Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, pregnant mares and young foals

Abstract

The envelope glycoprotein D of equine herpesvirus 1 (EHV-1 gD) has been shown in laboratory animal models to elicit protective immune responses against EHV-1 challenge, and hence is a potential vaccine antigen. Here we report that intramuscular inoculation of EHV-1 gD produced by a recombinant baculovirus and formulated with the adjuvant Iscomatrix™ elicited virus-neutralizing antibody and gD-specific ELISA antibody in the serum of over 90% of adult mixed breed horses. The virus-neutralizing antibody responses to EHV-1 gD were similar to those observed after inoculation with a commercially available killed EHV-1/4 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. Isotype analysis revealed elevated gD-specific equine IgGa and IgGb relative to IgGc, IgG(T) and IgA in horses inoculated with EHV-1 gD or with the whole virus vaccine. Following inoculation of pregnant mares with EHV-1 gD, their foals had significantly higher levels of colostrally derived anti-gD antibody than foals out of uninoculated mares. The EHV-1 gD preparation did not induce a significant mean antibody response in neonatal foals following inoculation at 12 h post-partum and at 30 days of age, irrespective of the antibody status of the mare. 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.

Introduction

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal deaths and neurological disease in horse populations worldwide (Allen and Bryans, 1986, Crabb and Studdert, 1995). Extensive research efforts have been directed at developing vaccines to EHV-1 and the closely related equine herpesvirus 4 (EHV-4), and commercial vaccines (Allen, 2002) have been either live attenuated or inactivated whole virus preparations. However both EHV-1 and EHV-4 continue to circulate in horse populations vaccinated with Duvaxyn™, an inactivated EHV-1/4 vaccine. In some cases foals as young as 11 days of age are being infected from their dams who have been previously vaccinated (Foote et al., 2004). Although modified live viruses employed as vaccines may offer protection against various disease manifestations of EHV-1 (Patel et al., 2003a, Patel et al., 2003b), there is nonetheless a concern regarding their safe use.

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 for immunogenicity 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). Inoculation with EHV-1 glycoprotein D (gD) expressed by a recombinant baculovirus (Love et al., 1993) or as plasmid DNA led to protective responses in mice (Tewari et al., 1994, Ruitenberg et al., 1999). Inoculation of mice with EHV-1 gD DNA followed by recombinant protein (prime-boost) was more effective than inoculation with either protein or DNA alone (Ruitenberg et al., 2000b). However, the efficacy of such approaches in horses is not yet proven, and there have been few reports of testing recombinant glycoproteins in the horse (Audonnet et al., 1999, Ruitenberg et al., 2000a).

Here we describe the antibody response in horses to inoculation with full length gD of EHV-1 strain HVS25A produced in insect cells by a recombinant baculovirus (Love et al., 1993) and delivered with the ISCOM-related adjuvant Iscomatrix™ (Cox et al., 1997). The recombinant EHV-1 gD (EHV-1 gDr) has been shown by Western blotting to be similar in molecular mass to gD from EHV-1-infected mammalian cells, with multiple forms due partly to glycosylation (Love et al., 1993). Antibody responses to intramuscular inoculation of EHV-1 gDr were compared with those elicited by EHV-1 gD DNA, by DNA followed by recombinant protein and the whole virus EHV-1/4 vaccine Duvaxyn™. We also investigated antibody responses of mares inoculated late in gestation with EHV-1 gDr and the 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 EHV-1 gDr could elicit detectable antibody responses in such young foals.