Hendra: why not just go ahead and vaccinate? - web series by holistic equine veterinarian, Dr Christine King

An experimental vaccine challenge study was published in 2014, in which 10 horses were exposed to 2 million units of live Hendra virus (HeV), isolated from the 2008 outbreak in Redlands, Queensland. The virus was administered to the horses via the nose and mouth, either 3 weeks or 7 months after vaccination (which consisted of 2 doses of HeV vaccine, given 3 weeks apart).

The 'control' group consisted of 4 additional horses who were not vaccinated; they were exposed to the same amount of virus.

All 10 vaccinated horses remained healthy for the 7 to 9 days after viral challenge (the length of the observation period in this study), whereas all 4 unvaccinated horses became ill between days 5 and 7 after viral challenge.

But the devil, as they say, is in the details...

Funding

This study was co-funded by the CSIRO, Zoetis, the US Department of Health and Human Services, and the US National Institutes of Health (NIH). Five of the 21 authors (24%) were affiliated with Zoetis, which in research circles means paid by Zoetis.

That's not a problem in itself. Drug and vaccine manufacturers should be the ones paying to prove the effectiveness of their products (i.e., the onus is on the company to prove that its product works as claimed). However, money can influence the outcome, with negative or nonsignificant results being buried in favour of the publication of positive results, and studies deliberately designed to ensure the desired outcome.

The burying of negative results or results that don't achieve statistical significance is a huge problem in medical research, as is the deliberate creation of study designs that practically guarantee the desired outcome. I'm not saying that either of these things happened here, but I do think it's noteworthy that Zoetis had 5 fingers in this pie. I always read research papers with the funding source in mind, and I encourage you to do the same.

But here's where it gets really interesting, and a bit weird...

One of the study authors (Christopher C Broder, PhD) invented the soluble forms of Hendra and Nipah virus G glycoproteins. As he was a US federal employee at the time (working at Uniformed Services University in Bethesda, Maryland), and still is, the patents are assigned to the US government (Department of Health and Human Services) and the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc.

Nipah virus (NiV) is so closely related to HeV that a subunit vaccine based on the G glycoprotein of one can be expected to protect against the other virus as well. Nipah virus is an important human pathogen in South and Southeast Asia, being transmitted to humans directly from bats or from infected pigs (who may be infected by bats, just as flying foxes can transmit HeV to horses). But why the US government has invested in the development of a HeV-NiV vaccine is beyond me. Perhaps it's simply to protect US military personnel serving in S/SE Asia. Or...

The US Centers for Disease Control (CDC) considers NiV a 'Category C' agent: an emerging pathogen that might be engineered for mass dissemination because of its availability, ease of production and dissemination, high mortality rate, or ability to cause a major health impact.

Holey socks, Batman! That's part of the origin story of the Hendra virus vaccine — and of this study.

Study horses

Although there were 10 horses in the vaccinated group, the subgroups contained as few as 3 horses each. The 10 vaccinated horses were divided into 7 horses who received the full dose (100 micrograms of HeV soluble G protein) and 3 horses who received a half dose (50 micrograms). And of the 7 horses who received the full dose, 4 horses were challenged with live virus 3 weeks after vaccination and the other 3 horses were challenged 7 months after vaccination.

Small number of 'subjects' (e.g., n=3) is a common problem in live-animal research, particularly equine research. Experimental studies involving live horses are very expensive to conduct, especially when the data collection period spans months. A further expense with this study is that HeV is a human pathogen with lethal potential, so the viral challenge portion of the study had to be conducted in a level 4 biosecurity facility (= $$$$). Lastly, "terminal" studies (in which the animals are killed at the end of the observation period) are generally capped at the number of animals sacrificed, particularly with domestic animal species (compared with laboratory animals such as rats and mice).

So, while I don't fault the study design in this regard, it's important to note that the conclusions of this study, and their real-world implications, are based on as few as 3 horses.

A key component to interpreting the findings of any study is how well the study group reflects the population it's meant to represent (in this study, that's horses who may, at some point, be naturally exposed to HeV).

Can 3 horses adequately represent the 500,000+ horses in Queensland and New South Wales? Or the ~ 750,000 horses in Australia (perhaps the more relevant population, given that the new HeV variant has been documented in southern states as well as in QLD and NSW)?

Three things suggest not:

1. We know nothing about the study horses. The published report does not specify their age, breed, gender, or history (although the report does suggest that they were all mares; and typically such studies are limited to adults).

Usually, the horses that end up in experimental studies such as this are rejects from the Thoroughbred or Standardbred racing industries, horses sold at general auction (including 'dogger' sales), and horses with chronic medical, behavioural, or soundness issues that are donated to research because they're not much good for anything else.

The study horses are generally kept in very basic housing, often in solitary confinement (certainly true for part of this study, for biosecurity reasons among others) or thrown together in a paddock, whether or not they all get along.

And for what it's worth, this study was conducted in Geelong, Victoria.

How well does the study group represent the general horse population in Australia, or even just in QLD and NSW? How well does it represent well loved and well cared-for horses belonging to conscientious horse owners? How well does the study protocol reflect the management and environmental conditions under which horses are naturally exposed to HeV?

How well does the study group represent immature horses (any horse less than 5 years of age), senior horses (20 years and older), pregnant or lactating mares, breeding stallions, and performance horses (i.e., the various stresses of training, competition, boarding, and travel)? How well do such studies consider the life history and immune competence of the individual horse? How well do they consider the effects of stress on immune response to vaccination?

How well does the study group represent other equid species, such as donkeys, the few mules we have in Australia, and the zebras in our zoos? What about our significant brumby (feral horse) population? What about draft breeds and the littlies (small ponies and Miniature horses)? I could go on...

The stark reality is that, other equids aside, such studies assume that a horse is a horse, and as long as it's clinically healthy (no obvious signs of illness) and not too bad to handle, it can be included (and yes, 'it' is the correct pronoun in conventional circles for any animal ... don't get me started!). But that is a very big, and often a very flawed, assumption, as we shall see...

2. There was considerable variation in HeV-specific antibody titres among the horses, even within the same subgroup (same vaccine dose, same interval between vaccination and viral challenge, same housing, same diet, etc.). I'll discuss the titres in more detail in a bit.

A titre is one way we measure or report the amount of specific antibodies, in this case antibodies against HeV, in the bloodstream. Antibodies are targeted proteins produced by the immune system in response to viruses and other pathogens (disease-causing agents). Their particular conformation is highly specific to the pathogen involved, making them good markers of a specific immune response to the particular pathogen (or to the vaccine targeting that pathogen).

Can we safely extrapolate from such limited data to the general horse population in Australia, or even just in QLD or NSW? In particular, can we safely assume than the lowest protective titre reported in this study (16–32) is indeed the lower limit of protection in the general horse population under real-world conditions of exposure? Because that's what happened on the basis of this study. However ...

3. The study didn't challenge any vaccinated horses with titres less than 16. Based on this one study, involving as few as 3 horses in a group, a HeV-specific antibody titre of 16 or 32 (depending on who you read) has been accepted as the lower limit of protection against HeV.

Acceptance of this threshold, along with "abundance of caution" thinking, has led to the current vaccination requirements (third dose 6 months after the second, and thereafter annual boosters for life).

However, a titre of 16 was simply the lowest titre challenged. We have no way of knowing from this study whether horses with a titre of 8 or 4, or even 2, are protected against natural challenge in real-world situations.

As to that (natural challenge, real-world situations)...

Viral challenge

If 2 million units of live virus sounds like a lot, you're right; it is.

The researchers even stated, "In assessing the field significance of this observation [i.e., the real-world relevance of their results], the following must be noted: the experimental horses were exposed to considerably higher levels of HeV than have been recovered from flying foxes..."

Why did they use such a large dose of virus? Probably because they needed to show that it was enough to cause illness in unvaccinated horses. This study was primarily designed for vaccine approval, so proving vaccine efficacy in the face of serious viral challenge was their main objective.

It's good to know that the vaccine is effective in preventing illness, even in the face of massive viral challenge. But...

All of the horses in this study, including those who were vaccinated, were euthanised ("put down") between days 6 and 9 post-challenge, so we don't know what would have happened beyond that point.

Would some of the unvaccinated horses have survived and recovered? Unlikely, given the massive dose of HeV delivered directly into their nose and mouth, but certainly possible. As I discussed regarding the first HeV outbreak in 1994, not all exposed horses became ill and died; some became ill but recovered, while others didn't even become ill.

Would some of the vaccinated horses have become ill at a later time? The incubation period in horses naturally infected with HeV is reported to range from 5 to 16 days.

The main objective of this study was to prove vaccine efficacy in relation to unvaccinated horses, so the study design ended data collection for all of the horses once the unvaccinated horses became seriously ill with signs of HeV infection. But the fact remains that this study doesn't prove vaccine efficacy beyond 9 days after viral challenge.

Would lower titres be protective against lesser challenge? This is a very important question, with real-world relevance. But we have no way of knowing from this study — which is the only one published to date that challenges HeV immunity using a known dose of a live HeV isolated from a natural outbreak — because they didn't look.

Viral shedding

Before I go on, I want to briefly discuss the 'viral shedding' component of this study, because it's important in the 'politics' of HeV vaccination (vaccinate horses to protect people) and the management of sick horses who are unvaccinated or whose vaccination status is overdue, lapsed, or unknown. (These horses are often considered HeV cases until proven otherwise.)

In addition to monitoring the horses' health following viral challenge, the researchers tested numerous body fluids and tissues for the presence of HeV, using PCR.

Polymerase chain reaction (PCR) is a type of lab test that is used to detect the presence of genetic material, in this case a portion of the HeV (its N gene). Note that PCR does not distinguish between live (infective) and dead virus. It simply tests for the presence of any of the genetic material specified by the particular test (e.g., HeV N gene), even if just fragments of the virus remain.

None of the vaccinated horses challenged 3 weeks after the second vaccine dose had any viral material in their body fluids (oral, nasal, or rectal swabs, urine, manure, or blood) nor in any of the tissues sampled after death (post-mortem).

One of the 3 horses challenged 7 months after vaccination had a small amount of viral genetic material (HeV N gene) detectable in her nasal swabs between 2 and 7 days after challenge. However, the authors didn't consider the amount of material significant, and the mare was negative on PCR for all other body fluids and tissues. In other words, she was not infected with HeV.

Remember that viral challenge involved administering 2 million units of live HeV directly into the horse's nose and mouth. This one horse had a small amount of HeV N gene in her nasal swabs on days 2, 3, 4, and 7, but not on days 5, 6, or 8. This is not the pattern of viral infection, but rather it may reflect delayed clearance of her nasal passages.

The authors stated, "We conclude that the level and pattern of virus replication in the 1 vaccinated horse do not meet the epidemiological criteria presently associated with transmission of infection to humans."

There's a saying in US politics: "[s/he] said the quiet part out loud," a political indiscretion in which the person inadvertently told the truth and revealed the end-game which is usually kept quiet. You or I might talk instead about letting the cat out of the bag...

The push to get more horses vaccinated against HeV, and to relentlessly booster even if the horse doesn't need it, is as much about protecting people from infected horses as it is about protecting the horses themselves, perhaps even more so. Protecting people from HeV infection is a worthy goal; I certainly don't want to be infected with HeV. But is over-vaccination of horses really the best policy we can come up with?

In the 4 unvaccinated horses, nasal swabs were PCR-positive for the HeV N gene from day 2 post-challenge onward, although the quantity of N gene varied considerably among horses. It wasn't until day 5, 6, or 7 that blood, urine, manure, and oral swabs were also positive.

In short, not only did HeV vaccination protect against illness, it prevented meaningful shedding of virus in the body fluids of all 10 vaccinated horses.

Vaccination protocol

Vaccination in this study consisted of 2 doses of HeV vaccine, given 3 weeks apart. The current vaccination protocol requires a third dose, to be given 6 months after the second, and thereafter annual boosters for life.

Note that this abbreviated vaccination protocol (2 doses instead of 3) was effective, even against massive viral challenge, 7 months later.

So, why is the third dose (the booster given 6 months after the second dose) necessary? Beats me.

It was most likely added because HeV antibody titres were lower 7 months after the second dose than they were 3 weeks after the second dose. But... well... duh! Of course they were! That's how humoral immunity works. Bodies favour economy of their limited resources, so titres gradually drop over time unless the system is challenged again, whether through natural exposure or vaccine booster, at which point titres rapidly increase again.

Bearing in mind that it would be unwise to make too much of a study involving only 3 horses, I do wonder: is the 6-month booster really necessary? All three horses challenged 7 months after their second vaccine dose remained healthy in the face of a massive dose of live virus, and none were shedding meaningful amounts of virus. So, what more would a booster 6 months after their second vaccine dose have done for them?

(More on this third dose on the next page, which discusses the field study of the HeV vaccine.)

Supported in part by this study, Zoetis added a third dose to the primary vaccination series (2 doses, 3–6 weeks apart). But based on what data?

In their 2014 application for approval to the Australian Pesticides and Veterinary Medicines Authority (APVMA), Zoetis asserted the following:

Onset of immunity: 3 weeks after primary vaccination course. (In this application document, the primary course consisted of 2 doses, 3 weeks apart. The current product leaflet advises 2 doses, 3–6 weeks apart.)

In other words, they assert that the horse has immunity 3 weeks after the second vaccine dose. That's true, in that the vaccine challenge study did indeed show both high HeV antibody titres and protection from viral challenge at this timepoint in 7 horses (4 given the full dose, 3 given the half dose).

However, the statement is misleading because it implies that it takes a full 3 weeks after the second dose for immunity to begin ("onset of immunity"). That was not supported by any published data at the time and it has since been shown to be untrue.

In the field study published in 2018 (detailed on the next page), most horses had titres after the first vaccine dose that were well above the threshold considered to be protective based on this experimental vaccine challenge study (which found a titre of 16–32 to be protective).

In the field study, the average titre after the first vaccine dose, measured just before the second dose, was 52. All of the horses enrolled in the study were unvaccinated and all had undetectable HeV antibodies (titre less than 8) at the start. The titre after the first vaccine dose ranged from less than 8 to 4,096, and almost 60% had a titre of 64 or higher. After just one dose of HeV vaccine.

However, 3 of the initial 57 horses (5%) in the field study were "nonresponders" (titre below 8 after the first vaccine dose). It wasn't until after the second dose that all of the horses had titres of 32 or higher.

So, while a second dose is advisable as a 'blanket' recommendation, and it is supported by immunological principles (priming and then reminding the system to ensure a good humoral response and to establish immunological memory), a third dose 6 months after the second for every horse is harder to justify.

Zoetis also claims the following in their application:

Duration of immunity: 6 months after primary vaccination course

The product leaflet that comes with the HeV vaccine states the following: "Duration of immunity has not been demonstrated for more than 6 months following the primary vaccination course." Elsewhere, it states that "A third dose 6 months after the second primary dose is also required." However, the two statements are not supported by data that clearly establish the need for a third dose 6 months after the second.

In fact, the vaccine challenge study showed protection, and thus presumably protective titres, 7 months after the second vaccine dose.

Why is Zoetis (and many equine vets) being so stiff-necked about the vaccination protocol required in order for a horse to be considered currently, fully, or properly vaccinated? If titres are adequate to prove immunity post-vaccination (e.g., the field study), then why are they not adequate to prove immunity in place of boosters in your horse? Couldn't the third dose be optional, based on the individual's response to the second dose? And couldn't all subsequent annual boosters likewise be optional based on individual response to the previous dose? That would follow immunological principles as well.

Perhaps we should now take a look at the actual titres reported in this vaccine challenge study, and in a 2021 real-world titre study involving privately owned horses. Because things get even more interesting...

Protective titres

In this experimental vaccine challenge study, none of the 10 vaccinated horses became ill or shed significant amounts of virus, but individual responses to vaccination were highly variable.

Blood samples were taken immediately before viral challenge and HeV-specific antibody titres were measured in duplicate for each horse (i.e., 2 samples per horse, just to double-check the first result).

In the 7 horses challenged 3 weeks after the second vaccine, individual titres ranged from 128 to over 4,096 (the upper limit of the assay, so who knows how high they actually went). That's more than a 10-fold difference among the horses.

Titres are based on serial dilutions. In this case, each successive dilution is 2x that of the one before, so the maths are a bit odd. There are 5 'doublings' between 128 and 4,096.

In the 3 horses challenged 7 months after the second vaccine, the titres were much more consistent; in all 3 horses the pre-challenge titre was 16–32. However, these 3 horses "were selected from 29 vaccinated horses in a larger field efficacy and safety study on the basis of temperament and for having the lowest serum neutralization titers in the group at the time."

In other words, these 3 horses were not randomly selected from the group, nor even deliberately selected from the mid-range of the group; they were deliberately selected from the bottom of the titre range. So, they're not likely to be representative of the titres expected in the general horse population 7 months after the second vaccine dose.

Here's where things get interesting: in a 2021 study which analysed the results of titre testing in 332 privately owned horses, the median titre in the 27 horses who had received only 1 or 2 doses of HeV vaccine was 32.

The median is a statistical contrivance; it represents the value that divides the group into equal halves, with 50% at or above it and 50% at or below it. For example, 'median income' means that 50% of people earn at least that much, and 50% of people earn only that much or less. Median and average (the 'mean' in statistical parlance) are the same in normally distributed groups, where the range of individual values forms a nice, neat bell-shaped curve. But in real-world groups, the median and average are often quite different.

That median titre (32) is a bit misleading, as it is not the same as the average. The middle 50% of the group is perhaps a more useful value: they had titres of 16–128.

For the scientists, that's the interquartile range (25th and 75th percentiles) for the horses who received only 1 or 2 vaccine doses.

But here's where it gets really interesting: the interval between their last vaccine dose and titre testing in the horses who'd received only 1 or 2 vaccine doses ranged from 1,327 to 2,393 days — or 3.6 to 6.6 years. Years! Years!!

In other words, 50% of the horses vaccinated only once or twice still had a HeV antibody titre of at least 32 (the median value) 3+ years later!

That is real-world data, collected by equine vets from client-owned horses, for the purpose of checking HeV antibody titres.

It must be noted that 3 horses (11%) had titres below 4 (the lower limit of quantitation in that study). At the other end of the spectrum, 3 horses (11%) had titres of 1,024 or more 3+ years later! After just 1 or 2 doses of the vaccine.

Now, that's impressive. I think we can state without risk of contradiction that the HeV vaccine is highly effective at eliciting a HeV-specific antibody response that is durable.

So, why is Zoetis (and many equine vets) being so stiff-necked about us all rigidly adhering to their prescribed vaccine schedule?

A generous reading is that this may simply be "abundance of caution" thinking. A conventional colleague in the US recently stated the conventional mindset rather succinctly when he asserted that "It's better to over-vaccinate than under-vaccinate."

But that statement is premised on two important conditions: (1) that there is a high risk of serious illness in "under-vaccinated" individuals, and (2) that the vaccine has a very low rate of adverse effects, particularly when it is repeated in individuals with already high titres.

There is a third consideration here: it fails to take immunological memory into account. Even when the titre falls to below a certain 'protective' threshold or below the limit of detection for that particular test, the immune system retains its memory of how to respond if it has ever encountered that pathogen before, whether through natural exposure or vaccination.

Is it really better to over-vaccinate than to under-vaccinate? Perhaps in some circumstances and in some individuals, but not in all.

Dose

Here's another interesting bit of data from the experimental vaccine challenge study: even the half dose of HeV vaccine (50 micrograms instead of 100 micrograms per dose) protected against illness and prevented viral shedding in body fluids following viral challenge 3 weeks after vaccination.

Of the 4 horses who received the full dose of vaccine and were challenged 3 weeks after the second dose, 2 horses had titres of 512–1,024 and the other 2 horses had titres of more than 4,096.

Of the 3 horses who received the half dose, 1 horse had titres of over 4,096 (remember that the titres were run in duplicate, so each horse had 2 titre values). Another had titres of 2,048–4,096; and the third horse had titres of 128–256. None of these horses became ill or had detectable virus in any of their body fluids or tissues after challenge. Alas, we don't know what might have happened if these horses had been challenged 7 months after vaccination.

This experimental study used a huge dose of HeV to 'challenge' the vaccine. It's good to know that even half the recommended dose protected the horses from illness and prevented them from shedding HeV.

But, why are we using the higher vaccine dose (100 micrograms/dose)? Would half that amount be better tolerated by more horses without compromising their immunity to HeV in real-world situations?

While we're at it, wouldn't an intranasal HeV vaccine provide better protection, be better tolerated by horses (fewer and milder vaccine reactions), and perhaps be better accepted by horse owners, than the injectable vaccine? After all, the nose and mouth are the natural routes of HeV infection, the virus' first port of call. Intranasal vaccines are used in the US to prevent equine influenza and strangles (a bacterial infection caused by Streptococcus equi). They provide superior protection compared with the traditional intramuscular vaccines because they target the local immune response in the upper airways, which is the first line of defense against respiratory pathogens such as influenza virus, Strep. equi, and HeV.

Further thoughts

In the absence of any further viral challenge, antibody titres decline fairly predictably over time, so the higher the starting point, the longer protective levels of antibody persist in the bloodstream. This is a key aspect to duration of immunity, which determines the recommended booster interval for a particular vaccine. (More on that with the next study.)

It's to be expected that titres would be lower several months after vaccination than those measured just 3 weeks after vaccination. However, this small study showed that a titre of 16–32 was protective against illness in the face of massive challenge with HeV 7 months after the second dose, at least in these 3 horses.

Would lower titres have been equally protective? We don't know, as 16 was the lowest titre challenged in this study. It is entirely possible that horses with lower titres who are challenged with more realistic amounts of virus would likewise be protected from serious illness.

A titre of 16–32 also prevented viral shedding in 2 of the 3 horses challenged 7 months after vaccination, and limited viral shedding to an insignificant amount in the third horse. In fact, it's not even accurate to call it "shedding" when a small amount of HeV N gene was found on a nasal swab — not in nasal discharge dripping from the horse's nose or on the side of the feed tub or water bucket; on a cotton swab wiped across her nasal membranes.

It would be good to know how the horses' antibody titres changed in the week or so after viral challenge. Unfortunately, no such data were collected in this study.

In the 2018 study of diagnostic methods I mentioned in relation to the development of naturally acquired immunity (all samples obtained before a HeV vaccine was available), 9 of the 11 horses with two or more blood samples showed an increase in HeV antibody titre between paired samples taken about 1 week apart.

The tenth horse showed a decrease (from > 32 to 16) and the eleventh horse had persistently high titres (2,048 in 4 samples taken over 2 weeks).

We don't know where any of these horses were in the timeline between viral exposure, infection, and recovery, but in 10 of these horses it's a fairly safe bet that they were mounting an active immune response to current viral challenge.

Duration of immunity is about more than maintenance of high antibody titres; it's also about cell-mediated immunity, which is broader and both more immediate and enduring than antibody production, yet it was not evaluated in this study.

Here is what the researchers who conducted the field study had to say about this experimental vaccine challenge study:

"Under laboratory conditions [i.e., this experimental vaccine challenge study], 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. The reasons for this include the rapid time-frame over which extensive mucosal [oral and nasal] exposure to infective fluid occurs under experimental conditions and the fact that protection will depend upon the development of an anamnestic [cell memory] response, in addition to pre-existing antibody levels."

Read on...

Last updated 30 May 2022.

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