Figure 1

Figure 1. Histologic and immunohistologic findings in Hendra virus–infected horse tissueA) Hematoxylin and eosin staining shows systemic vasculitis affecting the lungB) Immunohistologic examination, using polyclonal rabbit anti-Nipah N protein, indicates Hendra virus antigen...

Vaccine efficacy in immunized horses was assessed against the clinical, virologic, and pathologic features of HeV infection in 4 unvaccinated control horses. Infection characteristics for 3 of these unvaccinated animals have been described (6); data from the fourth control animal was gathered as part of the current work. In that fourth control, onset of fever accompanied by a rising heart rate was noted on postchallenge day 6. On postchallenge day 7, the horse became clinically depressed, its temperature and heart rate continued to rise, and it was euthanized. Gross postmortem findings included pleural thickening and moderate dilation of the lymphatic vessels on the ventral 10 cm of the cardiac lung lobes. Histologic examination revealed systemic vasculitis affecting the lung (Figure 1, panel A), spleen, kidney, nasal epithelium, lymph nodes, and brain; alveolitis; and lymphadenitis. HeV antigen was identified in endothelial cells and vascular walls within lung, brain (Figure 1, panel B), nasal epithelium, lymph nodes, spleen, kidney, liver, myocardium, salivary gland, pharynx, small intestine, uterus, ovary, and adrenal gland, as well as in myocardial fibers and glomeruli.

Viral RNA from this fourth control horse was detected in nasal swabs collected on postchallenge day 3 (Table 2; summarized in Table 3) and also in blood collected immediately before the onset of fever. After onset of fever, but before development of other clinical signs of illness, HeV RNA was also detected in the oral swab sample. On the day of euthanasia, genome was detected in oral and nasal swab samples, blood, rectal swab, and urine samples; however, virus was not reisolated from any sample collected before postmortem examination. Viral RNA was detected in all tissues sampled at postmortem examination except cerebrospinal fluid. Reisolation of virus was attempted for all tissues: HeV was recovered from lung, submandibular lymph node, small intestine, large intestine, and adrenal gland.

In a series of vaccine efficacy studies, 10 horses were immunized with HeVsG glycoprotein and then exposed to an otherwise lethal dose of HeV by the oronasal route. Each study also included a pathogenicity control for the virus inoculum. In the first of these, the pathogenicity control was the fourth control horse described above. Together with historical data gathered from 3 horses following their exposure to HeV under equivalent experimental conditions (5), data from this horse completed the requirements of the Australian Pesticides and Veterinary Medicines Authority for defining the horse infection model. In subsequent studies, guinea pigs or ferrets were used as pathogenicity controls to maximize the number of vaccinated horses that could be accommodated in the BSL-4 facility. These animals duly displayed signs, lesions, tissue antigen and viral genome distribution, and virus reisolation data consistent with acute HeV infection.

Figure 2

Figure 2. Scatter plot showing quantitation of the Hendra virus N gene in nasal swab samples from 1 vaccinated horse (V9) and 4 control horses (C1–C4); controls were challenged but not vaccinatedDays represent...

In contrast to unvaccinated control horses, vaccinated horses remained clinically healthy during the observation period after exposure to HeV. Following elective euthanasia at the time of predicted peak viral replication, there was no gross or histologic evidence of HeV infection in vaccinated horses; all tissues examined were negative for viral antigen by immunohistochemistry; and viral genome was not recovered from any tissue, including nasal turbinates, pharynx, and guttural pouch (Table 3). For 9 of 10 vaccinated horses, viral RNA was not detected in daily nasal, oral, or rectal swab specimens or from blood, urine, or feces samples collected before euthanasia, and virus was not reisolated from any of these clinical samples. For 1 (V9) of 3 horses exposed to HeV 6 months after completing the vaccination course, low viral gene copy numbers were detected in nasal swab samples collected on postchallenge days 2–4 and 7 (Figure 2); this finding was consistent with self-limiting local replication. Virus was not reisolated from these samples.

Serum neutralization titers before HeV challenge ranged from 128/256 to >4,096 for horses V1–V7 when challenged 21 days after the second vaccination and from 16 to 32 for horses V8–V10 when challenged 6 months after the second vaccination (Table 3). At the time of euthanasia, no rise in antibody titer was detected in any vaccinated horse following exposure to HeV.

The formal launch of the HeV horse vaccine in November 2012 represents the culmination of multiple studies conducted in several animal infection models over the course of many years. Studies using Nipah virus in cats (16,17) and monkeys (18) and HeV in ferrets (15) provided strong evidence that a HeVsG glycoprotein subunit–based vaccine could prevent not only disease but often infection in animals exposed to otherwise lethal doses of Nipah virus or HeV. Where evidence of low-level virus replication did occur in secretions, it was transient and unaccompanied by the development of clinical illness, and virus was not isolated from the secretions.

The henipavirus surface-expressed G glycoprotein has the critical role of initiating infection by binding to receptors on host cells, and antibodies directed against this protein can neutralize virus (19). Earlier reports have shown that passive immunotherapy with antibody to the G or F glycoprotein of HeV or Nipah virus alone can prevent fulminating disease (20): G glycoprotein–specific human monoclonal antibody prevented Nipah virus disease in ferrets (21) and HeV infection in African green monkeys (22); and F or G glycoprotein–specific monoclonal or polyclonal antibodies prevented HeV and Nipah virus disease in hamsters (23–25). Thus it is likely that, as seen for other paramyxoviruses with a viremic infection phase (e.g., measles and mumps), antibodies to the G and F glycoproteins play a major role in protection provided by HeVsG glycoprotein vaccination (26–28).

In the studies reported here, we show that 2 doses of a commercially formulated HeVsG glycoprotein subunit–based vaccine prevented infection in 7 of 7 horses exposed to HeV at least 21 days after the second vaccine dose; this finding is in contrast to that for unvaccinated control horses. Similar results were obtained for 2 of 3 horses exposed to HeV 6 months after vaccination. In the third horse, which also remained clinically healthy, evidence of HeV replication was limited to low-level transient detection of viral genome (but not virus) from the nasal cavity. In assessing the field significance of this observation, the following must be noted: the experimental horses were exposed to considerably higher levels of HeV than have been recovered from flying foxes (1), higher levels of viral genome were routinely found in the nasal secretions of nonimmunized horses, and all human infections have been acquired from animals in which clinical disease developed. It is reasonable to suggest that the higher transmission risk that is clearly associated with such horses is a consequence of not only increased viral load but also of the illness itself: it is the clinically ill horse that promotes increased human–animal contact through diagnostic investigations and administration of nursing care. We conclude that the level and pattern of virus replication in the 1 vaccinated horse do not meet the epidemiologic criteria presently associated with transmission of infection to humans.

In previous henipavirus vaccine efficacy studies in cats and ferrets, a neutralizing antibody titer of 32 was shown to be protective against the development of clinical disease (17). In the horse efficacy studies, the 3 horses with prechallenge antibody titers of 16 or 32 were similarly protected from clinical illness. However, we caution that any correlation between antibody titer at the time of exposure to virus and levels of subsequent protection against infection and disease is unlikely to be linear; it is possible that animals with even lower titers will have epidemiologically meaningful protection against HeV exposure occurring in the field, not least because of stimulation of immunological memory. Additional studies assessing the duration of protection are planned, and the outcome of these will further inform recommendations regarding booster vaccination.

As expected, initial uptake of the HeVsG glycoprotein subunit–based vaccine was strongest in the area with the highest perceived risk for HeV infection, namely coastal Queensland, Australia. In other regions where HeV infection of horses has not been reported, there is understandably more uncertainty regarding the value of vaccination as part of horse preventative health programs. Any reluctance to vaccinate horses against HeV that is based on assessment of risk is probably exacerbated by several factors, including the novelty of the vaccine roll-out process to the Australian horse industry, a (mistaken) perception that fast-tracking vaccine release involved overlooking key safety and efficacy issues, the lack of published data on safety in pregnant mares, reluctance of certain industry sectors to vaccinate because of import restrictions on HeV-seropositive horses, and cost. Although it is likely that each of these barriers will diminish over time, our experiences may assist the development of road maps to guide the future release of vaccines against BSL-4 pathogens that are associated with highly sporadic disease events and where the decision to vaccinate is in the hands of the persons whom vaccination was designed to protect.

Several recently emerged zoonotic viruses, including HeV, Nipah, Ebola, and Marburg viruses, are classified as BSL-4 agents because of their ability to cause severe illness or death in humans and because there have been no effective vaccines or postexposure treatments to protect against the diseases they cause. The vaccine against HeV (Equivac HeV) is a commercially deployed vaccine developed against a BSL-4 agent and is the only licensed treatment for henipavirus infection.

Development of vaccines against BSL-4 agents for use in humans requires that the US Food and Drug Administration implement the animal rule, which requires that such vaccines first be tested for efficacy in at least 2 animal models (29). As a veterinary vaccine, Equivac HeV did not need to meet this requirement, and it was both cheaper and faster to produce than a vaccine intended for human use. At the same time, the vaccine is expected to provide a substantial health benefit to humans. In so doing, this vaccine encapsulates the spirit of a One Health approach, not just in terms of the interconnectedness of human and animal health but also with respect to environmental health. One consequence of the recent HeV outbreaks was a move to eradicate bat populations, despite their crucial environmental roles in pollination and reduction of the insect population. Successful deployment of the HeV vaccine, with a targeted reduction in the risk for acute disease events in horses and humans, should help reduce the current momentum toward the setting of control policies with potential adverse effects on the environment. Furthermore, the increasing evidence for henipaviruses and henipa-like viruses in bats in other areas (30–32) raises the possibility of future henipavirus outbreaks. The current HeVsG glycoprotein vaccine technology provides a platform for the rapid development of related vaccines to counter future emergent threats.