo were identical in appearance and volume for each participant in each cohort.

Procedures The m102.4 drug substance was manufactured at the National Biologics Facility (NBF) located at the University of Queensland (Brisbane, QLD, Australia) with a recombinant Chinese hamster ovary cell line. A parent cell line was obtained from the US National Institutes of Health (Bethesda, MD, USA) and then subcloned and adapted to a serum-free and protein-free medium that was commercially available (CD FortiCHO, ThermoFisher Scientific, Waltham, MA, USA). Cells were expanded from a qualified master cell bank using a conventional seed train, followed by a fed-batch bioreactor process. The clarified harvest containing secreted m102.4 was subjected to a two-step purification process including MabSelect SuRe LX protein A and mixed-mode Capto adhere chro­ matography (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The process included two viral inactivation or removal steps, a low pH hold, and nanofiltration (Viresolve Pro Modus, MilliporeSigma, Burlington, MA USA). m102.4 was formulated at a concentration of 10 mg/mL in phosphate buffered saline pH 7∙3 and aseptically dispensed into 10 mL vials at Sypharma (Melbourne, VIC, Australia). The final product complied with specifications commonly accepted by regulatory agencies for identity, purity, potency, and safety. Before clinical studies, Good Laboratory Practicecompliant, repeat-dose, toxicology studies were done in Sprague-Dawley rats at vivoPharm (Melbourne, VIC, Australia). m102.4 was administered to the rats once per week for 4 weeks at doses of 0 mg/kg, 10 mg/kg, 30 mg/kg, and 100 mg/kg. The antibody was well tolerated and the no-observed-adverse-effect level (NOAEL) was found to be 100 mg/kg, the highest dose tested. On the basis of animal models of henipavirus challenge,1,22–24,26 a target dose of 20 mg/kg was selected as the maximum dose to be tested. Treatment recipients in the five cohorts were dosed with four concentrations

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See Online for appendix

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77 patients assessed for eligibility

37 excluded 9 ineligible 23 withdrawals 10 time constraints 4 personal or family illness 9 no reason given 5 eligible but not dosed

40 enrolled to five cohorts

40 randomly assigned to receive m102.4 or placebo

30 received m102.4 6 in cohort 1 (single dose of 1 mg/kg on day 1) 6 in cohort 2 (single dose of 3 mg/kg on day 1) 6 in cohort 3 (single dose of 10 mg/kg on day 1) 6 in cohort 4 (single dose of 20 mg/kg on day 1) 6 in cohort 5 (two doses of 20 mg/kg, on days 1 and 4)

10 received placebo 2 in cohort 1 2 in cohort 2 2 in cohort 3 2 in cohort 4 2 in cohort 5

40 included in safety and tolerability analyses

Figure 1: Trial profile

of m102.4. Cohort 1 received single intravenous doses of 1 mg/kg or placebo (0∙9% saline solution) and cohort 2 received 3 mg/kg or placebo, infused in a total volume of 100 mL of 0∙9% saline solution. Cohort 3 received single intravenous infusions of 10 mg/kg or placebo and cohort 4 received 20 mg/kg or placebo, in a total volume of 500 mL of 0∙9% saline. Cohort 5 received two intra­ venous infusions of 20 mg/kg or placebo, in 500 mL of 0∙9% saline, 72 h apart. All infusions were given over a 1-h period using a peristaltic infusion pump. Sentinel pairs were dosed 7 days before the other participants in their cohort to detect any serious adverse advents in accordance with the European Medical Agency first-in-man guidelines.27 Participants in cohorts 1–4 were domiciled for a total period of 3∙5 days for initial safety and tolerability monitoring and to facilitate blood sampling for pharma­ cokinetics, virus neutralisation, and immuno­ genicity analysis. Participants received the m102.4 monoclonal antibody or placebo on day 1, and follow-up assessments were scheduled on days 4, 6, 8, 15, 29, 43, 57, 85, and 113. Participants in cohort 5 were domiciled for a total period of 6∙5 days, receiving a single dose of m102.4 or placebo on days 1 and 4. Follow-up visits to the clinical site were scheduled on days 7, 9, 11, 18, 25, 32, 39, 53, 67, 95, and 123. For all cohorts, the final safety assessment was scheduled to occur within a maximum period of 16 weeks after the last infusion. 4

The safety and tolerability of m102.4 across all cohorts was assessed according to the following specific assess­ ments: physical examination, clinical laboratory tests, vital signs, electrocardiogram (ECG), signs and symp­ toms of tolerability, infusion site reactions, and adverse events. Details of assessments made at each follow-up visit are provided in the appendix (pp 22–39). Dose escalation from one cohort to the next followed review and approval of safety and tolerability data for at least six participants per group. Additionally, blinded pharmacokinetic data for cohort 1 were reviewed to confirm the estimation of m102.4 elimination half-life. Initiation of dosing in cohort 5 followed review of available unblinded pharma­cokinetic data for cohorts 1–4 (appendix pp 4, 20). The concentration of m102.4 in serum samples for the estimation of pharmacokinetic profiles was measured with an ELISA developed and validated at vivoPharm. Briefly, serum m102.4 that was bound to Hendra virus soluble G protein coated on a plate was detected with a radish peroxidase custom rabbit anti-m102.4 horse­ conjugate (Thermo Fisher Scientific, Waltham, MA, USA) followed by the addition of 3,3’,5,5’-tetramethylbenzidine substrate (Sigma Aldrich, St. Louis, MO, USA). The validated assay met criteria in the relevant US guidelines28 and had a working range of 1∙7–163∙2 ng/mL, which corresponded to the lower and upper limits of quantitation. All collected samples for all cohorts were diluted within this working range and quantitated via standard curve interpolation, and pharmacokinetic parameters were calculated using non-compartmental techniques. We also took serum samples before infusion and at nominal post-dose timepoints for the assessment of the presence of anti-m102.4 antibodies and for the measurement of m102.4 Hendra virus and Nipah virus neutralisation. To assess the presence of anti-m102.4 antibodies in serum samples (collected on days 1, 29 [cohorts 1–4] or 39 [cohort 5], and 113 [cohorts 1–4] or 123 [cohort 5]), an antidrug antibody ELISA was developed and validated. A bridging assay design was used in which m102.4, previously coupled either with biotin or horseradish peroxidase, was mixed with serum or positive control (a custom anti-m102.4 rabbit polyclonal antibody [IVMS Veterinary Services, Giles Plains, SA, Australia]) to form a complex that was captured onto a streptavidin coated plate. The assay procedure included an acidification step to dissociate drug–antibody com­ plexes. The validated assay met criteria consistent with the US guidelines28 and had a sensitivity of 100 ng/mL. Human serum samples were considered positive if both the screening cut-point (decided statistically on the basis of a 95% upper confidence limit from negative normal donor sera) and specificity confirmation cut-point (where excess m102.4 was added) were exceeded. Virus neutralisation was measured with an assay designed and done by the Australian Animal Health

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Cohort 1 (1 mg/kg; n=6)

Cohort 2 (3 mg/kg; n=6)

Cohort 3 (10 mg/kg; n=6)

Cohort 4 Cohort 5 (20 mg/kg; n=6) (2 ×20 mg/kg; n=6)

Pooled placebo (n=10)

Overall (n=40)

Sex Female

5 (83%)

2 (33%)

1 (17%)

3 (50%)

2 (33%)

3 (30%)

16 (40%)

Male

1 (17%)

4 (67%)

5 (83%)

3 (50%)

4 (67%)

7 (70%)

24 (60%)

Age (years)

29∙8 (9∙1; 18–42)

30∙5 (5∙1; 23–36)

24∙7 (3∙9; 21–32)

27∙7 (5∙5; 21–35)

26∙6 (5∙0; 20–37)

28∙0 (6∙7; 18–44)

Weight (kg)

71∙5 84∙9 74∙7 67∙7 (15∙2; 51∙3–95∙7) (14∙1; 65∙2–98∙0) (10∙8; 60∙4–86∙4) (7∙3; 58∙2–77∙0)

80∙5 (12∙1; 67∙1–98∙5)

79∙3 (8∙0; 71∙8–95∙6)

76∙7 (11∙9; 51∙3–98∙5)

Body-mass index (kg/m²)

24∙6 (5∙3; 19∙5–33∙9)

26∙0 (4∙4; 20∙3–32∙9)

26∙3 (3∙4; 22∙6–32∙7)

25∙2 (3∙9; 19∙5–33∙9)

26∙6 (5∙3; 21∙8–31∙3)

24∙0 (4∙0; 19∙9–29∙5)

29∙7 (10∙9; 20–44)

23∙1 (2∙5; 19∙7–26∙2)

Race White

5 (83%)

5 (83%)

5 (83%)

6 (100%)

6 (100%)

9 (90∙0%)

Other*

1 (17%)

1 (17%)

1 (17%)

0 (0%)

0 (0%)

0 (0%)

36 (90∙0%) 3 (7∙5%)

Pacific islander

0 (0%)

0 (0%)

0 (0%)

0 (0%)

0 (0%)

1 (10%)

1 (2∙5%)

Data are n (%) or mean (SD; range). *Includes Asian, Aboriginal, American indigenous.

Table 1: Baseline characteristics

Laboratory located at the Commonwealth Scientific and Industrial Research Organisation (Geelong, VIC, Australia). Briefly, sera from five participants in cohort 4 were tested against Hendra virus and Nipah virus to assess the ability of circulating m102.4 to neutralise each virus in samples at four different timepoints after infusion (12 h, 24 h, day 4, and day 8). Serum samples were diluted to achieve an endpoint for neutralisation. Control plates were set up using m102.4-spiked media plus 10% normal human serum to achieve final con­ centrations of 50–0∙4 µg/mL. Samples of Hendra virus (isolate Hendra virus/Australia/Horse/2008/Redlands) and Nipah virus (isolate Nipah virus/Bangladesh/ human/2004/Rajbari, R1) were obtained from the CSIRO Australian Animal Health Laboratory (Geelong, VIC, Australia) and diluted to contain 100 times the median tissue culture infectious dose of the viruses and added to the required wells. Serum, cell, and virus controls were included. A sus­pension of Vero cells was added to every well at a concentration of 4 × 10⁵ cells per mL. Plates were incubated at 37°C in a humid atmosphere containing 5% CO2 for 45–60 min. Cells were examined after 3 days under an inverted microscope for cytopathic effect.

Outcomes The primary outcome was the safety and tolerability of single and multiple intravenous doses of m102.4 in healthy human participants. All adverse events and treatment-emergent adverse events were reported for participants from dosing until the end of the study. Definitions of adverse events and treatment-emergent adverse events are given in the trial protocol (appendix pp 51–53). Secondary outcomes were the pharmacokinetic profiles and immunogenicity of single and multiple doses. Calculated pharmacokinetic values were maximum observed drug concentration (Cmax), time to maximum observed drug concentration (Tmax), area under the drug

concentration-time curve from before infusion to last measurable concentration (AUC0–last), elimination halflife, clearance, and volume of distribution. An additional objective was to explore the pharma­ codynamics of m102∙4 against the Hendra and Nipah viruses (ie, virus neutralisation and presence of anti-m102.4 antibodies).

Statistical analysis Statistical analysis comprised summary descriptive statistics of the five dose groups and placebo group. Given the exploratory nature of the study, the sample size was not based on power calculations, but was consistent with the typical sample size used for similar studies. Descriptive statistics were done with SAS version 9.4. Pharmacokinetic data were analysed with Phoenix WinNonlinSoftware version 6.3, with pharmacokinetic listings, tables, and figures generated using SAS version 9.4. This study is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12615000395538.

Role of the funding source The funders of the study played no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Results Between April 15, 2015, and Feb 18, 2016, of 77 individuals assessed for eligibility, 40 were allocated into five cohorts (figure 1). All enrolled participants received their full assigned treatments and completed the study according to schedule apart from two participants in cohort 4, one of whom did not attend their day-43 visit and the other did not attend their day-85 visit. Baseline

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Cohort 1 Cohort 2 Cohort 3 Cohort 4 (1 mg/kg; n=6) (3 mg/kg; n=6) (10 mg/kg; n=6) (20 mg/kg; n=6)

Cohort 5 (2×20 mg/kg; n=6)

Pooled placebo (n=10)

Overall (n=40)

≥1 TEAEs Participants TEAEs

5 (83%)

5 (83%)

6 (100%)

6 (100%)

6 (100%)

9 (90%)

37 (93%)

12

10

14

13

11

26

86

Participants with serious TEAEs

0

0

0

0

0

0

0

Participants with any TEAE leading to discontinuation of study drug

0

0

0

0

0

0

0

Participants

1 (17%)

0

2 (3%)

1 (17%)

1 (17%)

1 (10%)

6 (15%)

TEAEs

1

0

3

3

1

1

9

Gastrointestinal disorders

General disorders and administration site conditions Participants

1 (17%)

2 (3%)

3 (50%)

0

1 (17%)

4 (40%)

11 (28%)

TEAEs

1

3

3

0

1

7

15

Participants

4 (67%)

2 (33%)

2 (33%)

3 (50%)

2 (33%)

5 (50%)

18 (45%)

TEAEs

5

2

2

4

2

6

21

Participants

0

0

0

0

2 (33%)

1 (10%)

3 (8%)

TEAEs

0

0

0

0

2

1

3

Infections and infestations

Investigations

Metabolism and nutrition disorders Participants

0

0

1 (17%)

0

0

0

1 (3%)

TEAEs

0

0

1

0

0

0

1

Musculoskeletal and connective tissue disorders Participants

0

0

0

0

0

2 (20%)

2 (5%)

TEAEs

0

0

0

0

0

3

3

Nervous system disorders (headaches) Participants

1 (17%)

1 (17%)

2 (33%)

4 (67%)

4 (67%)

3 (30%)

15 (38%)

TEAEs

1

1

2

6

4

4

18

Participants

0

1 (17%)

0

0

0

1 (10%)

2 (5%)

TEAEs

0

1

0

0

0

1

2

Participants

0

0

0

0

0

1 (10%)

1 (3%)

TEAEs

0

0

0

0

0

1

1

Psychiatric disorders

Renal and urinary disorders

Respiratory, thoracic and mediastinal disorders Participants

2 (33%)

1 (17%)

0

0

0

2 (20%)

5 (13%)

TEAEs

2

1

0

0

0

2

5

Skin and subcutaneous tissue disorders Participants

2 (33%)

1 (17%)

2 (33%)

0

1 (17%)

0

6 (15%)

TEAEs

2

2

2

0

1

0

7

Surgical and medical procedures Participants

0

0

1 (17%)

0

0

0

1 (3%)

TEAEs

0

0

1

0

0

0

1

Data are number of participants (%) or number of TEAEs. TEAE=treatment-emergent adverse events.

Table 2: Summary of adverse events

characteristics did not vary between treatment groups (table 1). Overall, the dosage of m102.4 was safe and well tolerated. No deaths, life threatening events, serious adverse events, or adverse events leading to study 6

discontinuation occurred. Across all cohorts, 37 (93%) of 40 participants had at least one treatment-emergent adverse event, with a total of 86 treatment-emergent adverse events reported (table 2). The occurrence of treatment-emergent adverse events was similar across all

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Cohort 1 (1 mg/kg) Cohort 2 (3 mg/kg) Cohort 3 (10 mg/kg) Cohort 4 (20 mg/kg) Cohort 5 (2 × 20 mg/kg)

10 000

Serum concentration (µg/mL)

1000

100

10

1

0·1

0·01

0

150

300

450

600

750

900

1050

1200

1350

1500

1650 1800 1950

2100

2250 2400 2550

2700 2850 3000

Nominal time after dose (h)

Figure 2: Mean m102.4 concentrations in serum from healthy adults (six treated participants per cohort), by nominal time and treatment group Data are mean (SD). M102.4 concentrations were determined by ELISA.

active-treatment and placebo groups. The most frequently reported events were infections and infestations, classed as mild to moderate; 13 of such events events were reported in 11 (46%) of 24 single-dose participants, two events in two (33%) of six repeat-dose participants in cohort 5, and six events in five (50%) of ten in the pooled placebo group. Eight (67%) of 12 participants dosed in cohorts 4 and 5 compared with four (22%) of 18 participants dosed in cohorts 1–3 and three (30%) of ten participants in the pooled placebo group reported headaches. All treatment-emergent adverse events were resolved upon follow-up without specific intervention. Of all treatment-emergent adverse events, 29 events in 20 participants were considered treatment-related, occurring in three (50%) of six participants in cohorts 1 and 5, two (33%) of six participants in cohorts 2 and 3, five (83%) of six participants in cohort 4, and five (50%) of ten participants in the pooled placebo group. The most common treatment-related, treatment-emergent adverse events were headaches (ten participants: one each in cohorts 1–3, four in cohort 4, two in cohort 5, and one in the pooled placebo group), intravenous catheter site bruising (three participants: two in cohort 3 and one in the pooled placebo group), and elevated alanine aminotransferase (ALT) concentrations (three partici­ pants: two in cohort 5 and one in the pooled placebo group). Of the 29 treatment-related, treatment-emergent adverse events, 24 were rated as mild and five as moderate. Moderate treatment-emergent adverse events included two headaches (one each in cohorts 4 and 5), one intravenous catheter site bruising in cohort 3, lethargy in a participant receiving placebo, and reduced appetite in

cohort 3. There were no severe treatment-emergent adverse events (table 2). No clinically significant changes in haematological or coagulation parameters were observed in any participant. Three participants had elevated ALT concentrations, two in cohort 5 and one in the pooled placebo group. The participants in cohort 5 had elevated ALT concen­ trations at day 4 (one participant) and 11 (the other participant); both findings following m102.4 admin­ istration were considered to be mild and treatmentrelated. ALT concentrations returned to normal after day 11 without intervention and were not considered to be clinically significant (appendix p 1). No other clinically significant abnormalities in biochemical parameters, urinalysis, vital signs, or ECG findings were observed. One clinically significant abnormal physical examination finding was reported in one participant in cohort 3; pityriasis rosea was noted on the arms, legs, and abdomen on day 113 but was not assessed as treatment-related. No pregnancies were reported during the study period. Following a single dose of m102.4, the median Tmax was 8·0 h for cohort 1, 4·0 h for cohort 2, 2∙5 h for cohort 3, and 4∙1 h for cohort 4. Cmax and AUC0–last increased linearly with dose, with mean Cmax being 692 µg/mL and mean AUC0–last being 131  648 h  ×  µg/mL for cohort 4 (figure 2, table 3). Concentration profiles over time showed an initial rapid peak followed by a biexponential decline consistent with multicompartmental distribution kinetics. The median elimination half-life was similar for cohorts 2–4 (between 397·0 h and 467·0 h) but higher for cohort 1 (663·3 h). Quite low inter-individual variability of pharmacokinetic parameters was observed. In cohort 5,

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Cohort 1 (1 mg/kg; n=6)

Cohort 2 (3 mg/kg; n=6)

Cohort 3 (10 mg/kg; n=6)

Cohort 4 (20 mg/kg; n=6)

Cohort 5 (first 20-mg/kg dose [day 1]; n=6)

Cohort 5 (second 20-mg/kg dose [day 4]; n=6)

Cmax Mean (SD; µg/mL)

29∙9 (5∙8)

Coefficient of variation

19∙5%

Median Tmax (range; h)

130∙4 (38∙9) 29∙8%

357∙2 (72∙7) 20∙3%

692 (104∙7) 15·1%

8∙0 (4∙0–12∙0)

4∙0 (4∙0–8∙0)

2∙5 (1∙0–12∙1)

4·1 (1∙0–8∙0)

5528∙7 (953∙0)

21 874∙2 (4942∙0)

79 102∙2 (15 061∙2) 131 648·3 (18 344∙0)

834∙3 (150∙6) 18∙0%

1076∙7 (187∙7) 17∙4%

6∙0 (1∙0–12∙1)

2∙5 (1∙0–8∙0)

33 082∙4 (6222∙7)

337 574∙6 (50 058∙1)

AUC0–last Mean (SD; h × µg/mL) Coefficient of variation Median t1/2 (range; h)

17∙2%

22∙6%

19∙0%

13·9%

663∙3 (474∙3–735∙1)

466∙3 (382∙8–522∙3)

397∙0 (333∙9–491∙8)

466·7 (351∙0–889∙6)

18∙8% ··

14∙8% 472∙0 (385∙6–592∙0)

Clearance Mean (SD; mL/h/kg) Coefficient of variation

0∙18 (0∙03) 16∙4%

0∙14 (0∙03)

0∙13 (0∙03)

21∙3%

20∙6%

95∙2 (29∙5)

75∙1 (11∙6)

31∙0%

15∙4%

0∙15 (0∙02)

··

15·5%

··

14∙6%

0∙06 (0∙001)

··

40∙5 (6∙6)

··

16∙3%

Vz Mean (SD; mL/kg) Coefficient of variation

167∙3 (54∙1) 32∙3%

112·6 (36∙8) 32·7%

Pharmacokinetic data for monoclonal antibody m102.4 serum concentrations were calculated using all participants receiving m102.4 (n=30). Cmax=maximum observed serum concentration. Tmax= time to Cmax. AUC0–last =area under the m102.4 concentration curve from before dose administration to last measurable concentration. t1/2=elimination half-life. Vz=volume of distribution.

Table 3: Pharmacokinetic parameters for each cohort (six treated participants per cohort) of healthy adults treated with m102.4

the Tmax values were 6·0 h for the first dose and 2∙5 h for the second dose and the Cmax values were 834·3 µg/mL for the first dose and 1076·7 µg/mL for the second dose. All participants had negative antidrug antibody results at days 1, 29 (cohorts 1–4) or 39 (cohort 5), and 113 (cohorts 1–4) or 123 (cohort 5). Virus-neutralisation activity for both Hendra and Nipah viruses was found in all samples and timepoints tested. The viral neutralising titres ranged from 1/8 to 1/64 or more across all samples, corresponding to m102.4 concentrations of 2–10 µg/mL as measured by the pharmacokinetic ELISA. In four (80%) of five participant samples, the day-8 neutralising titre was within one or two doubling dilutions of the 12-h sample from the same participant. For the fifth participant sample, the day-8 neutralising titre was within three doubling dilutions of the 12-h sample for Hendra virus and one doubling dilution for Nipah virus. These results were considered to be within experimental variation, and therefore it was concluded that the m102.4 remained active for at least 8 days following monoclonal antibody administration.

Discussion In this phase 1 study we assessed the safety and tolerability of single and multiple intravenous doses of m102.4 in healthy human adults. All 40 participants completed the trial, with safety data indicating that m102.4 was safe and well tolerated. The maximum dose of 20 mg/kg was in line with the human equivalent dose conversion (based on body surface area) at the NOAEL of 100 mg/kg, assigned from the rat toxicology study of weekly doses for 4 weeks. Therefore, the maximum clinical dosing of 8

two times 20 mg/kg m102∙4 was supported by the nonclinical data and is a dose hypothesised to be pharma­ cologically relevant based on animal challenge data. Treatment-emergent adverse event reports were similar between placebo and active-treatment groups. There was an increase in treatment-related headaches recorded among participants receiving single-dose m102∙4 com­ pared with those administered placebo (67% vs 10%); there was no increase in treatment-related headaches among those with repeat dosing. Dose-related changes in ALT concentration were noted in three participants; however, these were mild abnormalities and were not considered clinically significant. There were no deaths or severe adverse events leading to withdrawals or premature discontinuations during the study. Pharma­ cokinetics based on those receiving active treatmen were linear, with the mean half-life ranging from 16∙5 days to 27∙6 days across cohorts 1–4. There was a small-to-moderate accu­ mulation of monoclonal antibody in those receiving repeat dose (cohort 5), with elimination kinetics that were similar to those receiving single-dose regimens. A variety of active immunisation strategies have been explored for infection with Hendra virus and Nipah virus involving various recombinant viral vector-based vaccines in animal challenge models.4 These studies showed the possibility of inducing a strong neutralising antibody response that could afford protection in livestock or an emergency-use scenario.4 Among the most notable studies was the identification of the m102.4, the first fully human monoclonal antibody that cross-neutralised in vivo all Hendra and Nipah virus isolates, including those from the Australian 1994 Hendra virus outbreak and

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the Malaysian and Bangladesh Nipah virus outbreaks. A single dose of m102.4 administered to ferrets 10 h after a lethal virus challenge was shown to be fully protective with no evidence of toxicity.20,22 In subsequent studies designed to closely reflect realworld human exposure, non-human primates were challenged with lethal doses of Hendra and Nipah viruses by an intratracheal route, then infused twice with m102.4 mAb (15 mg/kg), beginning at 1 day, 3 days, or 5 days after infection and again 2 days later. Untreated control animals succumbed to disease 8 days after infection, whereas those in the treatment groups recovered by day 16.23,24 Also tissue samples collected from treated animals had no evidence of infectious Hendra or Nipah virus at the termination of the study, showing the potential of m102.4 to treat Hendra and Nipah virus infections in human and animal populations. A potential challenge to developing antiviral therapies for RNA viruses is their heterogeneity and mutational instability. However, the m102.4 recognises epitopes that are highly conserved among virus variants, making it unlikely for the virus to escape neutralisation because of mutation activity even in the context of therapeutic dosing in an immunocompetent individual. Before this trial, 14 individuals received prophylactic high-dose m102∙4 on compassionate grounds following high exposure to Hendra virus (13 individuals in Australia) or Nipah virus (one individual in the USA; appendix p 1). Individuals were aged 8–59 years. No individual showed clinical or laboratory evidence of infection before or after m102.4 administration. The m102.4 administration was generally well tolerated; two individuals developed infusion-related febrile reactions that were related to an early production process of the antibody. Because of the available data and findings on m102.4, the 2018 outbreak of Nipah virus in Kerala, India, encouraged a number of organisations, including the Indian Council of Medical Research, the Henry M Jackson Foundation, WHO, and the US National Institute of Allergy and Infectious Diseases and US National Institutes of Health to work towards the development of an Nipah virus therapeutics protocol for the use of m102.4 in the event of another outbreak of Nipah virus in India or Bangladesh. To date, treatment for Nipah virus has been limited to supportive care and management of acute encephalitis syndrome and standard infection control practices to reduce person-to-person transmission.29 One pharma­ cological option that was used for postexposure prophylaxis during the Malaysian Nipah virus outbreak of 1999 was the antiviral ribavirin. Although this treatment was associated with a significant reduction in mortality due to acute encephalitis, treat­ment allocation was not randomised, and the lack of clinical trial safety data beyond short-term emergency administration limits its use to outbreak circumstances.30 The safety profile of both single and repeat dosing of m102.4 and the standard elimination pharmacokinetic profile presented in this

study underpin the current standard of care, in the context of compassionate emergency use, to offer a single 20-mg/kg dose of m102.4 to humans with high levels of exposure to Hendra virus and Nipah virus. It also supports its potential use for patients with established clinical manifestations of henipavirus infection; on the basis of African green monkey challenge studies,23,24 two doses separated by 48 hours would seem appropriate. The main limitation of our study is the small number of participants, which is not uncommon in phase 1 studies. Also, pharmacokinetic parameters were based on active treatment cohorts only consisting of 30 individuals. Future studies will be needed to ascertain the efficacy of m102.4 for treatment and prophylaxis against different viral strains of Nipah and Hendra viruses, particularly among populations living in settings where there is the potential for an outbreak. Also, although there is no evidence of escape mutants to m102.4, the potential for this cannot be ruled out with RNA viruses. It might be necessary to consider a cocktail of monoclonal antibodies with additional targets to combat the likelihood of diminished efficacy of m102.4. In summary, m102.4 single and double dosing 3 days apart appears to be safe and well tolerated when administered to healthy volunteers. Given that a 57% mortality rate has been observed from the seven known cases of human infection with Hendra virus, and that m102.4 has demonstrated protective efficacy in nonhuman challenge studies, m102∙4 is the most promising therapeutic opportunity available to date for addressing this unmet medical need. This study will inform future dosing regimens for m102.4 to achieve systemic efficacy against Hendra virus and Nipah virus as post-exposure prophylaxis. Contributors EGP was the lead investigator involved in study design, data collection and analysis, and interpretation. TM was responsible for preclinical and clinical monoclonal antibody production. SMM and PPG were responsible for study design. SE and PG were involved in clinical trial study design, conduct, supervision and data interpretation. MG was responsible for antibody quality review and data analysis. KLH, MLJ, BH, KH, and EH were responsible for antibody manufacture and project management. KDL, HC, DES, and SBL were involved in the planning and implementation phase of the clinical trial. MEG was the clinical trial coordinator for patient screening procedures. CdB was involved in analytical and stability testing of m102∙4 for clinical trial and animal toxicology studies. RK implemented virus neutralisation studies. MGS negotiated the transfer of preclinical materials and data on mAb from the USA to Australia. ILS and L-FW designed and analysed virus neutralisation studies. SBL reviewed and interpreted safety data from the trial. DSD contributed to trial design and supplied the cell line for production of antibody for trial. CCB supplied preclinical materials, including the master cell bank and data on monoclonal antibody, and contributed to study design and data analysis. All authors reviewed and approved the final manuscript. Declaration of interests CCB is a US federal employee. CCB and DSD are co-inventors on US patent numbers 7988971 and 8313746 (human monoclonal antibodies against Hendra and Nipah viruses), whose assignees are the USA as represented by the Henry M Jackson Foundation for the Advancement of Military Medicine (Bethesda, MD). All other authors declare no competing interests.

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Data sharing Data generated in this study will be shared as deidentified data. Study protocol, statistical analysis plan, and informed consent forms are available with this publication. Any additional data could be made available upon request to the corresponding author following review by the protocol team.

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10

Acknowledgments We thank the participants who generously gave their time to participate in this study and without whom the study would not have been possible. We acknowledge Roz Glazebrook and Wendy Morotti (Queensland Health, Brisbane, QLD, Australia) for initial coordination and preparation of trial related activity and support with maintaining the study; Terry Hurst (Q-Pharm, Brisbane, QLD, Australia) for initial preparation and planning of initial grant funding of this phase 1 clinical trial; Gary Crameri (CSIRO Australian Animal Health Laboratory, Geelong, VIC, Australia) for technical assistance with virus neutralisation studies. We thank Clinic Manager Sharon Rankine and the Q-Pharm clinic team. We thank Sonya Bennett (Executive Director) and Jeannette Young (Chief Health Officer) of Queensland Health, for ongoing support for this project, and securing a significant portion of the funding for this trial. The manufacture of m102∙4 drug substance was done at the National Biologics Facility (NBF), at the University of Queensland (Brisbane, QLD, Australia). The NBF is supported by Therapeutic Innovation Australia (Brisbane, QLD, Australia), which is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy programme. We thank Sharon Johnatty of SugarApple Communications (Cleveland, QLD, Australia) for providing medical writing support, which was funded by Queensland Health, in accordance with Good Publication Practice guidelines. References 1 Wang L-F, Mackenzie JS, Broder CC. Henipaviruses. In: Knipe DM, Howley PM, eds. Fields virology. Philadelphia: Lippincott Williams & Wilkins, 2013: 1070–85. 2 Halpin K, Young PL, Field HE, Mackenzie JS. Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol 2000; 81: 1927–32. 3 Chua KB, Koh CL, Hooi PS, et al. Isolation of Nipah virus from Malaysian island flying-foxes. Microbes Infect 2002; 4: 145–51. 4 Broder CC, Weir DL, Reid PA. Hendra virus and Nipah virus animal vaccines. Vaccine 2016; 34: 3525–34. 5 Geisbert TW, Feldmann H, Broder CC. Animal challenge models of henipavirus infection and pathogenesis. Curr Top Microbiol Immunol 2012; 359: 153–77. 6 Broder CC, Xu K, Nikolov DB, et al. A treatment for and vaccine against the deadly Hendra and Nipah viruses. Antiviral research 2013; 100: 8–13. 7 Wong KT, Ong KC. Pathology of acute henipavirus infection in humans and animals. Patholog Res Int 2011; 2011: 567248. 8 Playford EG, McCall B, Smith G, et al. Human Hendra virus encephalitis associated with equine outbreak, Australia, 2008. Emerg Infect Dis 2010; 16: 219–23. 9 Wong KT, Tan CT. Clinical and pathological manifestations of human henipavirus infection. Curr Top Microbiol Immunol 2012; 359: 95–104. 10 Murray K, Rogers R, Selvey L, et al. A novel morbillivirus pneumonia of horses and its transmission to humans. Emerg Infect Dis 1995; 1: 31–3. 11 International Society for Infectious Diseases. Hendra virus— Australia: (New South Wales) horse. 2019. http://www.promedmail. org/post/6531307 (accessed June 21, 2019). 12 Chua KB, Goh KJ, Wong KT, et al. Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 1999; 354: 1257–59.

13 Chattu VK, Kumar R, Kumary S, Kajal F, David JK. Nipah virus epidemic in southern India and emphasizing “One Health” approach to ensure global health security. J Family Med Prim Care 2018; 7: 275–83. 14 Sazzad HM, Hossain MJ, Gurley ES, et al. Nipah virus infection outbreak with nosocomial and corpse-to-human transmission, Bangladesh. Emerg Infect Dis 2013; 19: 210–17. 15 Mire CE, Satterfield BA, Geisbert JB, et al. Pathogenic differences between Nipah virus Bangladesh and Malaysia strains in primates: implications for antibody therapy. Sci Rep 2016; 6: 30916. 16 Luby SP. The pandemic potential of Nipah virus. Antiviral Res 2013; 100: 38–43. 17 WHO. WHO publishes list of top emerging diseases likely to cause major epidemics. 2015. https://www.who.int/medicines/ebolatreatment/WHO-list-of-top-emerging-diseases/en/ (accessed June 19, 2019). 18 Rottingen JA, Gouglas D, Feinberg M, et al. New vaccines against epidemic infectious diseases. N Engl J Med 2017; 376: 610–13. 19 Zhu Z, Dimitrov AS, Bossart KN, et al. Potent neutralization of Hendra and Nipah viruses by human monoclonal antibodies. J Virol 2006; 80: 891–99. 20 Zhu Z, Bossart KN, Bishop KA, et al. Exceptionally potent cross-reactive neutralization of Nipah and Hendra viruses by a human monoclonal antibody. J Infect Dis 2008; 197: 846–53. 21 Xu K, Rockx B, Xie Y, et al. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody. PLoS Pathog 2013; 9: e1003684. 22 Bossart KN, Zhu Z, Middleton D, et al. A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute Nipah virus infection. PLoS Pathog 2009; 5: e1000642. 23 Bossart KN, Geisbert TW, Feldmann H, et al. A neutralizing human monoclonal antibody protects African green monkeys from Hendra virus challenge. Sci Transl Med 2011; 3: 105ra3. 24 Geisbert TW, Mire CE, Geisbert JB, et al. Therapeutic treatment of Nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody. Sci Transl Med 2014; 6: 242ra82. 25 Queensland Government. World-first Hendra treatment one step closer. Oct 31, 2013. http://statements.qld.gov.au/ Statement/2013/10/31/worldfirst-hendra-treatment-one-step-closer (accessed June 19, 2019). 26 Luby SP, Broder CC. Paramyxoviruses: henipaviruses. In: Kaslow RA, Stanberry LR, Le Duc JW, eds. Viral infections of humans, epidemiology and control. New York: Springer Science+Business Media, 2014: 519–36. 27 European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on strategies to identify and mitigate risks for first-in-human and early clinical trials with investigational medicinal products. EMEA/CHMP/SWP/28367/07 Rev 1. 2017. https://www.ema.europa.eu/en/documents/scientific-guideline/ guideline-strategies-identify-mitigate-risks-first-human-earlyclinical-trials-investigational_en.pdf (accessed June 19, 2019). 28 US Food and Drug Administration. Guidance for industry: bioanalytical method of validation. 2001. http://www.labcompliance. de/documents/FDA/FDA-Others/ Laboratory/f-507-bioanalytical-4252fnl.pdf (accessed June 24, 2019). 29 Centers for Disease Control and Prevention. Nipah Virus (NiV)— treatment. https://www.cdc.gov/vhf/nipah/treatment/index.html (accessed July 16, 2019). 30 Banerjee S, Gupta N, Kodan P, et al. Nipah virus disease: a rare and intractable disease. Intractable Rare Dis Res 2019; 8: 1–8.

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