Equine herpesvirus 1 glycoprotein D expressed in E. coli provides partial protection against equine herpesvirus infection in mice and elicits virus-neutralizing antibodies in the horse

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Abstract

The envelope glycoprotein D of EHV-1 (EHV-1 gD) is essential for virus infectivity and entry of virus into cells and is a potent inducer of virus-neutralizing antibody. In this study, truncated EHV-1 gD (gDt) was expressed with a C-terminal hexahistidine tag in E. coli using a pET vector. Western blot analysis using an anti-gD monoclonal antibody demonstrated the presence of gDt bands at 37.5, 36, 29.5 and 28 kDa. The immunogenicity and protective efficacy of partially purified gDt was compared with gD expressed in insect cells by a recombinant baculovirus (Bac gD) using a BALB/c mouse model of EHV-1 respiratory infection. The proteins were also compared in a prime-boost protocol following an initial inoculation with gD DNA. gDt elicited similar levels of gD-specific antibody and neutralizing antibody compared with Bac gD and also provided a similar level of protection against EHV-1 challenge in mice. Inoculation of horses with gDt elicited EHV-1 gD-specific antibodies including virus-neutralizing antibody, suggesting that despite the lack of glycosylation, E. coli may be a useful vehicle for large scale production of EHV-1 gD for vaccine studies.

Introduction

The envelope glycoprotein D of EHV-1 (EHV-1 gD) is essential for virus infectivity due to its function in entry of virus into cells (Whittaker et al., 1992, Csellner et al., 2000), and it is one of the most potent inducers of virus-neutralizing antibody among the spectrum of EHV-1 proteins (Stokes et al., 1997). Monoclonal antibodies against EHV-1 gD provided passive protection against EHV-1 infection in a hamster model (Stokes et al., 1989), and a series of studies in mouse models have indicated that gD is a prime candidate as a component of subunit vaccines against EHV-1 (Tewari et al., 1994, Stokes et al., 1997, Zhang et al., 2000, Ruitenberg et al., 2001). To date most of these vaccine assessment studies on EHV-1 gD have used eukaryotic expression systems which allow for glycosylation, processing and folding that are likely to mimic those of natural infection. Using a recombinant baculovirus (Bac gD) to express full-length EHV-1 gD, we previously demonstrated protective effects in a mouse respiratory model, in association with neutralizing antibody and an inferred role for both CD4+ and CD8+ T-cells (Tewari et al., 1994). Similarly, a truncated form of EHV-1 gD expressed in insect cells generated protective responses associated with neutralizing antibody, in contrast to weaker effects from gD expressed as a β-galactosidase fusion protein in E. coli (Stokes et al., 1997). EHV-1 gD DNA expressed by a mammalian expression vector or in a prime-boost combination with baculovirus-expressed gD also resulted in accelerated clearance of challenge virus from lungs (Ruitenberg et al., 1999a, Ruitenberg et al., 2000b). A lowered prevalence of abortigenic effects of EHV-1 was observed in mice inoculated with gD DNA (Walker et al., 2000). Using truncated EHV-1 gD expressed in the yeast Pichia pastoris, strong protective responses were obtained in mice despite hyperglycosylation (Ruitenberg et al., 2001). However there is evidence that at least the neutralizing epitope(s) of EHV-1 gD may not require the entire three-dimensional processed molecule, as a 19-mer peptide near the N-terminus was shown to induce neutralizing antibody (Flowers and O’Callaghan, 1992). In addition a GST-gD N-terminal fusion protein expressed in E. coli induced increased rates of viral clearance in the mouse respiratory model in association with neutralizing antibody and T-cell responses (Zhang et al., 2000). A limited number of tests of EHV-1 gD immunogenicity in horses have been described using DNA (Ruitenberg et al., 2000a), a canarypox vector (Audonnet et al., 1999) or baculovirus-expressed gD (Foote et al., 2005). However, there have been no reports of testing E. coli-expressed gD products in horses. Here we report the characterization and partial purification of a truncated form of EHV-1 gD expressed with a C-terminal histidine tag in E. coli. The immunogenicity and protective efficacy of E. coli-expressed truncated gD was compared with EHV-1 gD expressed in insect cells by a recombinant baculovirus, gD DNA, and also with DNA followed by a recombinant protein boost, in a mouse model of EHV-1 respiratory infection. The antibody response of horses to the E. coli-expressed truncated EHV-1 gD was also investigated.

Section snippets

EHV-1 gD constructs

A truncated gD gene was generated by PCR using template DNA of the plasmid pRc/CMV, which carries the open reading frame of EHV-1 gD (Wellington et al., 1996a). Sequences encoding the N-terminal signal sequence and the C-terminal transmembrane region were deleted (Fig. 1) by the use of forward (5′-ATGTGCTGGATCCTGGAACATGCGAGA) and reverse (3′-AGGCTTTGTCTCGAGCTCTATGACCTC) primers, which were designed also to generate BamHI and XhoI (underlined) restriction sites, respectively. The PCR product was

Characterization of gDt

Following induction and analysis of different cell fractions, the gD products in the supernatant (‘soluble’ fraction) were shown by Western blotting to be similar to those in the total cell suspension (Fig. 2a). Expressed gDt appeared as two major products of 37.5 and 36 kDa with two other distinct bands at 29.5 and 28 kDa. These products, which were detected as early as 30 min, may represent two species, a 36 kDa product with the pelB leader sequence cleaved to yield the gD amino terminus, while

Discussion

Previous research by our group and others had demonstrated the vaccine potential of EHV-1 gD, but the recombinant gD used for those experiments were mostly glycosylated products of eukaryotic expression vectors. Here we showed that a truncated gD product expressed in E. coli, with a C-terminal hexahistidine tag, induced neutralizing antibody responses and provided similar levels of protection in a mouse model of EHV-1 disease to those provided by glycosylated gD expressed in insect cells. This

Acknowledgements

Grants from the Australian Research Council (LP0219675) and the Rural Industries Research and Development Corporation (UMA17A), and the support of Pfizer Animal Health Australia (formerly CSL Animal Health) are gratefully acknowledged. CUW was the recipient of a Macquarie University Postgraduate Scholarship, and GL received a scholarship from the New South Wales Racing Research Fund. We thank Professor David Hodgson for access to horses, Professor Michael Studdert and Dr. Carol Hartley for gG

References (30)

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