Article Sero-Monitoring of Horses Demonstrates the Equivac® HeV Hendra Virus Vaccine to Be Highly Effective in Inducing Neutralising Antibody Titres Kim Halpin * , Kerryne Graham and Peter A. Durr Australian Centre for Disease Preparedness (ACDP), Commonwealth Scientific and Industrial Research Organisation (CSIRO), 5 Portarlington Road, East Geelong, VIC 3219, Australia; [email protected] (K.G.); [email protected] (P.A.D.) * Correspondence: [email protected]; Tel.: +61-352275510 Abstract: Hendra virus (HeV) is a high consequence zoonotic pathogen found in Australia. The HeV vaccine was developed for use in horses and provides a One Health solution to the prevention of human disease. By protecting horses from infection, the vaccine indirectly protects humans as well, as horses are the only known source of infection for humans. The sub-unit-based vaccine, containing recombinant HeV soluble G (sG) glycoprotein, was released by Pfizer Animal Health (now Zoetis) for use in Australia at the end of 2012. The purpose of this study was to collate post-vaccination serum neutralising antibody titres as a way of assessing how the vaccine has been performing in the field. Serum neutralization tests (SNTs) were performed on serum samples from vaccinated horses submitted to the laboratory by veterinarians. The SNT results have been analysed, together with age, dates of vaccinations, date of sampling and location. Results from 332 horses formed the data Citation: Halpin, K.; Graham, K.; set. Provided horses received at least three vaccinations (consisting of two doses 3–6 weeks apart, Durr, P.A. Sero-Monitoring of Horses and a third dose six months later), horses had high neutralising titres (median titre for three or more Demonstrates the Equivac® HeV vaccinations was 2048), and none tested negative. Hendra Virus Vaccine to Be Highly Effective in Inducing Neutralising Antibody Titres. Vaccines 2021, 9, 731. Keywords: Hendra virus; Hendra virus vaccine; One Health; zoonoses https://doi.org/10.3390/vaccines 9070731 Academic Editors: Jessica Spengler 1. Introduction and Richard Bennett Hendra virus is a zoonotic, high consequence pathogen for which the natural reservoir is the four species of flying fox (Pteropus bat species) found on mainland Australia and Received: 6 May 2021 the primary spillover species is the horse, from which humans are secondarily infected [1]. Accepted: 12 June 2021 The virus emerged in Australia in 1994 in a racing stable in the Brisbane suburb of Hendra, Published: 2 July 2021 where a number of thoroughbred horses developed a mysterious illness. Thirteen horses died along with their noted trainer, Vic Rail. Another man, a strapper for Vic Rail, became Publisher’s Note: MDPI stays neutral infected but survived [2]. Five further outbreaks occurred between 1994 and 2004, and there with regard to jurisdictional claims in have been annual spillover events consistently since 2006, with most events occurring in the published maps and institutional affil- cooler months. Most of these have involved single horses infected in an outdoor grazing iations. environment. Four outbreaks have involved 3 horses each, and 5 outbreaks involved 2 horses each. Two outbreaks where >3 horses were involved have each occurred in a stable setting: one being the original case at a horse training facility and one at a veterinary clinic [2,3]. The spread of the virus between the stabled horses was a result of close contact Copyright: © 2021 by the authors. and assisted mechanical transmission of the virus [4]. Humans have also become infected Licensee MDPI, Basel, Switzerland. because of very close contact with infected horses [3]. In total, 7 humans have become This article is an open access article infected; the horse trainer and his strapper, three veterinarians, one veterinary nurse, distributed under the terms and and the husband of a veterinarian who assisted with a horse necropsy; of these, 4 deaths conditions of the Creative Commons occurred [3,5]. In 2011 alone there were 18 spillover events with 24 horses dying. This Attribution (CC BY) license (https:// cluster of events was concerning, particularly as each event represented a major threat creativecommons.org/licenses/by/ 4.0/). to the humans caring for the horses. Fortunately, no humans became infected in 2011; Vaccines 2021, 9, 731. https://doi.org/10.3390/vaccines9070731 https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, 731 2 of 13 however, the events of the year provided momentum for investment to produce a vaccine for horses which would also provide indirect protection for humans as well. A ferret model was used to assess the immunogenicity and protective efficacy of an experimental subunit vaccine based on a recombinant soluble version of the HeV attachment glycoprotein G (HeV sG) which had shown promising results in previous studies [6,7]. Doses of 100 µg or 20 µg of HeV sG vaccine were shown to completely prevent a productive HeV infection in ferrets and provided confidence that this vaccine would work in horses as well [8]. A subunit vaccine containing recombinant HeV sG glycoprotein was then formulated in a proprietary adjuvant (Pfizer Animal Health, now Zoetis) [9]. In a series of vaccine efficacy studies, 10 horses were immunized with HeV sG and then exposed to an otherwise lethal dose of HeV by the oronasal route [9]. Vaccinated horses remained clinically healthy during the observation period after exposure to HeV. The HeV horse vaccine (Equivac® HeV Hendra Virus Vaccine for Horses, Zoetis Australia Pty Ltd., Rhodes, NSW, Australia) was launched in November 2012, the first vaccine against a Biosafety Level-4 (BSL-4) agent to be licensed and commercially deployed [10]. The vaccine was initially released under a minor use permit pending registration with the Australian Pesticides and Veterinary Medicines Authority (APVMA), the government body responsible for the regulation of all agricultural and veterinary chemicals in Aus- tralia [11]. The permit enabled the vaccine to be used under strict conditions, including administration by a trained and accredited veterinary surgeon to horses that needed to be uniquely identified by a microchip, the details of which were to be also recorded on a national online registry. Several amendments have subsequently been made to the product labelling following the initial release, including six-monthly boosters and approval for use in pregnant mares. In August 2015, the vaccine was officially registered, with 12 monthly boosters approved in May 2016 [12]. The 1 mL vaccine contains not less than 100 µg of HeV sG glycoprotein antigen adjuvanted with 250 µg/dose of an immuno-stimulating complex and is administered by intramuscular injection [13]. The vaccination schedule involves a primary course of two doses 3 to 6 weeks apart, commencing as early as 4 months of age. A 3rd dose 6 months after the second dose is required, with further boosters to be administered every 12 months after the 3rd dose. All doses must be entered into an online registry at https://www.zoetis.com.au/vets-australia/ (accessed 30 March 2021) within two days of administration. All horses must be microchipped prior to administration of the first vaccine dose, and the microchip identifier also needs to be recorded in the online registry. Zoetis advises that foals born to vaccinated mares should commence vaccination at 6 months of age, and pregnant mares are not to be vaccinated during the first 45 days after conception or the two weeks prior to the expected foaling date. Since the vaccine’s release on 1 November 2012, Zoetis has sold over 750,000 doses. Zoetis has supplied details regarding sales from 2016–2019 where it sold 317,030 doses, representing 57,645 horses receiving their first dose (Supplementary Material Table S1). This is low compared to the vaccine for tetanus where approximately twice as many doses are sold per year (R. L’Estrange pers. com). A spike in Equivac® HeV Hendra virus vaccine sales in 2019 can be attributed to the identification of a Hendra virus infected horse in Scone in the state of New South Wales which expanded the distribution of Hendra virus cases southwards. Estimating the percentage of horses in Australia receiving at least one dose of the vaccine in one year, based on horse population estimates [14] can be approximated (Supplementary Material Table S2). If we assume most vaccine sales are in the HeV endemic states, vaccination coverage between years 2016 and 2019 ranged from 10.1% to 13%, which at a national level decreases to 6.9–8.9%. The serological response to the vaccine is measured using a serum neutralisation test (SNT), also known as a virus neutralisation test (VNT). Serial dilutions of serum are mixed with a standard amount of virus, incubated, and then placed onto cells. If there are antibodies in the serum, these neutralise the virus and it will not grow. An end point antibody titre can be measured in this test. Titres can range from 2 to >16,000, with the greater the amount of antibody in the serum, the higher the titre. Some horse owners Vaccines 2021, 9, 731 3 of 13 opt to have their horse’s post-vaccination titre assessed, which is more expensive than the vaccination but provides some information around how their horse has responded to previous vaccinations [15]. The only laboratory in Australia which can conduct this test is the Australian Centre for Disease Preparedness (ACDP). Since the vaccine was launched, the laboratory has conducted hundreds of HeV antibody titre tests on vaccinated horses. The Equivac® HeV Hendra virus vaccine for horses has been in use for more than 7 years. We collated post-vaccine serum neutralising antibody titres as a way of assessing how the vaccine has been performing in the field. The design of the study was a retrospec- tive exploratory data analysis using a data set which includes the age of the horse, dates of vaccinations, date of sampling and location as well as the post-vaccination SNT titre.