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by J M Whalley, C E Foote, S Raidal
September 2005
RIRDC Publication No 05/083 RIRDC Project No UMA 17A
Executive Summary
Equine herpesvirus 1 (EHV-1)
causes respiratory disease, abortion, neonatal deaths and myeloencephalitis
in horse populations worldwide. Extensive research has been directed at
developing satisfactory vaccines to EHV-1. These have been based mainly
on live or inactivated whole virus and several have been shown to reduce
the severity of respiratory disease and/or the risk of abortion. However
difficulties in achieving high levels of immunity in the field have been
illustrated by a survey of the antibody response to vaccination on a large
Thoroughbred stud farm when less than 50% of foals and less than 30% of
mares could be classified as responders to vaccination. Furthermore, we
have recently demonstrated by PCR that EHV-1 and EHV-4 continue to infect
foals as young as 11 days of age even when most of the mares on the farm
had been vaccinated. Envelope glycoproteins of EHV-1 have been shown to
play key roles in entry of the virus into cells, and to be strong targets
for the equine immune system. Therefore they are considered to be prime
candidates for potential new vaccines based on virus subunits or components.
Our group had previously shown that one of these glycoproteins, EHV-1 glycoprotein D (EHV-1 gD) could invoke antibody responses in adult horses, including neutralising antibody similar to those of a whole virus vaccine. However there had been no testing in mares or foals, and no experimental challenge to assess the vaccine potential of EHV-1 gD. At the same time we had available another key envelope glycoprotein, gB, which we considered also to be worth testing as a vaccine component.
Therefore the project had
the following aims:
Results
1. Immunogenicity of
envelope glycoproteins
Envelope glycoproteins D
and B of EHV-1 were previously cloned into recombinant baculoviruses which
allowed the correctly expressed glycoproteins to be produced in suspension
in insect cell culture. The glycoproteins were combined with the adjuvant
Iscomatrix ™ either singly (EHV-1 gDr or EHV-1 gB) together (EHV-1 gDBr).
The immunogenicity of these formulations at various doses was assessed
in adult horses, pregnant mares and very young foals, by measuring antibody
responses.
Inoculation of pregnant mares with EHV-1 gDr elicited a specific neutralizing antibody response, and foals born from these mares had greater levels of antibody than foals out of unvaccinated mares.
Inoculation of EHV-1 gBr also induced specific ELISA antibody responses in all horses tested. This provided the parameters for inoculations of mares and foals for subsequent challenge experiments.
2. Experimental challenge
model
An EHV-1 respiratory infection
challenge model was established at Murdoch University, Perth, WA.
Foals aged between two and four months of age were inoculated intranasally with an Australian strain of EHV-1 (HVS25A) and monitored or sampled over the subsequent four weeks. This is the first such experimental challenge using this virus. Clinical signs were observed in all foals from between two and four days post-challenge. Elevation in EHV-1 antibody was observed and EHV-1 DNA (indicating the presence of virus) was detected in nasal swabs from day three and in peripheral blood from day eight. All foals fully recovered by the conclusion of the sampling period. Later in the project similar protocols were used in EHV-1 challenge of mares post foaling. The parameters established provide a basis for subsequent testing of any vaccine for EHV-1 by experimental challenge of vaccinated horses.
3. Evaluation of subunit
vaccine candidates
Inoculation of weanling
foals with EHV-1 gDr and EHV-1 challenge
Thirteen foals were born from mares purchased for the experiment at Murdoch University. Foals were divided into two groups each of which was inoculated with two doses of the subunit formulation EHV-1 gDr at approximately four and five months of age. Following experimental challenge of the first group of foals at six months of age with EHV-1, there was a slight reduction in the number days of virus excretion as shown by analysis of nasal swab samples by PCR. Of the six foals in this group, the three vaccinated foals shed for 8 days, while the unvaccinated foals shed for 10, 11 and 12 days. Both vaccinated and control groups showed clinical signs, these results indicating that under the experimental conditions used the formulation did not prevent initial infection. The second group of foals were resistant to infectious challenge, and analysis of the serological and DNA data suggested that this was probably due to a recent prior infection with EHV- 1. This could have been from reactivation of latent virus in one of the mares. Although gD is probably the strongest elicitor of neutralising antibody, the EHV-1 gDr alone appeared not to prevent infection under these conditions. Cell-mediated immunity is likely to have a critical role in protection against herpesviruses. In earlier experiments following inoculation of mice with EHV-1 gB, low levels of neutralizing antibody combined with a level of protection suggested that cellmediated immunity may be contributing significantly to the protection conferred. Therefore a combination of EHV-1 gB and EHV-1 gD may target both arms of the equine immune system.
Accordingly with limited seasonal time in which to carry out experiments, EHV-1 gBr was incorporated into subsequent challenge experiments.
Inoculation with EHV-1 gDBr in mares and very young foals, and EHV-1 challenge In this study, mares and very young foals were inoculated with the combined formulation containing gD and gB (EHV-1 gDBr). Fourteen mares and foals were divided into 3 groups. Ten mares were inoculated at month 10 of gestation, while the remaining 4 mares remained as uninoculated controls.
At foaling, of the 10 inoculated mares, half of the newborn foals were also inoculated with EHV-1 gDBr and received a booster inoculation at 30 days of age. The remaining 5 foals born from inoculated mares remained uninoculated. All mares and foals were challenged with EHV-1 virus at approximately 60 days post partum. After experimental challenge, there was an expected increase in EHV-1 gG ELISA absorbance from around day 7 in mares and day 13 in foals, consistent with infection. However, mares inoculated with EHV-1 gDBr shed virus in nasal secretions for significantly fewer days compared to uninoculated mares. Foals inoculated with EHV-1 gDBr and born from inoculated mares shed virus for significantly fewer days than uninoculated foals born from both inoculated and uninoculated mares.
Challenge experiment in
weanling foals
In attempting to mimic the
on-farm situation, foals were retained from the previous challenge experiment.
Some were re-inoculated with EHV-1 gDBr, and subsequently weaned and all
were rechallenged with EHV-1. A slight reduction in number of times EHV-1
was detected in nasal swab samples and a slight decrease in severity of
nasal discharge in inoculated foals compared to uninoculated foals was
observed, but the differences were not statistically significant, probably
due to the relatively recent infection of the original challenge.
Outcomes and Implications
Outcomes
Implications
Publications arising
to-date
Related relevant publications
Equine Veterinary Journal
36, 341-345
Conference proceedings
and presentations
