Background
Nipah virus (NiV) is a zoonotic virus belonging to the henipavirus genus in the family
Paramyxoviridae. Hendra virus (HeV) is the only other recognised member of the henipavirus genus [
1], although there is increasing evidence of a range of henipa-like and other paramyxoviruses [
2‐
6], particularly in bats which are the reservoir host for both NiV and HeV. HeV and NiV are classified as BioSafety Level 4 (BSL-4) agents, a designation that reflects their ability to cause significant morbidity and mortality in humans as well as the absence of vaccines and post-exposure treatments. Since NiV was first identified [
7] outbreaks have occurred in humans on an almost annual basis with case fatality rates generally reported between 40% and 100% [
8]. Human-to-human transmission, although not yet observed for HeV [
9,
10], has been recorded in NiV outbreaks in Bangladesh. Here the local custom of family nursing infected members has resulted in extended exposure to infectious bodily secretions, particularly respiratory fluids [
11]. Documentation of human-to-human transmission in this highly virulent group of viruses confirms the possibility of sustained endemic human-to-human transmission and the attendant consequences.
Vaccination is an important component of outbreak preparedness and considerable data has now been accumulated on a subunit vaccine incorporating the G glycoprotein of HeV (HeVsG). The HeVsG vaccine has so far proved effective in preventing disease following exposure to both HeV and NiV. This is as predicted because the G glycoproteins of NiV and HeV share 83% amino acid identity [
12] and also host cell receptors - ephrin B2 and ephrin B3 [
13‐
15]. Cats vaccinated with HeVsG produced high levels of neutralising antibody to both HeV and NiV [
16]; neutralising antibody to surface glycoprotein has been shown to be particularly effective in protecting against infection with viruses like the henipaviruses that have a viraemic phase [
17‐
19]. Most vaccinated animals not only remained clinically well following exposure to virus but had no evidence of infection, including no boost in vaccinal antibody titres. Vaccination with HeVsG has also been shown to prevent NiV infection in cats and nonhuman primates, and HeV infection in ferrets [
16,
20‐
22] and horses. In this last case, HeVsG antigen forms the basis of a commercially available vaccine for HeV - the first vaccine licensed and deployed for use against a BSL-4 agent (Middleton et al., manuscript in preparation). Here we show that ferrets vaccinated with HeVsG were equally protected against NiV disease at 20 days post vaccination as against HeV [
22]. Furthermore, protection from disease persisted for over 12 months.
Results
Nipah virus challenge of immunized ferrets
The aim of this study was to determine whether a HeVsG based vaccine protected ferrets against disease caused by NiV and the duration of any vaccine-induced protection. Eight ferrets were exposed to an otherwise lethal dose of NiV at either 20 days post vaccination (study 1) or 14 months post vaccination (study 2).
Study 1: Two control ferrets 1–0 and 2–0 were given only adjuvant; 1–0 was febrile on day 5 pi and was euthanized on day 6 pi, while 2–0 was febrile on days 7 and 8 pi and was euthanized on day 10 pi. Two vaccinated ferrets, 4–4 and 7–100 were euthanized prior to exposure to virus for reasons unassociated with the scientific study. The remaining vaccinated ferrets, 3–4, 5–20, 6–20 and 8–100 remained clinically healthy and were electively euthanized on day 41 of the study.
Study 2: Two control ferrets, 9–0 and 10–0 became febrile and exhibited reduced playfulness by day 8 pi; both were euthanized. Ferret 15–100 was euthanized prior to exposure to virus for reasons unassociated with the scientific study. The remaining vaccinated ferrets, 11–4, 12–4, 13–20, 14–20 and 16–100 remained clinically healthy and were electively euthanized on day 455 (21 days pi) of the study.
Gross pathology, histopathology and immunohistochemistry
Study 1. Control ferrets 1–0 and 2–0 both had histological lesions including widespread deposition of NiV antigen consistent with acute NiV infection as previously described [
23]. No significant histological lesions were detected in the remaining 4 ferrets, 3–4, 5–20, 6–20 and 8–100 and NiV antigen was not detected in any of their tissues.
Study 2. Control ferrets 9–0 and 10–0 both had histological lesions including widespread deposition of NiV antigen consistent with acute NiV infection as previously described [
23]. No significant histological lesions were detected in the tissues of vaccinated ferrets, 11–4, 12–4, 13–20, 14–20, and 16–100 and NiV antigen was not detected in any of their tissues.
Viral RNA detection and virus isolation
Viral RNA detection results for study 1 are shown in Table
1. Viral RNA was found in all fluids, swabs and tissues of control ferrets 1–0 and 2–0 and virus was re-isolated from tissues collected from them post mortem. C
t results for positive samples and tissues are shown in Table
2. Where virus was isolated from viral RNA positive samples the titre was generally not greater than 10
5 TCID
50/ml. These data confirm that virus exposure was sufficient to induce serious infection in the naïve animal. In contrast viral RNA was not detected in any swabs, fluids or tissues collected from the 4 vaccinated ferrets either during the immediate post-exposure period or at post mortem examination; this included ferret 3–4 which had received the lowest dose (4 μg) of HeVsG.
Table 1
Study 1: Genome detection in ferrets challenged at day 20 post vaccination
1-0
| <2 | 6 | PM | PM | PM | PM | PM | 13/13 | 8/13 |
2-0
| <2 | 10 | 6, 8, PM | 8, PM | 8, PM | 8, PM | PM | 13/13 | 6/13 |
3-4
| 64, 64 | 21 | - | - | - | - | - | 0/13 | ND |
4-4
| NC | na | NC | NC | NC | NC | NC | NC | ND |
5-20
| 256, 256 | 21 | - | - | - | - | - | 0/13 | ND |
6-20
| 256, 512 | 21 | - | - | - | - | - | 0/13 | ND |
7-100
| NC | na | NC | NC | NC | NC | NC | NC | ND |
8-100
| 256, 512 | 21 | - | - | - |
-
| - | 0/13 | ND |
Table 2
C
t
values in genome positive samples in studies 1 and 2
Oral swab
| D6 | - | 31.4 |
-
| 32.6 |
-
|
-
|
| D8 |
-
| 34.8 |
-
|
-
|
-
|
-
|
| PM | 33.6 | 30.5 | 38.0 | 31.9 |
-
|
-
|
Rectal swab
| D6 | - | - | - | 35.8 |
-
|
-
|
| D8 |
-
| 37.5 |
-
|
-
|
-
|
-
|
| PM | 33.3 | 31.2 |
-
| 35.4 |
-
|
-
|
Blood
| D6 |
-
|
-
|
-
| 36.6 |
-
|
-
|
| D8 |
-
| 32.9 | 38.3 |
-
|
-
|
-
|
| PM | 30.2 | 30.1 |
-
| 26.5 |
-
|
-
|
Nasal wash
| D6 |
-
|
-
|
-
| 29.4 |
-
| 34.1 |
| D8 |
-
| 36.4 |
-
|
-
|
-
| 32.3 |
| PM | 28.0 | 29.6 | 31.9 | 31.9 |
-
|
-
|
Urine
| PM | 31.0 | 29.1 | 34.7 | 31.9 |
-
|
-
|
Tissues
| PM | 118-34.7 | 18.0-30.9 | 18.6-36.6 | 16.4-37.8 |
2
33.8 |
-
|
Viral RNA detection results for study 2 are shown in Table
3; the pattern of genome detection and virus re-isolation from the control ferrets 9–0 and 10–0 confirmed an otherwise lethal exposure to NiV. Viral genome was not detected in any swabs, fluids or tissues from 3 of the 5 vaccinated ferrets. In one animal (ferret 14–20), viral genome was detected in nasal washes at day 6 and day 8 pi and, in another (ferret 13–20), from the bronchial lymph nodes at post mortem. In neither case was virus re-isolated from these samples. C
t results for positive samples and tissues are shown in Table
2. Where virus was isolated from viral RNA positive samples the titre was generally not greater than 10
5 TCID
50/ml
Table 3
Study 2: Genome detection in ferrets challenged at 434 days post vaccination
9-0
| <4, <4 | 8 | PM | - | PM | PM | PM | 13/13 | 10/13 |
10-0
| <4, <4 | 8 | 6, PM | 6,PM | 6, PM | 6, PM | PM | 13/13 | 10/13 |
11-4
| 32, 128 | 20 | - | - | - | - | - | - | ND |
12-4
| 32, 64 | 20 | - | - | - | - | - | - | ND |
13-20
| 16, 16 | 20 | - | - | - | - | - | 1/13 | 0/13 |
14-20
| 64, 128 | 20 | - | - | - | 6,8 | - |
-
| - |
15-100
| NC | na | NC | NC | NC | NC | NC | NC | NC |
16-100
| 64, 64 | 20 | - | - | - | - | - | - | ND |
Post-challenge serology
In both studies sera were collected at the time of exposure to virus; day 6, 8 and 10 pi, and at euthanasia. There was no clear cut evidence of an anamnestic antibody response in either group.
Conclusions
As the human population expands into previously untouched environments there are increasing reports of viruses that have co-existed for long periods of time in their reservoir hosts ‘spilling over’ to emerge as novel human infections. The recent example of such an agent, human immunodeficiency virus (HIV), demonstrates the devastating economic and social impact that these events may have on human populations. Although the virus was not identified until 1981, HIV is thought to have made the initial jump from chimpanzees into humans at the beginning of the 20
th century. By 2010, 34 million people were living with acquired immunodeficiency syndrome (AIDS) and in the same year 2.4-2.9 million people were newly infected with HIV. HeV and NiV were also discovered relatively recently – HeV was isolated and identified in 1994 when it caused the death of 20 horses and 1 human [
24] and NiV in 1998–9 after an outbreak in pigs and humans in Peninsular Malaysia and Singapore [
7]. They were identified as paramyxoviruses but were sufficiently distinct to be assigned to a new genus,
Henipavirus, within the
Paramyxoviridae[
25,
26]. Their large genome was atypical as was their pathogenicity for a range of species, with reported mortality rates in humans of up to 100% for NiV and 57% for HeV. All human cases of HeV infection to date have resulted from close contact with secretions of infected horses either late in incubation period, during terminal illness, or at post mortem examination. There is no known instance of transmission directly to humans from the reservoir host, the bat, or of human-to-human transmission of HeV. In contrast, NiV in Bangladesh can be transmitted directly from bats to people and this has been linked epidemiologically to the consumption of contaminated palm sap; human-to-human transmission also occurs here and is thought to be facilitated by families nursing sick relatives with attendant copious exposure to infected bodily fluids [
27]. Repeated spillover events into human (and other animal populations) have been documented for both HeV and NiV [
8] suggesting persistence of the environmental circumstances that facilitated the initial emergence event. Spillover is thus likely to be ongoing, and it is conceivable that a future incident with increasing host adaptation might result in establishment of HeV or NiV in human populations, with an impact exacerbated by high mortality rates and no pre-existing immunity.
The sporadic occurrence of viral spillover events creates major challenges for emergency disease preparedness activities. However, in the 15–20 years since HeV and NiV were first identified substantial progress has been made in the development of vaccines and therapeutics for the prevention and treatment of infection with these viruses. The HeVsG vaccine, which prevents HeV disease in horses, is the first to be registered for use against a BSL-4 agent, and a therapeutic monoclonal antibody is currently being assessed for human use [
23,
28]. While the focus in Australia is on management of the infection risks posed by HeV infection, studies have shown that the HeVsG vaccine provides equally powerful cross-protection against NiV infection in cats and nonhuman primates.
Our current studies indicate that at the time of onset of protection the HeVsG vaccine can reliably protect ferrets from acute NiV disease and also prevent infection – providing so-called sterilizing immunity - consistent with earlier studies using HeV [
22]. Here we have also shown, in the first duration of protection study in an animal model, protection from disease persists at least 14 months after vaccination in ferrets. Recovery of viral genome (but not live virus) from the nasal washes of one animal and from the bronchial lymph nodes of another, in the absence of a rise in antibody titre, are consistent with self-limiting local virus replication at a level insufficient to generate an anamnestic immune response or to sustain a transmission event.
The animal numbers reported here are necessarily small due partly to the limitations of the BSL4 facility but also because 3 ferrets were euthanized before challenge leaving 3 groups in the 2 studies with a group size of 1. While no conclusion can be drawn from a group size of 1 there were 3 remaining groups where n = 2, the 20 μg group in both studies and the 4 μg group in study 2. Data from these groups indicated that the vaccine induced protection sufficient to suppress the course of a human pandemic persists for at least 12 months. The data from the reduced groups also support this conclusion.
Importantly, in the event of a spillover leading to sustained human-to-human transmission of HeV or NiV, proof of concept studies already exist for the sG subunit vaccine in two animal models of which one is a non-human primate. This is relevant to the US FDA animal rule that states where a medical countermeasure cannot be evaluated in humans, in vivo evaluation of a vaccine or therapeutic may be translated from the outcomes of work in two animal models. Finally, the experimental vaccine given to ferrets uses a formulation that is in clinical trials in humans.
Recrudescence in the form of encephalitis has been documented for both HeV and NiV and is thought to be due to persistence of the virus in some form within the central nervous system. A farmer from Mackay, Australia developed encephalitis 13 months after apparent recovery from acute meningencephalitis caused by HeV and died with evidence of HeV in the brain as detected by PCR, electron microscopy and immunohistochemistry [
29]. In the initial NiV outbreak in Malaysia 7.5% of survivors went on to develop relapsing encephalitis and 3.4% suffered late-onset encephalitis months to years after recovery from the initial infection [
30,
31]. Recent studies have shown that HeV can infect the mouse brain via an anterograde route of infection, probably along the olfactory nerve [
32] as also suggested for NiV in pigs [
33]. This suggests that an effective henipavirus vaccine will need to suppress the initial phase of replication in the upper respiratory tract (summarized in [
8]) to a level that prevents infection of olfactory sensory neurons as well as preventing the onset of viremia.
The HeVsG subunit vaccine has proved highly effective in suppression of virus replication and disease prevention, with duration of protection that is sufficient to make the formulation attractive to industry. A vaccine incorporating the HeVsG antigen has now been released for use in horses and its application and observed effectiveness, along with ongoing work in animal models including nonhuman primates, will enable a more rapid response to any future henipavirus spillover events that threaten to cause large scale outbreaks in humans.
Competing interests
CCB is a United States federal employee and an inventor on pending United States patents and Australian patent 2005327194, pertaining to soluble forms of Hendra and Nipah G glycoproteins; assignees are The United States of America as represented by the Department of Health and Human Services (Washington, DC), Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (Bethesda, MD).
Authors’ contributions
JAP designed the study, processed animal tissues, carried out virus isolation and serology and drafted the manuscript. RK processed animal tissues, carried out real-time PCR, virus isolation and serology. RA and JH carried out the animal infection studies. FL read histopathology and immunohistopathology slides. JRW extracted viral RNA from animal tissues and fluids. JP processed tissues for histopathology and immunohistopathology. LFW conceived the study and edited the manuscript. CCB conceived the study, provided funds and edited the manuscript. DM conceived and designed the study, supervised animal infection studies, provided veterinary pathology expertise and edited the manuscript. All authors read and approved the final manuscript.