Hendra antibody tests

guidelines for vets, farm managers, and event organisers

Following is the text of a PowerPoint presentation I made on using serology (antibody testing) to document proof of immunity instead of relying exclusively on proof of vaccination to keep horses and humans safe.


“When the risk and consequences of a systemic vaccine-associated adverse event exceeds the risk and consequences from the disease, it is rational to attempt to predict immunity by serologic testing to either eliminate vaccination for the disease in question or extend the revaccination interval for as long as possible. ”

AAEP Guidelines for serology in horses with adverse events from vaccination. American Association of Equine Practitioners (AAEP), 2020.


Many owners don’t want their horses vaccinated against Hendra virus (HeV)

  • concern about vaccine safety is prevalent and outweighs concern about the virus itself
  • other reasons include low incidence of HeV (perception of low risk) and cost

Many owners of vaccinated horses don’t want to keep up with annual HeV boosters

  • concerns include vaccine safety, cost, and whether annual boosters really are necessary for protection
  • horses who have had vaccine reactions or other adverse effects of the HeV vaccine are an important subset; these horses should be boostered with great care, at an extended interval, or not at all

Hendra antibody tests can be used as proof of immunity rather than relying solely on proof of vaccination


Antibodies are specific proteins the immune system produces in response to a pathogen (disease-causing agent) or vaccine

  • Hendra virus (HeV) is an example of a pathogen
  • the HeV vaccine contains a protein (‘G glycoprotein’) HeV has on its outer surface that the virus uses to attach to the horse’s cells in order to gain entry

Antibodies are designed to protect against disease when the body next encounters that pathogen

  • the HeV vaccine stimulates production of ‘neutralising’ antibodies against HeV’s G glycoprotein
  • these antibodies protect the horse against HeV infection and infectivity — illness and transmission


However, the HeV vaccine causes adverse effects in some horses

  • they may react to the G glycoprotein (the core vaccine component) itself or to one or more of the additives...
  • the proprietary adjuvant (a substance added to 'goose' the immune response to the core component), which can result in an exaggerated inflammatory or allergic response
    • the manufacturer doesn't say which adjuvant(s) is used, but a research paper documenting development of a HeV soluble G protein subunit vaccine discussed these two adjuvants [1]:
    • Alhydrogen™ (aluminium hydroxide gel), which boosts the antibody-mediated immune response to the core vaccine component
    • CpG, which is a short, single-stranded, synthetic DNA molecule that is used in vaccines as an immunostimulant
    • CpG mimics the presence of pathogens such as viruses; it has a 'pathogen-associated molecular pattern' (PAMP), so the body reacts to it as it would to invaders such as viruses
    • over-reaction to such vaccines can occur in horses whose immune systems are already primed to recognise and respond vigorously to PAMPs, whether natural (virus) or synthetic (vaccine)
  • thiomersal (aka thimerosal), a mercury-containing preservative and potential allergen

[1] Broder CC, Weir DL, Reid PA. Hendra virus and Nipah virus animal vaccines. Vaccine 2016; 34: 3525–3534.


However, the HeV vaccine causes adverse effects in some horses

  • vaccine site reactions and other adverse effects are more common than reported
  • with any vaccine, adverse effects are not always recognised as such, and most vaccine reactions are not reported

a Class Action lawsuit against Zoetis (HeV vaccine manufacturer) is currently in federal court

  • it essentially claims that Zoetis overstated the risks of HeV and understated the risks of the HeV vaccine
  • potential for harm (including death) is a pervasive reason why horse owners are reluctant or refuse to have their horses receive the HeV vaccine or continue with annual boosters

There is a way for us to have our cake and eat it, too ...


A field study showed that annual boosters are unnecessary in most horses [1]

  • the protective threshold for HeV antibodies is yet to be determined
  • in an experimental vaccine-challenge study, horses with titres of at least 16 (the lowest level tested) were protected [2]
  • those horses were exposed to 2 million units of HeV (from the 2008 Redlands outbreak) via the nose and mouth
  • the authors noted that “the experimental horses were exposed to considerably higher levels of HeV than have been recovered from flying foxes...” [2]
  • it is likely that much lower titres are protective in real-world situations
  • this is the only HeV vaccine-challenge study published to date; it didn't look at titres below 16, so as these studies are really expensive, we're unlikely to have definitive answers any time soon

[1] Tan RHH, Hodge A, Klein R, et al. Virus-neutralising antibody responses in horses following vaccination with Equivac® HeV: a field study. Australian Veterinary Journal 2018; 96: 161–166.

[2] Middleton D, Pallister J, Klein R, et al. Hendra virus vaccine, a One Health approach to protectin horse, human, and environmental health. Emerging Infectious Diseases 2014; 20: 372—379.


What is a titre?

  • the process of measuring the amount of an antibody in a sample (e.g., blood or serum) involves titration, which is where ‘titre’ comes from
  • titration in this instance involves a series of 2-fold dilutions or ‘doublings’ (2, 4, 8, 16, 32, 64, and so on) up to the point at which the antibody is no longer detectable in the sample
  • this particular assay involves neutralisation of live HeV, so it is called a virus-neutralising (VN) or serum-neutralising (SN) titre
  • the higher the titre, the more dilutions it took before the antibody was no longer detected, so...
  • the higher the titre, the greater the amount of that antibody there was in the sample; and...
  • the higher the titre, the longer it takes for the titre to drop below a protective level


In the field study, most horses had titres well above the current threshold (16) after the primary immunisation series (VAC 3 in the graph below)

Most horses had titres well above the current threshold after the first dose



— average (mean) values
— range (min to max)
— range just before vaccine given
— threshold of 16


What this graph doesn’t show is how high the values actually went ...

The upper limit of the test was 8,192. Any values above this range were recorded as 8,192. How high they actually went, we don't know. The lower limit was 8. Any values below this range were recorded as 'undetectable', even though the horse may have had HeV antibodies in its blood and adequate cell-mediated immunity to protect against natural exposure to HeV.


— upper and lower limits of detection


"Under laboratory conditions, the horses with serum-neutralising antibody titres (SNT) as low as 16 were protected from infection. It is possible that protection from field exposure to virus may also occur in immunised horses with lower (or even undetectable) titres."

Tan RHH, Hodge A, Klein R, et al. Virus-neutralising antibody responses in horses following vaccination with Equivac® HeV: a field study. Australian Veterinary Journal 2018; 96: 161–166.


The first annual booster was unnecessary in most horses

Titres remained high after the first annual booster

The second annual booster was unnecessary in most horses

The second annual booster caused only a modest increase in titre compared with the increase after the first annual booster


We don’t know what happens beyond this study period, but it is likely that the primary immunisation series ± the 1st annual booster is protective for years

This study also shows us that proof of immunity is a reasonable alternative to proof of vaccination


Testing for the presence of HeV antibodies in the horse’s blood allows us to tailor HeV vaccination for optimal safety, effectiveness, and vaccine use

  • identify the horses with zero or low titres and vaccinate/booster just those individuals
  • monitor the progressive decline in titre and booster only when it nears or dips below the accepted threshold (currently 16, but likely to be less than 8)
  • identify the horses with persistently zero/low titres despite vaccination (poor or non-responders) and target them for extra HeV precautions (e.g., strict avoidance of flying foxes and sick horses)
  • these individuals are probably protected by vaccination (which also induces cell-mediated immunity), despite having low circulating antibody levels, but it’s better to be safe than sorry


It helps horse owners economise without compromising horse and human health

  • testing costs money, but so does unnecessary vaccination
  • testing avoids the expense and health consequences of adverse effects (vet visits, medications, time off from training, missed events, potential for permanent debility/death) when a booster is not needed
  • in horses at risk for vaccine reaction or other adverse effects, testing allows the vet and horse owner to weigh the risk vs benefit of vaccinating/boostering using the horse’s own data


Proof of immunity helps vets, farm managers, and event organisers ensure both the participation and safety of the horses and people in their care

  • mandating annual HeV vaccination excludes many horses and their owners from veterinary services, boarding/training facilities, and equestrian events
  • mandating annual HeV vaccination is not the only way to ensure that horses and people are protected from HeV
  • mandating annual HeV vaccination is unjustified in horses with demonstrable immunity against HeV


Currently, there are two types of HeV antibody tests:

1. Virus neutralisation (VN) test
2. Enzyme-linked immunosorbent assay (ELISA)