evaluating equine immune responses to new ehv-1 vaccine … · ehv-1 dna in nasal swab samples from...

Evaluating Equine Immune Responses to New EHV-1 Vaccine Candidates A report for the Rural Industries Research and Development Corporation by J M Whalley, C E Foote, S Raidal September 2005 RIRDC Publication No 05/083 RIRDC Project No UMA 17A

ii

© 2005 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 146 1 ISSN 1440-6845 Evaluating equine immune responses to new EHV-1 vaccine candidates Publication No. 05/083 Project No. UMA-17A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details A/Prof J. M. Whalley Department of Biological Sciences Macquarie Univerisity, Sydney 2109 Phone: (02) 9850 8200 Fax: (02) 9850 8245 Email: [email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4819 Fax: 02 6272 5877 Email: [email protected]. Website: http://www.rirdc.gov.au Published in September 2005 Printed on environmentally friendly paper by Canprint

iii

Foreword The RIRDC Horse Research and Development Program has been consolidated into two main areas, one of which is Disease Prevention, Diagnosis and Treatment. The objectives and research of this project are consistent with, and contribute to, these overall strategies for the equine industry. The serious diseases and impact of equine herpesvirus-1 (EHV-1) on reproductive efficiency and respiratory health in horse populations have made the control of this virus one of the major goals of equine infectious disease research throughout the world. Despite the widespread use of whole virus vaccines, both equine herpesviruses 1 and 4 (EHV-1 and EHV-4) continue to circulate in vaccinated populations of horses. In some cases, foals as young as 11 days of age are being infected from their dams who have been previously vaccinated. This report describes development towards potential new vaccines for EHV-1. Firstly the immunogenicity of a formulation containing EHV-1 envelope glycoproteins was tested by measuring antibody responses of mares and young foals. Secondly, in order to provide a system whereby these and other vaccines could be assessed, an experimental EHV-1 respiratory challenge model of infection was established in mares and foals, using an Australian isolate of EHV-1. This model was then used to determine whether the immune responses to the glycoprotein formulation could provide protection against EHV-1 infection. This project was funded by RIRDC from industry revenue which is matched by funds provided by the Australian Government This report, an addition to RIRDC’s diverse range of over 1500 research publications, forms part of our Equine R&D program, which aims to assist in developing the Australian horse industry and enhancing its export potential. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop Peter O’Brien Managing Director Rural Industries Research and Development Corporation

iv

Acknowledgments The authors gratefully acknowledge the following: Rural Industries Research and Development Corporation (Horse R &D), NSW Racing Research Fund, The University of Sydney and Macquarie University, Australian Research Council, and CSL Animal Health (Pfizer Animal Health) for their financial support; and the managers, veterinarians and staff at participating farms for ongoing support and assistance. We thank Gordana Pecenpetelovska for real-time PCR determinations, Dr James Gilkerson for assistance with early immunogenicity studies, Professor Michael Studdert and Dr Carol Hartley for gG ELISA antigen, and Dr George Allen for monoclonal antibodies. The co-authors of this report are Dr Caroline Foote, Department of Biological Sciences Macquarie University, Sydney, and Dr Sharanne Raidal, School of Veterinary and Biomedical Science, Murdoch University, WA We wish to acknowledge the outstanding contributions to this work from our late colleague and friend Daria Love, of the University of Sydney, who was a joint Principal Investigator on the original proposal. Without her remarkable insights and energy much of this research could not have been achieved, both in this and in previous RIRDC-supported projects. We are forever grateful that she was part of our team.

v

Contents Foreword ................................................................................................................................................ iii Acknowledgments .................................................................................................................................. iv Executive Summary .............................................................................................................................. vii

Background ....................................................................................................................................... vii Results ............................................................................................................................................... vii Outcomes and Implications ................................................................................................................ ix Publications arising to-date ................................................................................................................ ix Conference proceedings and presentations ......................................................................................... x

1. Introduction ......................................................................................................................................... 1 Epidemiology of EHV-1 ..................................................................................................................... 1 Current vaccination against EHV-1..................................................................................................... 2 Envelope glycoproteins as vaccine candidates.................................................................................... 2

2. Objectives............................................................................................................................................ 4 1. Immunogenicity of EHV-I glycoprotein formulations.................................................................... 4 2. Development of experimental challenge model .............................................................................. 4 3. Subunit vaccine assessment............................................................................................................ 4

3. Methodology ....................................................................................................................................... 5 Study designs....................................................................................................................................... 5 Preparations for inoculation ................................................................................................................ 5 Sample collection ................................................................................................................................ 5 Challenge virus.................................................................................................................................... 5 Analysis of samples............................................................................................................................. 6 1. Immunogenicity of EHV-I glycoprotein formulations.................................................................... 7 2. Development of experimental challenge model ............................................................................ 12 3. Subunit vaccine assessment........................................................................................................... 16

5. Discussion of Results ........................................................................................................................ 30 6. Implications and Recommendations ................................................................................................. 32 7. References ......................................................................................................................................... 33

vi

Figures Fig 1. Cycle of Silent Infection By EHV-1 ............................................................................................. 1 Fig 2. PCR products ................................................................................................................................ 2 Fig 3. gD ELISA (a and b) and neutralizing antibody (VN) (c and d) responses of mares and foals (

= VN titre > 128)............................................................................................................................. 9 Fig 4. EHV-1 gD ELISA (a) and neutralizing antibody titres (b) of individual horses inoculated with

2 x 106 cells EHV-1 gDr ( VN titre = 178)............................................................................... 11 Fig 5. EHV-1 gB ELISA antibody absorbances of individual horses inoculated with 2 x 106 cells

EHV-1 gBr .................................................................................................................................... 11 Fig 6. Rectal temperatures of foals during the challenge period. Foals 1 – 3 received the higher dose

of EHV-1 virus, foals 4 and 5 received the lower dose................................................................. 14 Fig 7. EHV-1 gG ELISA absorbances of foals during the challenge period........................................ 15 Fig 8. EHV-1 gD (a and b) and gB (c and d) ELISA absorbances of individual mares and foals

following inoculation. ................................................................................................................... 20 Fig 9. EHV-1 gG ELISA absorbances of mares (a) and foals (b) following challenge ....................... 21 Fig 10. EHV-1 gD (a) and EHV-1 gB (b) ELISA data of individual foals pre-inoculation, at weaning

(2 weeks post-inoculation) and challenge (3 weeks post-inoculation).......................................... 27 Fig 11. Mean EHV-1 gG ELISA absorbances of groups of foals prior to and following experimental

challenge........................................................................................................................................ 27

Tables Table 1. Inoculation (In) and sample collection (S) schedule for mare and foal study.......................... 7 Table 2. Mean EHV-1 gD, EHV-1 gG and EHV-4 gG ELISA absorbances in mare and foal study .... 8 Table 3. Doses of inoculum for the EHV-1 gDr and gBr immunogenicity studies (cells = recombinant

baculovirus-infected cells) ............................................................................................................ 10 Table 4. EHV-1 positive nasal swab (NS) and PBMC samples from all foals. Foals 1 – 3 received

approximately 4 x 107 pfu/ml EHV-1; foals 4 and 5 received approximately 1.34 x 107 pfu/ml .. 12 Table 5. Clinical signs of EHV-1 infection recorded for each foal during the study period............... 13 Table 6. EHV-1 detected in nasal swab samples from inoculated and uninoculated foals in Group 1 16 Table 7. Inoculation (In) and sample collection (S) schedule for mare and foal challenge study........ 17 Table 8. Sampling schedule for EHV-1 challenge experiments............................................................ 18 Table 9. Scores allocated to clinical signs of infection ........................................................................ 18 Table 10. Mean EHV-1 gD, gB and gG ELISA absorbances of mares and foals prior to challenge... 19 Table 11. Detection of EHV-1 in nasal swab samples and PBMCs...................................................... 22 Table 12. EHV-1 DNA in nasal swab samples from individual mares and foals on each day post-

challenge. If a sample on either the morning or afternoon sampling was positive that day is highlighted..................................................................................................................................... 23

Table 13. Mare and foal study: clinical sign data.................................................................................. 24 Table 14. History of foals and design of weanling study ..................................................................... 25 Table 15. Mean EHV-1 gD, gB and gG ELISA absorbances of foals pre-inoculation, and at weaning

and challenge................................................................................................................................. 26 Table 16. Detection of EHV-1 in nasal swab samples and peripheral blood mononuclear cells .......... 28 Table 17. Weanling study: clinical signs data ...................................................................................... 28

vii

Executive Summary Background 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:

1. To test the immunogenicity of EHV-1 envelope glycoproteins D and B in mares and young foals

2. To develop an experimental challenge protocol in Australia for assessment of equine herpesvirus 1 (EHV-1) vaccines, including EHV-1 glycoprotein formulations

3. To use the challenge protocol to evaluate immune responses and the ability of subunit vaccine candidates to protect against EHV-1 infection

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

viii

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 cell-mediated 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 re-challenged 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.

ix

Outcomes and Implications Outcomes

• An experimental challenge model in horses along with suitable protocols and parameters were established using an Australian strain of equine herpesvirus 1

• A trial vaccine formulation based on the EHV-1 envelope glycoproteins B and D induced strong antibody responses in mares, and reduced the number of days of virus shedding following respiratory challenge

• Although no antibody responses were observed in very young foals, nonetheless these animals showed reduced amount of virus shedding following challenge

• A real-time PCR test was developed for EHV-1 (and EHV-4) DNA. This can be used to detect these viruses both in challenge experiments and in clinical samples

Implications • The development of the experimental EHV-1 challenge model in Australia is a useful adjunct

for independent assessment of EHV-1 vaccines • The results suggest that it is indeed possible to induce partial protection in very young foals

through vaccination, and while the inoculation of the EHV-1 glycoproteins did not prevent infection, it did reduce the amount of virus shed with the potential to thereby reduce the prevalence of infection

• A modified vaccine strategy is suggested where the pregnant mare and the young foal is the main target for vaccination

• Protective immune responses to the subunit formulation could be enhanced by the inclusion of components which have been identified as stimulating cell-mediated immunity in the form of cytotoxic T-cells

Publications arising to-date

• Foote, C. E., Raidal, S. L., Pecenpetelovska, G., Wellington, J.E., Whalley, J.M. Inoculation of mares and very young foals with EHV-1 glycoproteins D and B reduces virus shedding following respiratory challenge with EHV-1. Manuscript submitted to Veterinary Immunology and Immunopathology

• Foote, C. E., Love, D. N., Gilkerson, J. R., Rota, J., Trevor-Jones, P., Ruitenberg, K. M., Wellington, J. E., Whalley, J. M. Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, including pregnant mares and young foals. Veterinary Immunology and Immunopathology, in press

Related relevant publications • Foote, C. E., Love, D. N., Gilkerson, J. R., Whalley, J. M. (2004). Detection of EHV-1 and

EHV-4 DNA in unweaned Thoroughbred foals from vaccinated mares on a large stud farm. Equine Veterinary Journal 36, 341-345

• Soboll, G., Whalley, J.M., Koen, M.T., Allen, G.P., Fraser, D.G., Macklin, M.D., Swain, W.F., Lunn, D.P., 2003. Identification of equine herpesvirus-1 antigens recognized by cytotoxic T lymphocytes. Journal of General Virology. 84, 2625-2634.

• Foote, C. E., Gilkerson, J. R., Whalley, J. M., Love, D. N. (2003). Seroprevalence of equine herpesvirus 1 in mares and foals on a large Hunter Valley stud farm in years pre- and post- vaccination. Australian Veterinary Journal 81, 283-288.

• Foote, C. E., Love, D. N., Gilkerson, J. R., & Whalley, J. M. (2002). Serological responses of mares and weanlings following vaccination with an inactivated whole virus equine herpesvirus 1 and equine herpesvirus 4 vaccine Veterinary Microbiology 88, 13-25.

x

Conference proceedings and presentations

• ’Antibody responses in horses to glycoprotein D of EHV-1’ JM Whalley, CE Foote, JR Gilkerson, S Raidal, and JE Wellington. 29th International Herpesvirus Workshop Reno USA 2004.

• ’Detection of EHV-1 and EHV-4 DNA in unweaned Thoroughbred foals from vaccinated mares on a large stud farm’. CE Foote, DN Love, JM Whalley British Equine Veterinary Association Congress, Birmingham, England, 2003.

• ’Serological responses of mares and weanlings following vaccination with an inactivated whole virus equine herpesvirus 1 and equine herpesvirus 4 vaccine’. CE Foote, DN Love, JM 24th Bain-Fallon Memorial Lectures, 2002

1

1. Introduction

Epidemiology of EHV-1 EHV-1 is a major cause of epidemic abortion, perinatal mortality, respiratory disease and occasionally neurological signs in horse populations throughout the world (Allen and Bryans 1986; Crabb and Studdert 1995). Abortion is the most economically crippling outcome of EHV-1 infection with loss of clients and large insurance costs. Neurological disease can affect horses of all ages including unweaned foals, and often results in the horse requiring euthanasia. Respiratory illness caused by EHV-1 and the closely related EHV-4 adversely affects racing performance and predisposes young horses to other respiratory pathogens, including strangles and rattles. Natural infection with EHV-1 and EHV-4 does not generally provide a sufficiently high level of long-term immunity to consistently protect against subsequent EHV disease. In common with other herpesviruses, EHV-1 and EHV-4 can establish a lifelong latent infection in their host (Baxi et al., 1995; Borchers et al., 1999) and horses may experience reactivation of disease throughout their lives. EHV-1 and EHV-4 also carry genes that may assist the virus to evade host immune mechanisms (Huemer et al., 1995; Hannant et al., 1999). Prior to the introduction of routine vaccination in Australia, a study of foals was conducted on a Thoroughbred stud farm in the Hunter Valley of New South Wales (Gilkerson et al., 1997). Strong serological evidence of infection of foals as young as 30 days of age was reported with a maximum incidence of EHV-1 infection at approximately three months of age. An earlier study of weanling foals on two Thoroughbred stud farms also provided epidemiological data to indicate that foals were experiencing EHV-1 infection both before and after weaning, many at less than 5 months of age (Gilkerson et al., 1998). The most likely source of infection prior to weaning was a reactivation of latent virus in a mare or mares with subsequent spread to susceptible mares and foals. These studies led us to the construction of a schematic cycle of endemic EHV-1 infection (Fig 1) (Gilkerson et al., 1999), with mares, unweaned foals and recent weanlings as the source of virus for each new crop of foals. Subsequent research indicated that a similar cycle exists for EHV-4 (Foote et al., 2004).

Fig 1. Cycle of Silent Infection By EHV-1

August

Sept

Jan

Feb

Nov

Oct

Mar

April

May

June

July

Mare to foal spread

(pre-weaning)

Foal to foal spread

(pre-weaning)

Foal to foal spread

(post-weaning)

Foals first infected

with EHV-1 30 - 120 days

Peak incidence of new cases in

December

Peak incidence of new cases in

April

Dec

Prevalence ofantibody positive foals

decreases

Fig 1. Cycle of Silent Infection By EHV-1

2

A: 68 d B: 19 d C: 19 d

A B C

Fig 2. PCR products: A - C : foals; numbers = days of age (d) 1, 4 -EHV-1 and -4 PCR standards

1 4

Subsequent to the introduction of the whole EHV-1/4 vaccine (Duvaxyn™) in 1997, we carried out further epidemiological studies. These demonstrated a continuation of the cycle of infection with serological evidence in mares and foals (Foote et al., 2003). Furthermore the detection of EHV-1 and EHV-4 DNA in nasal secretions by PCR (Fig 2) showed 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 (Foote et al., 2004). This was the first direct evidence of infection in this age group of foals in their natural environment and has serious implications for vaccine design and administration. Unless foals are protected against EHV-1 or EHV-4 infection at a very young age, they will become latently-infected carriers and therefore a potential source of outbreaks of EHV-1 and EHV-4 disease later in life. Fig 2. PCR products

Current vaccination against EHV-1 Extensive efforts have been directed at developing vaccines to EHV-1, and the substantial numbers of commercial vaccines (listed in Allen, 2002) have been either live attenuated, or inactivated whole virus preparations. Several of these have been shown to reduce the severity of respiratory disease and/or the risk of abortion, including DuvaxynTM, which is based on a combination of inactivated EHV-1 and EHV-4 (Heldens et al., 2001). However, our research indicates that less than 50% of foals and less than 30% of mares on a large stud farm could be classified as responders to vaccination (Foote et al., 2002). This vaccine is recommended not to be administered prior to five months of age, consistent with the reported interference of maternal antibody inhibiting responses to the vaccination (Wilson and Rossdale, 1998). Mares are recommended to be vaccinated at month 5, 7 and 9 of gestation. Envelope glycoproteins as vaccine candidates Immunological protection against EHV-1 disease is expected to require appropriate stimulation of multiple arms of the equine immune system, including production of EHV-1 specific antibodies in serum and on mucosal surfaces that can neutralize free virus, as well as induction of cytotoxic T lymphocytes to destroy EHV-1 infected cells (Allen et al., 1999). Extensive efforts are now underway to identify viral components that can provide such targeted immune responses. In view of their role in virus entry and as targets for immune responses in other herpesviruses, the envelope glycoproteins of EHV-1 have been investigated mainly using murine models of EHV-1 respiratory disease (Tewari et al., 1994; Osterrieder et al., 1995; Stokes et al., 1997; Kukreja et al., 1998; Packiarajah et al., 1998; Zhang et al., 1998) and EHV-1 abortion (Walker et al., 2000). Among these, EHV-1 glycoprotein D (gD) and glycoprotein B (gB) are required for virus infectivity (Csellner et al., 2000; Neubauer et al., 1997), are conserved across all EHV-1 isolates so far characterized and are involved in virus entry and cell-to-cell fusion. Inoculation of mice with EHV-1 gD expressed by a recombinant baculovirus (Love et al., 1993) led to rapid clearance of challenge virus, in association with high levels of ELISA and virus-neutralizing antibody. In addition to the serological response, cell-mediated responses also played an important role in protection in the mouse model (Tewari et al., 1994). Recombinant EHV-1 gD induced delayed-type hypersensitivity and lymphoproliferation to EHV-1 antigen. A protective role for T-cells was indicated by adoptive transfer of spleen cells from gD-immunized donors to recipients that

3

were challenged with EHV-1. DNA expressing EHV-1 gD also induced protective responses in mice (Ruitenberg et al., 1999). Mice inoculated with EHV-1 gD DNA followed by recombinant baculovirus EHV-1 gD (prime–boost) had significantly higher neutralizing antibody titres and a faster clearance of challenge EHV-1 than mice inoculated with either recombinant protein or DNA alone (Ruitenberg et al., 2000a). EHV-1 gD has been shown also to be one of several EHV-1 proteins to provide a target for equine cytotoxic T lymphocytes in some ponies (Soboll et al., 2003). Since antibody to recombinant EHV-1 gD also neutralizes EHV-4 (Love et al., 1993) it is likely that protective responses induced by recombinant EHV-1 gD against EHV-1 will also act against EHV-4. Vaccination of mice with baculovirus-expressed EHV-1 gB induced high levels of ELISA antibody but low levels of neutralizing antibody (Packiarajah et al., 1998). Upon challenge with EHV-1, mice had reduced clinical signs, rapid clearance of virus from the lungs and reduction in herpesvirus-induced pathology. Low levels of neutralizing antibody combined with the level of protection in mice suggested that cell mediated immunity to gB may have contributed to the protection conferred. Hence EHV-1 gB is also a potential vaccine candidate. There have been only a few reports of testing recombinant glycoproteins in the horse. Audonnet et al. (1999) reported that inoculation of horses with a canarypoxvirus expressing glycoproteins B, C and D of EHV-1 Kentucky D strain led to a reduction in virus shedding after experimental challenge and we have previously demonstrated that over fifty percent of horses inoculated with EHV-1 gD DNA developed increased ELISA antibody and EHV-1 neutralizing antibodies (Ruitenberg et al., 2000b). In research supported in part by RIRDC (project UMA 15A) we showed that intramuscular inoculation of EHV-1 gD produced by a recombinant baculovirus and formulated with the adjuvant IscomatrixTM elicited virus-neutralizing antibody and gD-specific ELISA antibody in the serum of over 90% of adult mixed breed horses (Foote et al., in press). The virus-neutralizing antibody responses to EHV-1 gD were similar to those observed after inoculation with a commercially available killed EHV-1/EHV-4 whole virus vaccine. Intramuscular inoculation of EHV-1 gD DNA encoded in a mammalian expression vector was less effective in inducing antibody responses when administered as the sole immunogen, but inoculation with EHV-1 gD DNA followed by recombinant EHV-1 gD induced increased gD ELISA and virus-neutralizing antibody titres in six out of seven horses. However, these titres were not higher than those induced by either EHV-1 gD or the whole virus vaccine. The ability of EHV-1 gD to evoke comparable neutralizing antibody responses in horses to those of a whole virus vaccine confirms EHV-1 gD as a promising candidate for inclusion in subunit vaccines against EHV-1, and provided the rationale for the current project. Here we describe experiments in pregnant mares and young foals that investigated the vaccine potential of EHV-1 gD (EHV-1 gDr) and EHV-1 gB (EHV-1 gBr) produced in insect cells by recombinant baculoviruses (Love et al., 1993; Munro et al., 1999) and delivered with the ISCOM-related adjuvant IscomatrixTM (Cox and Coulter, 1997). Following investigations of the immune responses to EHV-1 gDr in, pregnant mares and young foals, EHV-1 gBr was tested in adult horses and then combined with EHV-1 gDr as a bivalent subunit inoculum (referred to as EHV-1 gDBr). This inoculum was tested in mares, very young foals and weanling foals in experimental inoculation/challenge experiments.

4

2. Objectives

The overall aim was to further investigate the vaccine potential of EHV-1 envelope subunit proteins and at the same time to consider modified vaccine strategies. A series of experiments were carried out to examine the immunogenicity of recombinant EHV-1 proteins, in mares and foals. Parameters for an EHV-1 challenge model were established and a selected formulation of recombinant proteins was tested for protective capabilities against EHV-1 infection, in foals and in mares. Therefore the project had the following experimental objectives:

4. To test the immunogenicity of EHV-1 envelope glycoproteins D and B in mares and young foals

5. To develop an experimental challenge protocol in Australia for assessment of equine herpesvirus 1 (EHV-1) vaccines, including EHV-1 glycoprotein formulations

6. To use the challenge protocol to evaluate the ability of subunit vaccine candidates to protect against EHV-1 infection in mares and foals

These aims were addressed within the following studies: 1. Immunogenicity of EHV-I glycoprotein formulations (a) EHV-1 gDr in pregnant mares and very young foals We investigated antibody responses of mares inoculated late in gestation with the EHV-1 glycoprotein formulation EHV-1 gDr and measured maternal antibody levels acquired by their foals compared with foals from uninoculated mares. All foals were also inoculated at 12 h and 30 days of age to assess whether the EHV-1 gDr could elicit detectable antibody responses in such young foals. This study demonstrated the safe inoculation of pregnant mares and very young foals. (b) EHV-1 gBr Although we had tested the immunogenicity of EHV-1 gDr this had not been done for EHV-1 gBr. Here we investigated whether recombinant baculovirus-expressed EHV-1 gB (EHV-1 gBr) would elicit antibody responses in the horse. This also allowed an estimation of dose for vaccination studies. 2. Development of experimental challenge model In order to establish that we could infect horses with EHV-1 isolate HVS25A, foals were inoculated intranasally, and monitored and sampled for a period of several weeks. Parameters measured included a number of clinical signs and EHV-1 DNA in nasal secretions and blood. This formed the basis for challenge experiments of both foals and mares in assessment of the EHV-1 gDBr vaccine formulation. 3. Subunit vaccine assessment (a) Inoculation of weanling foals with EHV-1 gDr and experimental challenge In an initial study to determine efficacy of EHV-1 gD as a single vaccine antigen, foals were inoculated with EHV-1 gDr, and later challenged with EHV-1. Foals were then monitored using the parameters established. (b) Inoculation with bivalent EHV-1 gDBr and experimental EHV-1 challenge Mares and very young foals: Arising from the observations of antibody responses and maternal antibody indicated above, this study aimed to assess whether strategic inoculation of pregnant mares and very young foals with the dual subunit formulation EHV-1 gDBr provided protection against EHV-1 challenge. Weanling foals: To determine if EHV-1 gDBr offers protection against experimental challenge of weanling foals, we attempted to mimic the on-farm situation. Foals from the previous challenge experiment were retained, some were re-inoculated with EHV-1 gDBr, and subsequently weaned and all foals were re-challenged with EHV-1.

5

3. Methodology Study designs The detailed design and numbers of animals used in groups are given for each study in the Results section. Prior to each study the EHV-1 antibody status of each horse was determined using the gG ELISA assay (Crabb et al., 1995). Horses defined as EHV-1 ELISA antibody positive (Gilkerson et al., 1997) were distributed evenly between treatment groups. The EHV-1 and EHV-4 antibody status of each horse was also monitored over the period of each study to determine whether there was any occurrence of natural infection during this time. Preparations for inoculation EHV-1 gD (EHV-1 gDr) Sf9 insect cells were infected in a medium-scale fermenter (BioTech Australia, Sydney) with a recombinant baculovirus expressing the open reading frame of EHV-1 gD including the C-terminal anchor sequence (Love et al., 1993). Infected cells were harvested, washed with phosphate-buffered saline (PBS) and stored at -20°C prior to use. The infected cell pellet was suspended in PBS in the presence of leupeptin protease inhibitor and treated with beta-propiolactone to inactivate baculovirus. Prior to i. m. inoculation infected cells were mixed with the adjuvant Iscomatrix™ (CSL Limited, Australia) in PBS. EHV-1 gB (EHV-1 gBr) Sf9 insect cells were infected as above with a recombinant baculovirus expressing the open reading frame of EHV-1 gB (Munro et al., 1999) and inoculum was prepared as described for EHV-1 gDr. EHV-1 gD/gB (EHV-1 gDBr) EHV-1 gDr and gBr were combined and mixed with Iscomatrix™ in PBS. Sample collection Serum Blood samples were collected by jugular venepuncture into non-additive vacutainer tubes (Becton Dickinson) and allowed to clot at room temperature. Serum was removed, transferred to 5 ml sterile screw-capped tubes and stored at -20°C. Peripheral Blood Mononuclear Cells (PBMCs) Blood samples were collected into lithium heparin tubes (Becton Dickinson) and centrifuged for 5 minutes. PBMCs were lysed using an ammonium chloride solution, washed with PBS and stored at -20°C. Nasal swabs Nasal swabs were collected using sterile wooden swabs (Transwab, Biolab Scientific) and were transferred to 1 ml of DMEM supplemented with 2% FBS. Each nasal swab sample was divided into two aliquots that were stored at -20°C.

Challenge virus

EHV-1 for challenge experiments was prepared by infecting semi-confluent rabbit kidney (RK13) cell monolayers at a multiplicity of infection of ~0.01 and incubating in DMEM containing 2% FBS, at 34°C until CPE was > 90%. Infected cell cultures were centrifuged at 2500g for 10 min at 4°C. Supernatant fluid containing extracellular virus was aliquoted and stored at -80°C. EHV-1 titres of aliquots were determined by plaque assay on RK13 cells.

6

Analysis of samples Enzyme Linked Immunosorbent Assays (ELISAs) For assaying antibody using the ELISAs described below, samples were tested in order of collection from each horse over the study period. Hence individual horses were tested on the same day to avoid day-to-day test variation while control samples were used to exclude plate-to-plate variation. Serum from a known EHV-1 infected horse with a high antibody titre and a pooled serum from presuckle newborn foals with no IgG were incorporated as positive and negative controls respectively. EHV-1 gD ELISA For initial studies inclusion bodies (Harlow and Lane, 1988) from E. coli cells expressing EHV-1 pEX gD (Love et al., 1992) were used as antigen in a gD-specific ELISA assay (Ruitenberg et al., 2000b). Prior to the challenge studies a gD-specific sandwich ELISA was developed using a gD-specific monoclonal antibody 20C4 as primary antibody and a detergent extract of baculovirus-expressed gD as antigen, with a test serum dilution of 1/4000. For each serum a detergent extract of uninfected Sf9 cells were also tested as a negative control and the reading was subtracted from the gD ELISA reading. Cross-reacting antibodies to EHV-4 gD are likely to be detected also in these assays, consistent with sequence similarity to EHV-1 gD (Cullinane et al., 1993). EHV-1 gB specific ELISA In an assay similar to the gD sandwich ELISA, a gB-specific sandwich ELISA used monoclonal antibody 3F6 as primary antibody and baculovirus-expressed gB as antigen, with a test serum dilution of 1/4000. EHV-1 and EHV-4 gG ELISAs EHV-1- and EHV-4-specific antibodies were determined using the glycoprotein G (gG) type-specific ELISA (Crabb et al., 1995), with a test serum dilution of 1/100. Samples were tested in triplicate and classified as EHV-1 or EHV-4 antibody-positive if the mean absorbances were greater than 0.129 or 0.246 respectively (Gilkerson et al., 1997). Virus neutralization assay Sera were heat-inactivated at 56ºC for 30 minutes. Complement-independent virus-neutralizing antibody was determined by serial dilution in quadruplicate against 25 TCID50 of EHV-1 in RK13 cells, with titres expressed as the reciprocal of the highest serum dilution that prevented EHV-1 cytopathic effects in 50% of wells (Reed and Muench, 1938). Real-time polymerase chain reaction (PCR) for detection of EHV-1 DNA Nasal swab samples previously stored at -20°C were thawed, filtered and heated to 98°C in a waterbath for 20 min. DNA was extracted from peripheral blood mononuclear cells (PBMCs) using an Eppendorf Perfect gDNA Blood Mini Isolation Kit. Samples were extracted and tested in order of samples collected on each day, rather than in order of samples collected from each horse over the study period. Forward and reverse oligonucleotide primers used in the PCR were based on DNA sequences for the gene encoding glycoprotein C (gC) of EHV-1 as used in our previously developed gel-based test (Lawrence et al., 1994). SYBR Green I fluorescent dye was used to follow amplification in an ABI Prism 7000 on filtered nasal samples and PBMCs. Standard samples consisted of known concentrations of EHV-1 DNA from a Bacterial Artificial Chromosome. Linear and reproducible responses were obtained for standards, while melting curves were used to validate the specificity of the amplified products. The real-time data compared closely with the gel-based test for a set of clinical samples of known EHV status. Statistical Analysis Statistical analysis of data was performed using Minitab® and significant differences in mean values (P<0.05) were determined using a one-way ANOVA.

7

4. Results 1. Immunogenicity of EHV-I glycoprotein formulations (a) Inoculation of pregnant mares and very young foals with EHV-1 gDr The aim of this study was to investigate antibody responses of mares inoculated late in gestation with EHV-1 gDr and to measure maternal antibody levels acquired by their foals compared with foals from uninoculated mares. All foals were also inoculated at 12 h and 30 days of age to assess whether the EHV-1 gDr could elicit detectable antibody responses in such young foals. Design Fifteen Thoroughbred mares were evenly distributed between two groups. Mares 1–8 were inoculated with EHV-1 gDr (5 x 107 cells) at the tenth month of gestation while mares 9–15 remained uninoculated. All foals were inoculated with EHV-1 gDr (5 x 107 cells) at 12 hours following parturition (day 0) and again at one month (day 30) of age. The schedule is shown in Table 1. Table 1. Inoculation (In) and sample collection (S) schedule for mare and foal study

Day –30 0 30 60 Inoculated mares (1-8) In1 S1 S2 S3 S4 Uninoculated mares (9-15)

S1

S2

S3 All foals (1-15) In1 In2 S1 S2 S3

Antibody response of mares to EHV-1 gDr. Following a single inoculation of mares with EHV-1 gDr in the 10th month of gestation, there was a significant increase in mean serum antibody to gD (Table 2). This level was sustained for the three month duration. The mean gD ELISA absorbance for these mares was significantly greater than that of uninoculated mares at days 0, 30 and 60 after foaling. Individual gD ELISA data of mares are shown in Figure 3a. Five of the eight inoculated mares showed a strong response to EHV-1 gDr as determined by gD ELISA. Two of the other three mares had high pre-inoculation gD ELISA antibody. An increase in neutralizing titre was observed in sera of seven out of eight inoculated mares, with at least a four-fold increase observed in four inoculated mares (Fig 3c). No changes in mean EHV-1 gG or EHV-4 gG ELISA absorbances were observed within or between groups of mares throughout the study period (Table 2). All mares were EHV-4 seropositive at the time of inoculation. Serum antibody in foals. Mean gD ELISA absorbances of sera from foals are shown in Table 2. Foals out of mares that had been inoculated during the tenth month of gestation had significantly higher gD ELISA absorbances at day 0 compared with foals out of uninoculated mares. There was a significant decline overall in mean gD ELISA absorbances of foals out of uninoculated mares between day 0 and 60 (Table 2). Individual gD ELISA data (Fig. 3b) show that six foals (2 -7) from inoculated mares acquired similar high levels of anti-gD antibody to those of their dams. Foals 2, 5 and 6 acquired virus-neutralizing antibody titres greater than 128 at day 0 (Fig. 3d), consistent with levels attained by their dams (Fig. 3c) following inoculation of EHV-1 gDr. Two other mares (1 and 7) also had high virus-neutralizing titres following inoculation of EHV-1 gDr, although their foals did not passively acquire the same level of neutralizing antibody. Foals acquired a similar level of EHV-1 and EHV-4 gG antibody to their dams at day 0 with a decline over time (Table 2). Response of foals to gDr inoculation. Although all foals were inoculated with EHV-1 gDr 12 hours post-partum and again at 30 days of age, the mean gD ELISA absorbance declined with age (Table 2). Limited indications of antibody responses were observed in some cases, with foal 15 showing an increase in both gD ELISA and virus-neutralizing antibody, while foals 1, 5 and 6 had relatively steady gD ELISA antibody levels (Figs 3b and 3d).

8

Table 2. Mean EHV-1 gD, EHV-1 gG and EHV-4 gG ELISA absorbances in mare and foal study

Mean ELISA Absorbance gD EHV-1 gG EHV-4 gG MARES EHV-1 gDr inoculated Pre-inoc (day -30) 1.246abc 0.200 1.166 Post-foaling

Day 0 2.171ad 0.195 1.217 Day 30 2.001be 0.178 1.205 Day 60 2.092cf 0.183 1.220

uninoculated Day 0 1.025d 0.201 1.391 Day 30 1.108e 0.212 1.341 Day 60 1.107f 0.227 1.360

FOALS from inoculated mares

Day 0 2.018g 0.170 1.168 Day 30 1.725 0.074 1.144 Day 60 1.305 0.072 0.990

from uninoculated mares Day 0 0.700gh 0.164 1.360i

Day 30 0.496 0.164 1.357j

Day 60 0.393h 0.113 1.190ij

a P = 0.002; F = 13.43 e P = 0.001; F = 18.14 h P = 0.000; F = 9.97 b P = 0.001; F = 14.23 f P = 0.001; F = 17.87 i P = 0.001; F = 8.99 c P = 0.001; F = 14.66 g P = 0.047; F = 5.78 j P = 0.006; F = 10.14 d P = 0.000; F = 19.75

9

Fig 3. gD ELISA (a and b) and neutralizing antibody (VN) (c and d) responses of mares and foals ( = VN titre > 128)

0

20

40

60

80

100

120

140

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

0.5

1

1.5

2

2.5

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

0.5

1

1.5

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Inoculated mares Uninoculated mares


Foals from inoculated mares

490n

m a

bs

490n

m a

bs

VN

titre

V

N ti

tre

a

b

c

d

Foals from uninoculated mares

Foals from inoculated mares Foals from uninoculated mares

0

20

40

60

80

100

120

140

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Day -30 Day 0 Day 60

Fig 3. gD ELISA (a and b) and neutralizing antibody (VN) (c and d) responses of mares and foals ( = VN titre > 128)

10

Conclusions This mare and foal study describes the safe inoculation of EHV-1 gDr in pregnant mares late in gestation and the induction of a strong antibody response in the majority of mares. This resulted in enhanced passively acquired antibody titres in neonates, with foals born from inoculated mares having significantly higher levels of both anti-gD ELISA antibody and neutralizing antibody than foals out of uninoculated mares. In general the antibody levels in the foals after ingestion of colostrum closely reflected those of their dams. Hence, the inoculation of mares in late pregnancy with EHV-1 gDr represents a strategy that stimulates immune responses in the mare and at the same time transfers an increased amount of virus-specific maternal antibody. A later study assesses whether this passively acquired maternal antibody is sufficient to afford the neonate any protection against EHV-1 following experimental challenge. (b) Immunogenicity of EHV-1 gBr This study aimed to determine whether EHV-1 gBr would elicit antibody responses in the horse and also to estimate an appropriate dose for subsequent vaccination studies. We have included previous data for EHV-1 gDr, as a comparison with the gBr data. Design Thirty-two, and twenty-two horses of mixed breed (predominantly Thoroughbred) were recruited to examine the immunogenicity of EHV-1 gDr and gBr respectively and groups of horses were inoculated with high, medium and low doses or received no inoculation. The doses of inoculum used in each study are shown in Table 3. Serum samples were collected prior to and one month following each inoculation and were tested by ELISA assay for antibodies to EHV-1 gD or gB and EHV-1 gG. Table 3. Doses of inoculum for the EHV-1 gDr and gBr immunogenicity studies (cells = recombinant baculovirus-infected cells)

Group EHV-1 gDr study EHV-1 gBr study1 High dose 5 x 107 cells 2 x 107 cells Medium dose 1 x 107 cells 2 x 106 cells Low dose 2 x 106 cells 2 x 105 cells Control No inoculation No inoculation

Results At least 80% of horses inoculated with any dose of EHV-1 gDr or EHV-1 gBr showed an increase in gD or gB ELISA absorbance by at least 50% after one inoculation. Since all groups in each study responded to a similar extent no dose response effect was observed at the antigen concentrations used. Accordingly, 2 x 106 baculovirus-infected cells (the lowest dose tested for EHV-1 gDr) were used in subsequent preparations of the bivalent inoculum containing both recombinant EHV-1 gD and gB (EHV-1 gDBr). The responses observed by individual horses in the groups that were inoculated with 2 x 106 cells for EHV-1 gDr and gBr are shown in Figure 4a and 5 respectively. Virus neutralizing titres were determined for horses inoculated with EHV-1 gDr, and 22 of the 24 horses showed an increase in titre following inoculation. The virus-neutralizing titres of the individual horses in the group that received 2 x 106 cells EHV-1 gDr are shown in Figure 4b. There was no significant change in mean EHV-1 gG or EHV-4 gG ELISA absorbance in any group throughout the study periods indicating responses were due to inoculation and not a result of natural infection (data not shown).

1 The EHV-1 gBr immunogenicity study was conducted subsequent to the EHV-1 gDr immunogenicity study. As there was no observed dose response in the gD study, the gB doses were lowered as shown.

11

0.0

0.5

1.0

1.5

2.0

2.5

1 2 3 4 5 6 7 8

0

20

40

60

80

100

1 2 3 4 5 6 7 8

Fig 4. EHV-1 gD ELISA (a) and neutralizing antibody titres (b) of individual horses inoculated with 2 x 106 cells EHV-1 gDr ( VN titre = 178) Fig 5. EHV-1 gB ELISA antibody absorbances of individual horses inoculated with 2 x 106 cells EHV-1 gBr Conclusions Intramuscular injection of EHV-1 gBr produced a specific serum antibody response in a high percentage of horses tested. This was similar to the frequency and levels of of responses following one inoculation of EHV-1 gDr. The response to EHV-1 gDr was associated with an increase in virus-neutralizing antibody. Therefore from the above results, both the EHV-1 gDr and gBr were highly immunogenic and suitable for further studies for assessment as vaccine candidates.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1 2 3 4 5 6 7

490n

m a

bs

VN

titre

Pre-inoc (week 0) Post-inoc 1 (week 4) Post-inoc 2 (week 8)

Horse

a

b

Horse Fig 4. EHV-1 gD ELISA (a) and neutralizing antibody titres (b) of individual horses inoculated with 2 x 106 cells EHV-1 gDr ( VN titre = 178) EHV-1 gBr

490n

m a

bs

Fig 5. EHV-1 gB ELISA antibody absorbances of individual horses inoculated with 2 x 106 cells EHV-1 gBr

EHV-1 gDr

Horse

12

2. Development of experimental challenge model The aim of this study was to establish an EHV-1 infection model for use in experimental inoculation/challenge experiments. Design Five foals aged between two and four months of age were challenged intranasally with two doses of virus (EHV-1 HVS-25A). Three foals (1-3) were inoculated with approximately 4 x 107 pfu/ml EHV-1 while the remaining two foals (4 and 5) received approximately 1.34 x 107 pfu/ml. Due to its earlier foaling, foal 1 was experimentally challenged before the remaining 4 foals. Foals 2 and 3, and foals 4 and 5, were paddocked together. All these foals were kept isolated from other horses on the property. Nasal swabs, rectal temperature, heart and respiratory rate were collected or measured twice daily and blood samples (for sera and peripheral blood mononuclear cells (PBMCs)) were collected daily for a period of two weeks. Nasal and blood samples were also collected from mares daily for two weeks (data not shown). Mares and foals were then sampled twice weekly for a further two weeks. EHV-1 DNA in nasal swabs and PBMCs EHV-1 DNA was detected by PCR in nasal swab samples from day two in all foals and in peripheral blood mononuclear cells from day eight in four foals (Table 4). Table 4. EHV-1 positive nasal swab (NS) and PBMC samples from all foals. Foals 1 – 3 received approximately 4 x 107 pfu/ml EHV-1; foals 4 and 5 received approximately 1.34 x 107 pfu/ml

Foal 1 Foal 2 Foal 3 Foal 4 Foal 5 Day NS PBMC NS PBMC NS PBMC NS PBMC NS PBMC 1 am pm 2 am + pm + + + + + 3 am + + + + pm + + 4 am + + + + + pm + 5 am + + + + + pm + + + + 6 am + + + + pm + + + + 7 am + + + + pm + + ns ns 8 am + + + pm + + + 9 am + pm + 10 am + + + pm + 11 am + + + pm + 12 am + + + + pm + 13 am + + + pm + 14 am + pm 17 + + 21 + 24 28 Days positive 13 5 6 5 9 3 7 3 5 0

13

Clinical signs of infection Clinical signs were observed in all five foals including a serous to mucous nasal discharge and swelling of the submandibular lymph nodes. A detailed description of observed clinical signs are shown in Table 5. An increase in rectal temperature (RT) was observed in all foals. Individual rectal temperature data for all foals are shown in Figure 6. A mucous nasal discharge and a second elevation in rectal temperature occurred between day nine and 11 in three foals (two of which received the higher dose of virus) indicating the onset of a secondary bacterial infection. Table 5. Clinical signs of EHV-1 infection recorded for each foal during the study period

Day Foal 1 Foal 2 Foal 3 Foal 4 Foal 5 0 am

pm 1 am

pm nostril flare 2 am MND+, nostril flare++ SND+ SND+ SND+ SND+1/2

pm MND+, nostril flare++ MND++ SND+ SND++ COUGH 3 am MND++ MND+++ MND++ MND++ SND++

pm MND+ MND++ MND++ MND++ SND++ 4 am SND+ MND+++ SMLN+ MND+++ SMLN++ SND++ SMLN+ SND++

pm SND+ MND+++ SMLN+ MND++ SMLN++ SND++ 5 am SND+/- MND+++ SMLN++ MND++++ SMLN+++ SND+++ SND++

pm SND+ MND+++ SMLN+ MND++SMLN ++ SND++ SND++ 6 am MND+++++SMLN+MOD+++ MND++ SMLN++ MND++ SMLN+ SND++ SMLN+

pm SND+++ MND++ SMLN+ MND++++ SMLN++ MND+ MND+ SMLN+ 7 am SND++ MND++++ SMLN+ MND+++ SMLN+ MND++ SMLN+ MND++ SMLN+

pm MND+ MND+++ SMLN+ MND++ SMLN++ 8 am SND+ MND++++ SMLN+ MND+++++ SMLN+ SND+ SMLN+ SND+ SMLN+

pm SND+ MND++ SMLN+ MND++ SMLN+ SND+ SMLN+ SND+ SMLN+ 9 am SND+ MND+++ SMLN+ MND+++ SMLN++ SND+ SND+

pm SND+ d/ch on nose MND+++ SMLN+ MND++ SMLN+ SND++ SND++ 10 am dull (rain & cold) MND++++ SMLN+ MND+++ SMLN++ SND++

pm dull MND++++ SND++ SMLN++ SND++ SND+ 11 am dull, A+ MND++ SMLN+ MND++ SMLN++ SND+ SND+

pm brighter, A+ MND++ SMLN+ SND++ SMLN+ SND++ SMLN+ 12 am bright MND+++ SMLN++ MND+ MND+

pm trembling (cold & hail) S/MND++ SND++ SND+ SND+ 13 am v. bright MND+++ SMLN++ M/SND++ SMLN++ SND+

pm S/MND++ SND+++ SMLN++ SND+ SND++ 14 am v. bright S/MND+++ SMLN+ SND++ SMLN+ SND++ SND++

pm MND+ SMLN+ SND+++ SMLN+ SND++ SND+ SND – serous nasal discharge MND – mucous nasal discharge SMLN – submandibular lymph node MOD – mucous ocular discharge

14

Foal 1

3737.5

3838.5

3939.5

4040.5

41

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Day of challenge

RT

Foal 4

3737.5

3838.5

3939.5

4040.5

41

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Day of challenge

RT

Foal 2

3737.5

3838.5

3939.5

4040.5

41

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Day of challenge

RT

Foal 5

3737.5

3838.5

3939.5

4040.5

41

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Day of challenge

RT

Foal 3

3737.5

3838.5

3939.5

4040.5

41

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Day of challenge

RT

Fig 6. Rectal temperatures of foals during the challenge period. Foals 1 – 3 received the higher dose of EHV-1 virus, foals 4 and 5 received the lower dose. Haematology A reverse neutrophil:lymphocyte ratio was observed in Foals 2 and 5 on day 7 and from day 5 to 14 in Foal 3. Serology An elevation in anti-EHV-1 gG ELISA antibody was observed from approximately two weeks post challenge, consistent with successful infection. EHV-1 gG ELISA absorbances are shown for all foals during the challenge period in Fig 7.

15

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 19 23

Day of challenge

450n

m a

bsor

banc

e

Foal 1Foal 2Foal 3Foal 4Foal 5

Fig 7. EHV-1 gG ELISA absorbances of foals during the challenge period. Conclusions Five foals were used in this pilot experiment to test doses of infective challenge virus to be used in subsequent EHV-1 inoculation/challenge experiments. The virus HVS25A is a well-characterized Australian strain of EHV-1 and this is the first such experimental challenge using this virus. EHV-1 DNA was detected in nasal swab samples and PBMCs at an overall greater frequency in the three foals that received the higher dose of challenge virus. Also, clinical signs of infection appeared more severe in Foals 2 and 3 compared to Foals 4 and 5. It was interesting to note that EHV-1 DNA was detected at the greatest frequency from Foal 1, although this foal did not display severe clinical signs of infection. Due to the early foaling, Foal 1 was challenged separately, and it is possible that this may have influenced the severity of clinical signs as it was not in contact with any other foal excreting virus. Foals also displayed an increase in rectal temperature, and a secondary increase in rectal temperature was observed in three foals, indicative of a secondary bacterial infection subsequent to infective EHV-1 challenge. This study demonstrated the infectivity of EHV-1 strain HVS-25A in horses and the protocols established for experimental challenge provide a basis for subsequent testing of vaccines for EHV-1.

16

3. Subunit vaccine assessment (a) Inoculation of weanling foals with EHV-1 gDr and experimental challenge Design Thirteen mares and foals (Standardbred and Thoroughbred) were recruited. The mares and foals remained isolated for several months prior to the commencement of the study. Blood and nasal samples were routinely collected from mares and foals prior to the commencement of the study. The thirteen foals were placed into two groups, depending on age. Group 1 foals (6 foals) were born between 23/8/02 and 14/10/02, and Group 2 foals (7 foals) were born between 25/10/02 and 8/11/02. Half of each group of foals (three foals from Group 1 and four foals from Group 2) were inoculated with 1 ml of EHV-1 gDr at approximately 4 and 5 months of age. All foals were experimentally challenged with 3 ml of EHV-1 virus (1.3 x 107 pfu/ml) at approximately 6 months of age. The two groups were treated separately. Nasal swabs, rectal temperature, heart and respiratory rate were collected or measured twice daily from foals and blood samples were collected daily for a period of two weeks. Foals were sampled twice weekly for a further four weeks. Clinical signs All foals in Group 1 displayed typical clinical signs of EHV-1 infection as previously described (Table 5). Unexpectedly no foal in Group 2 displayed any clinical sign of infection subsequent to challenge. Detection of EHV-1 DNA in nasal swab samples EHV-1 was detected in nasal swab samples from all foals in Group 1. On average, inoculated foals shed virus on fewer occasions that uninoculated foals (Table 6). EHV-1 DNA was not detected in any foal in Group 2. Table 6. EHV-1 detected in nasal swab samples from inoculated and uninoculated foals in Group 1

Foal number 1 2 3 4 5 6 Inoc Inoc Inoc

Day 0 + + 1 + + + + + + 2 + + + + + + 3 + + + + + + 4 + + + + + + 5 + + + + + + 6 + + + + + + 7 + + + + + + 8 + + + + + 9 + + + 10 + + 11 + 12 + 13 14 +

Number of days 10 8 11 8 8 12 Serology All foals in Group 1 displayed an increase in EHV-1 gG ELISA absorbance approximately 14 days post-challenge (data not shown), consistent with infection. All foals in Group 2 had elevated levels of EHV-1 gG ELISA absorbance at the time of challenge.

17

Conclusions This study highlighted difficulties involved in carrying out experiments in “field” conditions. Group 1 foals appeared to shed virus for fewer days, but the loss of the Group 2 foals from the experiment prevented the planned statistical analysis. Group 2 foals were treated as a separate group and were inoculated and challenged subsequent to Group 1. At no time were horses in Group 2 in contact with horses in Group 1, both groups were separated by a number of paddocks. Group 2 horses were not handled by anyone who had been in contact with Group 1 horses during their challenge. At the time of challenge of Group 2, no horse displayed any clinical sign of infection, and EHV-1 DNA was not detected in any nasal swab sample irrespective of vaccination status. Further analysis of ELISA and PCR data revealed that Group 2 horses had experienced a “natural” EHV-1 infection just prior to experimental infective challenge, with high EHV-1 gG ELISA absorbances observed in all horses (mares and foals). One mare had an EHV-1 positive nasal swab sample approximately two weeks prior to challenge, and each foal showed individual positive nasal samples during the week following (one week prior to challenge). It is hypothesised that the initial Group 2 mare reactivated a latent EHV-1, and subsequently infected one (or more) of the foals in the group, with infection spreading through the group, rendering them immune to subsequent infective challenge. While the three inoculated foals appeared to shed virus for fewer days, it was decided that EHV-1 gBr would be included in the inoculum in subsequent challenges to maximise the potential of the available subunit formulations. (b) Inoculation with bivalent EHV-1 gDBr and experimental challenge

(i) Mares and very young foals Design Fourteen pregnant mares (Thoroughbred and Standardbred) were recruited and distributed evenly into three groups based on pre-existing gD and gB ELISA antibody. Group 1 and 2 mares were inoculated with EHV-1 gDBr at month 10 of gestation and within 12 hours of foaling, while Group 3 mares were not inoculated. Group 1 foals only were inoculated with EHV-1 gDBr at 12 hours and 30 days post-partum. All mares and foals were experimentally challenged with EHV-1 at approximately 60 days post partum. A schedule for this study is shown in Table 7. Table 7. Inoculation (In) and sample collection (S) schedule for mare and foal challenge study

Day Day –30

Foaling Day 0

Day 30

Day 60

Group 1 (5 mares and foals) Mares

In1 &S1

In2 & S2

S3

S4 & Challenge

Foals In1 & S1 In2 & S2 S3 & Challenge Group 2 (5 mares and foals) Mares

In1 &S1

In2 & S2

S3

S4 & Challenge

Foals S1 S2 S3 & Challenge Group 3 (4 mares and foals) Mares

S1

S2

S3

S4 & Challenge

Foals S1 S2 S3 & Challenge Following experimental challenge, mares and foals were monitored for a period of 14 days. Samples were collected from each mare and foal as shown in Table 8. Clinical signs of infection including nasal discharge, lymph node enlargement, ocular discharge and rectal temperature were also recorded twice daily and were allocated a clinical score depending on severity (Table 9).

18

Table 8. Sampling schedule for EHV-1 challenge experiments Sample type Times collected Analysis Serum Daily EHV-1 gG ELISA assay to determine

antibody responses to infection

Peripheral blood mononuclear cells (PBMCs)

Daily EHV-1 DNA detection by real-time PCR

Nasal swabs Twice Daily EHV-1 DNA detection by real-time PCR

Table 9. Scores allocated to clinical signs of infection

Nasal discharge Lymph node enlargement Ocular discharge Observation Score Observation Score Observation Score Serous + 1 + 1 Serous + 1 Serous ++ 2 ++ 2 Serous ++ 2 Serous +++ 3 +++ 3 Serous +++ 3 Mucous + 4 Mucous + 4 Mucous ++ 5 Mucous ++ 5 Mucous +++ 6 Mucous +++ 6 ELISA antibody responses toEHV-1 gDBr inoculation Mares There was no significant difference in mean gD, gB and gG ELISA absorbances of groups of mares prior to inoculation. By the first day of experimental challenge, inoculated mares had significantly higher levels of mean gD ELISA antibody compared to uninoculated mares (P = 0.013; F = 6.54; Table 10) and also higher observed levels of gB antibody. There was no change in gG ELISA absorbances prior to challenge indicating that responses observed were a result of inoculation of EHV-1 gDBr and not a result of natural infection. Individual gD and gB ELISA absorbances of mares are shown in Fig 8a and 8c respectively. Foals Foals born from inoculated mares had higher mean gD and gB ELISA absorbances at foaling (samples collected after ingestion of colostrum) and challenge compared to foals born from uninoculated mares (Table 10). gD and gB ELISA data for individual foals are shown in Fig 8b and 8d respectively. Generally, foals acquired comparable levels of maternal ELISA antibody to their dams, except for one foal (foal 2). Despite the inoculation of Group 1 foals (1-5) at 12 hours post-partum (Day 0), there was no increase in gD or gB ELISA absorbance. EHV-1 gG ELISA antibody response to experimental infection Mares Following experimental EHV-1 challenge there was an expected increase in EHV-1 gG ELISA absorbance in all groups of mares from approximately 7 days post-challenge (Fig 9a). Foals A delayed response in EHV-1 gG ELISA antibody was observed in the three foal groups, with increases in ELISA antibody not occurring until approximately 13 days post-challenge (Fig 9b). These increases in gG ELISA antibody confirmed that the experimental infection of mares and foals was successful.

19

Table 10. Mean EHV-1 gD, gB and gG ELISA absorbances of mares and foals prior to challenge Mean ELISA Absorbance gD gB gG MARES Group 1 EHV-1 gDBr inoculated Pre-inoc (day -30) 0.190 0.119 0.082

Post-foaling Day 0 1.205 0.576 0.093 Day 60 (Challenge) 0.901 0.357 0.037

Group 2 EHV-1 gDBr inoculated Pre-inoc (day -30) 0.235 0.173 0.014


Group 3 uninoculated Day -30 0.165 0.122 0.183


FOALS Group 1 EHV-1 gDr inoculated from inoculated mares

Day 0 0.734 0.477 0.000 Day 60 0.367 0.188 0.005

Group 2 uninoculated from inoculated mares

Day 0 0.835 0.525 0.000 Day 60 0.364 0.193 0.005

Group 3 uninoculated from uninoculated mares

Day 0 0.203 0.214 0.245 Day 60 0.064 0.100 0.162

20

Fig 8. EHV-1 gD (a and b) and gB (c and d) ELISA absorbances of individual mares and foals following inoculation.

0

0.5

1

1.5

2

2.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

0.5

1

1.5

2

2.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Uninoculated mares

Fig 8. EHV-1 gD (a and b) and gB (c and d) ELISA absorbances of individual mares and foals following inoculation. Mares 1-10 were inoculated at day -30 and 0, foals 1-5 were inoculated at Day 0 and 30 ( Mare and foal 14 entered study 2 weeks prior to challenge date, hence no prior information available)

a

b

c

d

Inoculated mares

Inoculated foals from inoculated mares

Uninoculated foals from inoculated mares

Uninoculated foals from uninoculated mares


Inoculated foals from inoculated mares

Uninoculated foals from inoculated mares

Uninoculated foals from uninoculated mares

gD ELISA

gB ELISA

Day -30 Day 0 Challenge (~Day 60)

490n

m

490n

m

490n

m

490n

m

21

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Mares and foals vaccinatedMares vaccinated onlyControls

Fig 9. EHV-1 gG ELISA absorbances of mares (a) and foals (b) following challenge

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Mares and foals vaccinatedMares vaccinated onlyControls

Fig 9. EHV-1 gG ELISA absorbances of mares (a) and foals (b) following challenge (day 0 = day of infection; mares and foals monitored for a period of 14 days)

a

b

Mares

Foals

Day after challenge

Day after challenge

450n

m a

bs

450n

m a

bs

22

EHV-1 DNA in nasal swabs and PBMCs Table 11 shows the number of times that EHV-1 DNA was detected using real-time PCR in nasal swab samples from each horse. On average, inoculated mares shed virus on significantly fewer occasions than uninoculated mares (P = 0.041; F = 4.33) and inoculated foals shed virus on significantly fewer occasions than uninoculated foals (P = 0.030; F = 4.90). Table 12 diagrammatically represents detection of EHV-1 in nasal swabs from each mare and foal on each day of challenge. If either the morning or afternoon sample collected on the day was positive, that day is highlighted in green. EHV-1 was detected on significantly fewer days in both inoculated mares (P = 0.044; F = 4.21) and inoculated foals (P = 0.018; F = 5.97) compared to uninoculated mares and foals. EHV-1 was detected in PBMC samples from mares and foals irrespective of inoculation status (Table 11). Table 11. Detection of EHV-1 in nasal swab samples and PBMCs

Treatment Group Horse ID EHV-1 positive nasal swabs (from a total of 28

samples from each horse)

EHV-1 positive PBMC (days)

Mares Foals Mares

Foals

1 Mares and foals 1 0 2 1 1 inoculated 2 0 3 5 8 3 2 2 2 4 4 5 4 3 1 5 6 1 4 4 Mean 2.6 2.4 3 3.6 s.d. 2.8 1.1 1.6 2.9

2 Mares inoculated, 6 6 5 0 1 foals uninoculated 7 1 5 0 4 8 0 9 2 3 9 4 6 4 5 10 1 15 4 7 Mean 2.4 8.0 2 4 s.d. 2.5 4.2 2 2.2

3 Mares and foals 11 8 8 4 7 uninoculated 12 9 8 2 1 13 8 2 1 2 14 5 14 3 0 Mean 7.5 8.0 2.5 2.5 s.d. 1.7 4.9 1.3 3.1

23

Table 12. EHV-1 DNA in nasal swab samples from individual mares and foals on each day post-challenge. If a sample on either the morning or afternoon sampling was positive that day is highlighted.

ARES Inoculated Inoculated Uninoculated Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0 1 + 2 + + + 3 + + + + + + 4 + + + + + + + + + 5 + + + + + + + 6 + + + + + 7 + + + + 8 + + 9

10 + + 11 + 12 13 + 14

Total 0 0 2 4 4 4 1 0 4 1 5 6 6 4 Mean 2 days 2 days 6 days

FOALS Inoculated Uninoculated Uninoculated Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0 +1 1 + + + 2 + + + 3 + + + + + 4 + + + + + + + + + 5 + + + + + + + + + + 6 + + + + + + + + 7 + + + + + + + + 8 + + + + + + 9 + + +

10 + + + + + 11 + + 12 + 13 14

Total 2 3 2 3 1 4 5 5 6 10 6 6 2 9 Mean 2 days 6 days 6 days Day 0 = day of challenge

1 Positive sample was collected on afternoon of challenge

24

Clinical signs of infection following experimental challenge Table 13 describes the total clinical scores for each animal for nasal discharge, lymph node enlargement and ocular discharge over the challenge period. Inoculated mares had a significantly lower mean clinical score for nasal discharge than uninoculated mares (P = 0.018; F = 5.90) indicating that on average, inoculated mares had less pronounced nasal discharge than uninoculated mares. Foals born from inoculated mares also had less severe clinical signs of nasal discharge compared to foals out of uninoculated mares however the difference was not statistically significant (P = 0.107; F = 2.76). No other significant differences in clinical scores were noted between the three treatment groups for either mares or foals. Table 13. Mare and foal study: clinical sign data

MARES Rectal temperatures Nasal discharge

Lymph node

Ocular discharge

Treatment Horse ID Mean s.d. Days abnormal Total clinical score 1 1 37.1 0.6 0 21 0 0

(inoculated) 2 37.6 0.5 0 26 33 0 3 37.0 0.6 0 32 18 0 4 36.9 0.7 0 28 11 7 5 36.9 0.6 0 20 20 0 Mean 37.1 0 25 16 1

2 6 37.3 0.4 0 26 0 0 (inoculated) 7 37.8 0.5 0 23 0 0

8 37.3 0.6 0 35 2 0 9 37.4 0.7 0 17 7 4 10 37.4 0.6 0 22 1 0 Mean 37.4 0 25 2 1

3 11 37.3 0.5 0 32 0 0 (un- 12 37.3 0.5 0 35 0 0

inoculated) 13 37.1 0.4 0 37 15 0 14 37.0 0.6 0 53 18 0 Mean 37.2 0 39 8 0

FOALS Rectal temperatures Nasal discharge

Lymph node

Ocular discharge

Treatment Horse ID Mean s.d. Days abnormal Total clinical score 1 1 38.6 0.3 3 5 15 11

(inoculated) 2 38.7 0.5 7 26 36 0 3 38.6 0.3 5 24 23 0 4 38.5 0.8 7 14 14 1 5 38.4 0.5 1 25 14 0 Mean 38.6 5 19 20 2

2 6 38.5 0.4 3 20 22 0 (un- 7 38.8 0.5 10 20 16 3

inoculated) 8 38.5 0.5 2 22 20 0 9 38.9 0.4 13 23 17 0 10 38.6 0.4 9 22 20 0 Mean 38.7 7 21 19 1

3 11 38.6 0.4 3 21 16 0 (un- 12 38.9 0.5 10 32 12 0

inoculated) 13 38.4 0.3 2 37 27 0 14 38.5 0.2 2 26 18 0 Mean 38.6 4 29 18 0

25

Conclusions In this study, mares and very young foals were inoculated with EHV-1 gDBr and were subsequently challenged intranasally with EHV-1. At the first day of experimental challenge, inoculated mares had significantly higher levels of gD ELISA antibody compared to mares that had not been inoculated. They also had higher levels of gB ELISA antibody, although the difference was not statistically significant. Foals that were born from these inoculated mares had higher levels of gD and gB ELISA antibody 12 hours post-partum and at the first day of challenge. The difference in antibody levels did not reach statistical significance, most likely due to the small number of animals in each group. Following the inoculation of Group 1 foals, there was no increase in either gD or gB ELISA absorbance. These results are consistent with the previous study described in Part 1 of this report. 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. Inoculated mares shed virus in nasal secretions for significantly fewer days compared to uninoculated mares. Inoculated foals born from inoculated mares shed virus for significantly fewer days than uninoculated foals born from both inoculated mares and uninoculated mares. This suggests that pre-existing colostral antibody levels acquired by inoculation of the mare is not sufficient in affording protection in the neonate and that active immunization is required. It may however result in a reduction in severity of nasal discharge. Foals born from inoculated mares, irrespective of their inoculation status appeared to show less severe nasal discharge than foals born from uninoculated mares. The difference approached statistical significance. Further work with larger numbers of animals would be required to validate this suggestion. Inoculation of mares however did result in a significant reduction in the severity of nasal discharge. There were no other statistical significant differences observed between groups of animals. Contrary to prior reports, these results suggest that it is indeed possible to induce partial protection in very young foals through vaccination, and while the inoculation did not prevent infection, it did reduce the amount of virus shed with the potential to thereby reduce the risk and prevalence of infection. (ii) Weanling foals In this study we aimed to investigate whether EHV-1 gDBr could offer protection against experimental challenge of weanling foals, who represent one of the peak targets for infection in the annual cycle. In attempting to mimic the on-farm situation, we kept the foals from the previous challenge experiment, re-inoculated some of them with EHV-1 gDBr, and subsequently weaned and re-challenged all foals with EHV-1. Design Group 1 foals (five inoculated foals born from inoculated mares) were inoculated for a third time at 6 months of age. Group 2 foals (five uninoculated foals born from inoculated mares) were inoculated for the first time at 6 months of age. Group 3 foals (three foals used in the previous experiment plus two that had also been previously experimentally infected with EHV-1 three weeks prior to previous experiment) remained as uninoculated controls. All foals were weaned 2 weeks post-inoculation and challenged 3 weeks post-inoculation. Sampling procedures post-challenge were the same as for the previous experiment (Table 8). A summary of the history of these foals and the experimental design of this study are shown in Table 14. Table 14. History of foals and design of weanling study

Foals Mare/foal study (section 3.4 pt 1) Weanling study 12 hours 30 days 60 days 6 mths 6 mths

+ 2 weeks 6 mths

+ 3 weeks Group 1 In1 In2 C1 In3, S W, S C2, S Group 2 C1 In1, S W, S C2, S Group 3 C1 S W, S C2, S

In = inoculation; C = challenge; S = sample; W = Weaned

26

ELISA antibody response to EHV-1 gDBr There was no significant difference in mean gD, gB or gG ELISA absorbances of groups of foals prior to inoculation indicating an even distribution of foals in the three groups (Table 15). Following inoculation of foals with EHV-1 gDBr, there was a significant difference in mean gD ELISA absorbance between group 2 (inoculated) and 3 (uninoculated) foals at weaning (P = 0.022; F = 5.31) and at challenge (P = 0.021; F = 5.46). The difference in mean gB ELISA absorbance between these two groups also approached significance at weaning (P = 0.052; F = 3.83) and challenge (P = 0.055; F = 3.72). There was no change in mean EHV-1 or EHV-4 gG ELISA absorbances prior to challenge indicating that increases in gD and gB ELISA absorbance were the result of inoculation and not of natural infection. Individual gD and gB ELISA data for all foals are shown in Fig 10. Table 15. Mean EHV-1 gD, gB and gG ELISA absorbances of foals pre-inoculation, and at weaning and challenge

Mean ELISA Absorbance gD gB gG Group 1 EHV-1 gDBr inoculated; previously inoculated 12 h and 30 d

Pre-inoc 0.141 0.075 0.783 Weaning 0.752 0.296 0.791 Challenge 0.684 0.256 0.738

Group 2 EHV-1 gDBr inoculated; previously uninoculated

Pre-inoc 0.267 0.174 0.808 Weaning 1.333 0.632 0.758 Challenge 1.235 0.539 0.745

Group 3 uninoculated Pre-inoc 0.194 0.143 0.534 Weaning 0.192 0.129 0.474 Challenge 0.158 0.116 0.482

27

a

b

Fig 10. EHV-1 gD (a) and EHV-1 gB (b) ELISA data of individual foals pre-inoculation, at weaning (2 weeks post-inoculation) and challenge (3 weeks post-inoculation). Antibody response to challenge Following experimental EHV-1 challenge there was an increase in EHV-1 gG ELISA absorbance in all groups of foals from approximately 7 days post-challenge (Fig 11) as seen for the mares in the previous study.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Pre-inoc

Wean Chall(Day

0)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Group 1: inoculated, previously inoculated

Group 2: inoculated, not previously inoculated

Group 3: uninoculated

Fig 11. Mean EHV-1 gG ELISA absorbances of groups of foals prior to and following experimental challenge

0

0.5

1

1.5

2

2.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Pre-inoculation Weaning Challenge

gD ELISA

gB ELISA

Foal

Foal

Inoculated (previously inoculated at 12 h and 30 days)

Inoculated (previously

uninoculated) Uninoculated

Day of challenge

490n

m a

bs

Fig 11. Mean EHV-1 gG ELISA absorbances of groups of foals prior to and following experimental challenge

gG ELISA

28

EHV-1 in nasal swabs and PBMCs following challenge EHV-1 DNA was detected in nasal swab samples from six of ten inoculated foals (60%) and four of five (80%) uninoculated foals (Table 16). There was a slight decrease in the mean number of times EHV-1 DNA was detected in nasal swab samples from inoculated foals compared to uninoculated foals. EHV-1 DNA was also detected in PBMCs from two of 10 (20%) inoculated foals and two of five (40%) uninoculated foals. Table 16. Detection of EHV-1 in nasal swab samples and peripheral blood mononuclear cells

Treatment Group Horse ID EHV-1 positive nasal swabs (of 28)

EHV-1 positive

PBMC (days) 1 Foals inoculated 1 2 0 (previously inoculated) 2 1 1 3 0 1 4 0 0 5 2 0 Mean 1.0 0.4 s.d. 1.0 0.5 2 Foals inoculated, 6 0 0 (previously uninoculated) 7 1 0 8 7 0 9 1 0 10 0 0 Mean 1.8 0 s.d. 2.9 0 3 Foals uninoculated 11 0 0 12 1 0 13 4 0 14 5 2 15 7 1 Mean 3.4 0.6 s.d. 3.4 0.9

Clinical signs Foals inoculated with EHV-1 gDBr had a lower mean nasal discharge clinical score compared to uninoculated foals (Table 17). Table 17. Weanling study: clinical signs data WEANLINGS Rectal temperatures Nasal

discharge Lymph

node Ocular

discharge Treatment Horse

ID Mean s.d. Days

abnormal Total clinical score

1 1 38.2 0.4 1 34 14 2 (inoculated) 2 38.5 0.4 5 49 32 4

3 38.5 0.4 2 35 27 2 4 38.8 0.7 6 33 12 25 5 38.2 0.3 0 33 19 1 Mean 38.4 3 37 21 7

2 6 38.1 0.4 1 38 19 14 (inoculated) 7 38.2 0.7 5 32 4 4

8 38.3 0.6 5 26 19 0 9 38.5 0.4 3 22 11 3 10 38.5 0.3 4 50 55 0 Mean 38.3 4 34 22 4

3 11 38.4 0.3 2 21 11 5 (un- 12 38.1 0.4 3 56 15 16

inoculated) 13 38.3 0.7 4 81 27 0 14

15 38.1 38.6

0.4 0.3

1 8

40 61

10 19

14 8

Mean 38.3 4 52 16 9

29

Conclusions This study describes the inoculation of weanling foals with EHV-1 gDBr and subsequent EHV-1 challenge. In contrast to the same foals at a younger age in the previous study foals responded well to a single dose of EHV-1 gDBr and had higher levels of anti-gD and anti-gB ELISA antibodies at challenge compared with uninoculated foals. Inoculated foals who were not previously inoculated at a young age had significantly higher ELISA antibody levels 2 and 3 weeks post-inoculation compared to uninoculated foals. Inoculated foals that had been previously inoculated also had higher levels of gD and gB ELISA antibody than uninoculated foals, although the difference between those two groups was not significant. This suggests that prior inoculation of foals at a young age may reduce the antibody response to subsequent inoculation. There was however no statistical difference between the groups after challenge following analysis of parameters tested, with only 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 observed. These results confirm that antibody alone is not the sole mediator in protection and that other arms of the immune response play a vital role. In this study, we attempted to replicate the on-farm situation, where there is peak incidence of infection in foals aged less than 30 days and again at approximately 5-6 months of age, by re-using foals which had been previously challenged with EHV-1 (a few months prior to this study). However, it is difficult to reproduce the exact on-farm situation with respect to administering equivalent virus and it is likely that the dose of virus we administered was of substantially higher infectivity than virus available to foals in a natural situation. Hence, it is possible that through challenging our foals in the previous experiment at approximately 60 days of age with EHV-1, we produced foals with a greater degree of protection at weaning than would normally be the case, as all of the foals, including the control group, shed virus for fewer days than has been previously observed when experimentally infecting this age group of foals (approx 7 days; data not shown). Furthermore, all foals were EHV-1 gG seropositive at the time of challenge due to previous exposure to EHV-1. In a normal situation, on average 8% of foals are EHV-1 seropositive at the time of weaning (Foote et al., 2003). Nonetheless, while our results did not produce significant statistical differences between groups, the biological differences observed warrant further investigation of this preparation in this age group of foals.

30

5. Discussion of Results During the project conditions and expertise were developed for experimental respiratory challenge of foals and mares by an Australian isolate of EHV-1. The protocols and parameters established will be useful for assessments of any EHV-1 vaccine. The challenge model was then used to investigate the vaccine potential of a combination of the EHV-1 envelope glycoproteins D and B (EHV-1 gDBr). The results indicated that, following EHV-1 respiratory challenge, intramuscular inoculation of EHV-1 gDBr was associated with a reduction in viral shedding and a decrease in nasal discharge in lactating mares and in very young foals. These findings were made following extensive prior testing of the safety and immunogenicity of the EHV-1 gDBr formulation, in horses of all ages and including pregnant mares. Along with high levels of antigenicity observed for the EHV-1 gDBr formulation, the results support also the use of Iscomatrix ™ as a safe and effective adjuvant for horses. It was evident from the experiments that the EHV-1 gDBr formulation reduced but did not prevent a challenge infection. In the field there appears to be a brief window following EHV-1 infection where the animal is resistant to challenge (Allen et al., 1999) and we observed such an effect in our early challenge experiment in one group of foals. However the use of live virus vaccines that may mimic these responses raises difficult questions of safety for application, when the disease outcome can be so serious. Vaccines based on virion subunits such as EHV-1 gDBr invoke specific immune responses to an antigen without exposing the animal to the causal organism, removing safety concerns. They also allow for differential identification of vaccine-related responses, in contrast to existing whole virus vaccines. EHV-1 gD and gB are conserved across all EHV-1 isolates so far characterized, with virtually identical protein sequence, so that a vaccine based on these components should act against all EHV-1 strains. Since antibody to EHV-1 gD also neutralizes EHV-4 (Love et al., 1993) it is likely that protective responses induced by EHV-1 gDr against EHV-1 will also act against EHV-4. EHV-1 gB also shares most of its protein sequence with EHV-4 gB, with shared antigenic epitopes. In the horse, both mucosal and systemic antibody responses are likely to be factors associated with protective immunity to EHV-1 infection, in addition to cell-mediated responses including activation of cytotoxic T lymphocytes (CTLs) (Allen et al., 1999; Breathnach et al., 2001). Elevated circulating virus-neutralizing antibody such as is induced by EHV-1 gDr is likely to limit the systemic spread of EHV-1, and if present also in the upper respiratory tract should target incoming virus. EHV-1 gD and gB are essential for virus entry and cell-to-cell fusion (Wellington et al., 1996; Neubauer et al., 1997; Csellner et al., 2000). Therefore immune responses that target these proteins should in principle block early stages of infection. In this study we did not examine cell-mediated immune responses, but from the previous project we obtained data that showed that EHV-1 gDr elicited a similar balance of immunoglobulin isotypes to those following infection by EHV-1 (Sugiura et al., 1994, 1999) and EHV-4 (Mizukoshi et al., 2002). In theory this should represent the desirable spectrum of effective responses. In further rational for its inclusion of in a subunit vaccine, EHV-1 gD was shown to provide a target for equine CTLs in some ponies (Soboll et al., 2003). Another component which could be included in a multi-valent vaccine is the EHV-1 Immediate Early (IE) gene product, as peptides of this protein are targets for CTLs in horses of a commonly found genetic background (Sobell et al., 2003). This may enhance the cell-mediated response to vaccination, and the possibility of reducing viraemia. It has been suggested that after suckling, the foal is protected by maternal colostral antibody for the first few months of its life (Kendrick and Stevenson, 1979). Infection has been reported to occur less frequently in young foals born to vaccinated mares with foals becoming gradually more susceptible as maternal antibodies decrease (Rossdale and Scarnell, 1961; Lunn, 1997; van Maanen et al., 1992). Nonetheless our epidemiological data shows that early infection of foals is continuing despite

31

vaccination of mares with the currently available vaccine (Foote et al., 2004). It appears from the results of this present study that maternal colostral antibody to EHV-1 gD and gB does not provide protection in the neonate, as uninoculated foals with high levels of maternal antibody acquired from their inoculated dams were no more protected against EHV-1 challenge than uninoculated foals from uninoculated mares. Currently, vaccination against EHV-1 does not commence until the foal is approximately six months of age. Active immunization of newborns by natural infections or vaccination has been reported to be hampered by an immature immune system (Morein et al., 2002) and by passively-derived maternal immunity (van Oirschot et al., 1991; van Maanen et al., 1992) which has been suggested to induce immune tolerance to vaccination persisting for several months. Consequently, an aim of vaccination to-date has been to close the window of susceptibility to infection in young animals, the time between when maternal antibody diminishes and active immunization begins (Morein et al., 2002). In this study, we inoculated a group of very young foals with the trial subunit vaccine EHV-1 gDBr. Due to their age, these foals were naïve animals, and did not display increases in ELISA antibody response following inoculation. However after later challenge with EHV-1, the foals did shed virus for fewer days and exhibited less severe clinical signs than uninoculated controls, suggesting that these animals developed some cell-mediated immunity at a very young age. Despite careful farm management, routine handling of mares and foals and the mixing of paddock groups allow the introduction and spread of EHV disease (Bryans 1981). Epidemiological data shows that foals are being infected with EHV-1 at an early age and that the most likely source of infection is the mare (Gilkerson et al., 1997; Foote et al., 2003). These infections are continuing despite the introduction of widespread vaccination into Australia in 1997 (Foote et al., 2004). At present, mares are vaccinated in their 5th, 7th and 9th month of gestation while it is recommended that foals should receive their first vaccination at five months of age and no earlier than three months of age. This strategy leaves the lactating mare and very young foal susceptible to infection. Our results suggest it is possible to induce partial protection in very young foals through early vaccination with EHV-1 gDBr. While the inoculation did not prevent infection, it did reduce the number of days virus was shed thereby reducing the risk and prevalence of infection in a herd situation. With the aim of reducing infection in young foals through mare to foal contact, it may also be useful to vaccinate mares closer to foaling.

32

6. Implications and Recommendations

An experimental respiratory challenge model in foals and mares along with suitable protocols and parameters was established using an Australian strain of equine herpesvirus 1 (EHV-1). The establishment of this model in Australia is a potentially valuable development that could allow independent assessment of EHV-1 vaccines, either experimental or existing. A trial subunit vaccine formulation based on the EHV-1 envelope glycoproteins B and D (EHV-1 gDBr) induced strong antibody responses in mares, and reduced the number of days of virus shedding following respiratory challenge. The formulation EHV-1 gDBr was shown to be safe in horses of all ages and including pregnant mares. The results support the use of Iscomatrix ™ as a safe and effective adjuvant for horses. Although no antibody responses were observed in very young foals after inoculation with EHV-1 gDBr, nonetheless these animals had a reduced amount of virus shedding following challenge, compared to controls. This indicated that very young foals were indeed able to mount an immune response and that it is possible to induce partial protection in very young foals through vaccination. While the inoculation of EHV-1 gDBr did not prevent infection under the conditions used, a reduction in amount of virus shed is likely to reduce the prevalence of EHV-1 infection. A modified vaccine strategy is suggested where the pregnant mare and the young foal are the main target for vaccination. Therefore vaccination of the mare later in gestation and vaccination of very young foals should be further explored. Vaccines based on virion subunits, such as EHV-1 gDBr, are designed to induce specific immune responses to antigens without exposing the animal to the causal organism, a valuable safety feature for the animal and manufacturer. Subunit preparations allow for differential identification of vaccine-related responses to responses to natural infection and the use of specific viral subunits allows considerable flexibility in vaccine design. In addition the conserved glycoproteins gD and gB are present in all EHV-1 strains. Subunit preparations produced by recombinant systems are relatively inexpensive to produce. Another component which could be usefully included in a vaccine is the EHV-1 Immediate Early gene product, which appears to be a target for cytotoxic T cells in many horses. This may enhance the cell-mediated response to EHV-1 infection. This could be tested in the respiratory EHV-1 infection model established here. Further testing of the EHV-1 gDBr formulation could be warranted. While respiratory disease is one aspect of EHV-1 infection, abortion is probably the most widely recognized outcome both in Australia and worldwide. Additionally, the devastating effects of EHV-1 neurological disease are becoming increasingly realised. It would be important to investigate whether EHV-1 gDBr offers protection for horses against EHV-1 related abortion and neurological disease. Since the EHV-1 gD and gD are antigenically cross-reactive with EHV-4 gD and gB, there should also be protective effects against EHV-4, and challenge experiments with EHV-4 similar to those described here would determine if this were the case. During the project real-time PCR tests were developed for EHV-1 and EHV-4 DNA. These rapid and sophisticated tests can be used to detect these viruses both in subsequent challenge experiments and in clinical samples, and have wide potential application to the epidemiology of EHV-1 and EHV-4.

33

7. References

Allen, G.P. (2002). Respiratory infections by equine herpesvirus types 1 and 4. In: Lekeux, P. (Ed) Equine Respiratory Diseases, International Veterinary Information Service, New York.

Allen, G.P., Bryans, J.T. (1986). Molecular epizootiology, pathogenesis and prophylaxis of equine herpesvirus-1 infections. In Progress in Veterinary Microbiology and Immunology, pp 78 - 144. Edited by R. Pandey. Basel: S. Karger.

Allen, G.P., Kydd, J.H., Slater, J.D., Smith, K.C. (1999). Advances in understanding of the pathogenesis, epidemiology and immunological control of equine herpesvirus abortion. In: Wernery, J.W.U., Mumford, J., Kaaden, O.R. (Eds) Eighth International Conference of Equine Infectious Diseases, R&W Publication, Dubai, pp. 129-146.

Audonnet, J.C., Mumford, J.A., Jessett, D., Pardo, M.C., Taylor, J., Tartaglia, J., Minke, J.M. (1999). Safety and efficacy of a canarypox-EHV recombinant vaccine in horses. In: Wernery, J.W.U., Mumford, J., Kaaden, O.R. (Eds) Eighth International Conference of Equine Infectious Diseases, R&W Publication, Dubai, pp. 418-419.

Baxi, M., Efstathiou, S., Lawrence, G., Whalley, J.M., Slater, J., Field, H.J. (1995). The detection of latency-associated transcripts of equine herpesvirus 1 in ganglionic neurons. Journal of General Virology 76, 3113-3118.

Borchers, K., Wolfinger, U., Ludwig, H. (1999). Latency-associated transcripts of equine herpesvirus type 4 in trigeminal ganglia of naturally infected horses. Journal of General Virology 80, 2165-2171.

Breathnach, C.C., Yeargan, M.R., Sheoran, A.S., Allen, G.P. (2001). The mucosal humoral immune response of the horse to infective challenge and vaccination with equine herpesvirus-1 antigens. Equine Veterinary Journal 33, 651-657.

Bryans, J.T. (1981). Application of management procedures and prophylactic immunization to the control of equine rhinopneumonitis. In Proceedings of the 26th Annual Conference of the American Association of Equine Practitioners, pp. 259-272. Anaheim.

Cox, J.C., Coulter, A. R. (1997). Adjuvants – a classification and review of their modes of action. Vaccine 15, 248-256.

Crabb, B. S., Macpherson, C. M., Reubel, G. H., Browning, G. F., Studdert, M. J., Drummer, H. E. (1995). A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1. Archives of Virology 140, 245-258.

Crabb, B. S., Studdert, M. J. (1995). Equine herpesviruses 4 (equine rhinpneumonitis virus) and 1 (equine abortion virus). Advances in Virus Research 45, 153-190.

Csellner, H., Walker, C., Wellington, J.E., McLure, L.E., Love, D.N., Whalley, J.M. (2000). EHV-1 glycoprotein D (EHV-1 gD) is required for virus entry and cell-cell fusion, and an EHV-1 gD deletion mutant induces a protective immune response in mice. Archives of Virology 145, 2371-2385.

Cullinane, A.A., Neilan, J., Wilson, L., Davison, A.J., Allen, G., (1993). The DNA sequence of the equine herpesvirus 4 gene encoding glycoprotein gp17/18, the homologue of herpes simplex virus glycoprotein gD. Journal of General Virology 74, 1959-1964.

Foote, C.E., Gilkerson, J.R Whalley, J.M., Love, D.N. (2003). Seroprevalence of equine herpesvirus 1 in mares and foals on a large Hunter Valley stud farm in years pre- and post- vaccination. Australian Veterinary Journal 81, 283-288.

Foote, C. E., Love, D. N., Gilkerson, J. R., Rota, J., Trevor-Jones, P., Ruitenberg, K. M., Wellington, J. E., Whalley, J. M. Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, including pregnant mares and young foals. Veterinary Immunology and Immunopathology, in press

Foote, C.E., Love, D.N., Gilkerson, J.R., Whalley, J.M. (2002). Serological responses of mares and weanlings following vaccination with an inactivated whole virus equine herpesvirus 1 and equine herpesvirus 4 vaccine. Veterinary Microbiology 88, 13-25.

34

Foote, C.E., Love, D.N., Gilkerson, J.R., Whalley, J.M. (2004). Detection of EHV-1 and EHV-4 DNA in unweaned Thoroughbred foals from vaccinated mares on a large stud farm. Equine Veterinary Journal 36, 341-345.

Gilkerson, J. R., Love, D. N., Drummer, H. E., Studdert, M. J., Whalley, J. M. (1998). Seroprevalence of equine herpesvirus 1 in Thoroughbred foals before and after weaning. Australian Veterinary Journal 76, 677-682.

Gilkerson, J. R., Love, D.N., Whalley, J.M. (1997). Serological evidence of equine herpesvirus 1 (EHV-1) infection in Thoroughbred foals 30-120 days of age. Australian Equine Veterinarian 15, 128-134.

Gilkerson, J.R., Whalley, J.M., Drummer, H.E., Studdert, M.J., Love, D.N. (1999). Epidemiological studies of equine herpesvirus 1 (EHV-1) in Thoroughbred foals: a review of studies conducted in the Hunter Valley of New South Wales between 1995 and 1997. Veterinary Microbiology 68, 15-25.

Hannant, D., O'Neill, T., Ostlund, E.N., Kydd, J.H., Hopkin, P.J., Mumford, J.A. (1999). Equid herpesvirus-induced immunosuppression is associated with lymphoid cells and not soluble circulating factors. Viral Immunology 12, 313-321.

Harlow, E., Lane, D. (1988). Antibodies: A laboratory manual. New York: Cold Spring Harbor Laboratory.

Heldens, J.G.M., Hannant, D., Cullinane, A.A., Prendergast, M.J., Mumford, J.A., Nelly, M., Kydd, J.H., Weststrate, M.W., van den Hoven, R. (2001). Clinical and virological evaluation of the efficacy of an inactivated EHV1 and EHV4 whole virus vaccine (Duvaxyn EHV 1,4). Vaccination/challenge experiments in foals and pregnant mares. Vaccine 19, 4307-4317.

Huemer, H.P., Nowotny, N., Crabb, B.S., Meyer, H., Hubert, P.H. (1995). gp13 (EHV-gC): a complement receptor induced by equine herpesviruses. Virus Research 37, 113-126.

Kendrick, J. W., Stevenson, W. (1979). Immunity to EHV-1 infection in foals during the first year of life. Journal of Reproductive Fertility 27, Supplement 615-618.

Kukreja, A., Love, D.N., Whalley, J.M., Field, H.J. (1998). Study of the protective immunity of co-expressed glycoprotein H and L of equine herpesvirus-1 in a murine intranasal infection model. Veterinary Microbiology 60, 1-11.

Lawrence, G.L., Gilkerson, J.R., Love, D.N., Sabine, M., Whalley, J.M. (1994). Rapid, single-step differentiation of equid herpesviruses 1 and 4 from clinical material using the polymerase chain reaction and virus-specific primers. Journal of Virological Methods 47, 59-72.

Love, D. N., Bell, C.W., Pye, D., Edwards, S., Hayden, M., Lawrence, G.L., Boyle, D., Pye, T., Whalley, J.M. (1993). Expression of equine herpesvirus-1 glycoprotein-D by using a recombinant baculovirus. Journal of Virology 67, 6820-6823.

Love, D. N., Bell, C. W., Whalley, J. M. (1992). Characterization of the glycoprotein-D gene products of equine herpesvirus-1 using a prokaryotic cell expression vector. Veterinary Microbiology 30 387-394.

Lunn, D. P. (1997). Pediatric Immunology and Vaccination. Proceedings of the 43rd Annual Convention of the American Association of Equine Practitioners 43, 49-56.

Mizukoshi, F., Maeda, K., Hamano, M., Iwata, H., Matsumura, T., Kondo, T., Sugiura, T. (2002). IgG antibody subclass response against equine herpesvirus type 4 in horses. Veterinary Immunology and Immunopathology 88, 97-101.

Morein, B., Abusugra, I., Blomqvist, G., 2002. Immunity in neonates. Veterinary Immunology and Immunopathology 87, 207-213.

Munro, K.I., Wellington, J.E., Love, D.N., Whalley, J.M. (1999). Characteristics of glycoprotein B of equine herpesvirus 1 expressed by a recombinant baculovirus Veterinary Microbiology 68, 49-57.

Neubauer, A., Braun, B., Brandmuller, C., Kaaden, O.R., Osterrieder, N. (1997). Analysis of the contributions of the equine herpesvirus 1 glycoprotein gb homolog to virus entry and direct cell-to-cell spread. Virology 227, 281-294.

Osterrieder, N., Wagner, R., Brandmuller, C., Schmidt, P., Wolf, H., Kaaden, O. R. (1995). Protection against EHV-1 challenge infection in the murine model after vaccination with various formulations of recombinant glycoprotein gp14 (gB). Virology 208, 500-510.

35

Packiarajah, P., Walker, C., Gilkerson, J.R., Whalley, J.M., Love, D.N. (1998). Immune responses and protective efficacy of recombinant baculovirus-expressed glycoproteins of equine herpesvirus 1 (EHV-1) gB, gC and gD alone or in combinations in BALB/c mice. Veterinary Microbiology 61, 261-278.

Reed, J., Muench, H. (1938). A simple method of estimating fifty percent endpoints. American Journal of Hygiene 27, 493.

Rossdale, P.D., Scarnell, J. (1961). Immunization of the new-born foal against tetanus. The Veterinary Record 73, 184-185.

Ruitenberg, K.M., Love, D.N., Gilkerson, J.A., Wellington, J.E., Whalley, J.M. (2000b). Equine herpesvirus 1 (EHV-1) glycoprotein D DNA inoculation in horses with pre-existing EHV-1/EHV-4 antibody. Veterinary Microbiology 76, 117-127.

Ruitenberg, K.M., Walker, C., Love, D.N., Wellington, J.E., Whalley, J.M. (2000a). A prime-boost immunization strategy with DNA and recombinant baculovirus-expressed protein enhances protective immunogenicity of glycoprotein D of equine herpesvirus 1 in naive and infection-primed mice. Vaccine 18, 1367-1373.

Ruitenberg, K.M., Walker, C., Wellington, J.E., Love, D.N., Whalley, J.M. (1999). DNA-mediated immunization with glycoprotein D of equine herpesvirus 1 (EHV-1) in a murine model of EHV-1 respiratory infection. Vaccine 17, 237-244.

Soboll, G., Whalley, J.M., Koen, M.T., Allen, G.P., Fraser, D.G., Macklin, M.D., Swain, W.F., Lunn, D.P. (2003). Identification of equine herpesvirus-1 antigens recognized by cytotoxic T lymphocytes. Journal of General Virology 84, 2625-2634.

Stokes, A., Cameron, R.S., Marshall, R.N., Killington, R.A. (1997). High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2K(k)) murine model. Virus Research 50, 159-173.

Sugiura, T., Kondo, T., Imagawa, H. (1999). Immunoglobulin isotype responses in getah virus, equine infectious anaemia and herpesvirus-1 infections. In: Wernery, J.W.U., Mumford, J., Kaaden, O.R. (Eds) Eighth International Conference of Equine Infectious Diseases, R&W Publication, Dubai, p 220.

Sugiura, T., Kondo, T., Matsumara, T., Kamada, M. (1994). IgG subclass responses in equid herpesvirus-1 infected horses. In: Nakajima, H., Plowright, W. (Eds) Seventh International Conference on Equine Infectious Diseases, R & W Publication, Newmarket, p 350.

Tewari, D., Whalley, J.M., Love, D.N., Field, H.J. (1994). Characterization of immune responses to baculovirus-expressed equine herpesvirus type 1 glycoproteins D and H in a murine model. Journal of General Virology 75, 1735-1741.

van Oirschot, J.T., Bruin, G., de Boyer-Luytze, E., 1991. Maternal antibodies against equine influenza virus in foals and their interference with vaccination. Journal of Veterinary Medicine B 38, 391-396.

van Maanen, C., Bruin, G., de Boer-Luijtze, Smolders, G., de Boer, G.F. (1992). Interference of maternal antibodies with the immune response of foals after vaccination against equine influenza. Veterinary Quarterly 14, 13-17.

Walker, C., Ruitenberg, K.M., Love, D.N., Whalley, J.M. (2000). Immunization of BALB/c mice with DNA encoding equine herpesvirus 1 (EHV-1) glycoprotein D affords partial protection in a model of EHV-1-induced abortion. Veterinary Microbiology 76, 211-220.

Wellington, J.E., Lawrence, G.L., Love, D.N., Whalley, J.M. (1996). Expression and characterization of equine herpesvirus 1 glycoprotein D in mammalian cell lines. Archives of Virology 141, 1785-1793.

Wilson, W.D., Rossdale, P.D. (1998). Effect of age on the serological responses of thoroughbred foals to vaccination with an inactivated EHV-1/EHV-4 vaccine. In Eighth International Conference on Equine Infectious Diseases p 428. Edited by U. Wernery, J.F. Wade, J.A. Mumford, O.R. Kaaden. Newmarket, U.K.: R & W Publications.

Zhang, Z., Smith, P.M., Target, E.B., Osterrieder, N., Jennings, R., O'Callaghan, D.J. (1998). Protective immunity against equine herpesvirus type-1 (EHV-1) infection in mice induced by recombinant EHV-1 gD. Virus Research 56, 11-24.

evaluating equine immune responses to new ehv-1 vaccine … · ehv-1 dna in nasal swab samples from...

Documents