Equine herpesvirus 1 glycoprotein D expressed in Pichia pastoris is hyperglycosylated and elicits a protective immune response in the mouse model of EHV-1 disease

Abstract

Equine herpesvirus 1 glycoprotein D (EHV-1 gD) has been shown in mouse models and in the natural host to have potential as a subunit vaccine, using various expression systems that included Escherichia coli, baculovirus and plasmid DNA. With the aim of producing secreted recombinant protein, we have cloned and expressed EHV-1 gD, lacking its native signal sequence and C-terminal transmembrane region, into the methylotrophic yeast Pichia pastoris. The truncated glycoprotein D (gD) gene was placed under the control of the methanol inducible alcohol oxidase 1 promoter and directed for secretion with the Saccharomyces cerevisiae α-factor prepro secretion signal. SDS-PAGE and Western blot analysis of culture supernatant fluid 24 h after induction revealed gD-specific protein products between 40 and 200 kDa. After treatment with PNGase F and Endo H, three predominant bands of 34, 45 and 48 kDa were detected, confirming high mannose N-linked glycosylation of Pichia-expressed gD (Pic-gD). N-terminal sequence analysis of PNGase F-treated affinity-purified protein showed that the native signal cleavage site of gD was being recognised by P. pastoris and the 34 kDa band could be explained by internal proteolytic cleavage effected by a putative Kex2-like protease. Pic-gD, when used in a DNA prime/protein boost inoculation schedule, induced high EHV-1 ELISA and virus neutralizing antibodies and provided protection from challenge infection in BALB/c mice.

Introduction

Equine herpesvirus 1 (EHV-1) is endemic in horse populations world-wide (Matumoto et al., 1965, Bagust et al., 1972). Disease is characterised by a mild rhinitis, abortion and occasional neurological signs (Allen and Bryans, 1986, Crabb and Studdert, 1995). EHV-1 also establishes a latent infection in the nervous system (Slater et al., 1994), from which it may be reactivated and cause new outbreaks of infection. Presently horses may be vaccinated with either modified live or inactivated whole virus vaccines to limit EHV-1 disease. However, it has proved difficult to produce vaccines that prevent infection (Gilkerson et al., 1997).

Current approaches to new vaccines include investigation of the potential of viral envelope glycoproteins as subunit vaccines. Equine herpesvirus 1 glycoprotein D (EHV-1 gD) (Audonnet et al., 1990, Flowers et al., 1991, Whalley et al., 1991) has been shown, using recombinant proteins produced in Escherichia coli or a baculovirus expression system, to elicit protective immune responses against EHV-1 challenge in a mouse model of EHV-1 respiratory disease (Tewari et al., 1994, Stokes et al., 1997, Packiarajah et al., 1998, Zhang et al., 1998). In addition, we have shown that intramuscular (i.m.) inoculation of EHV-1 gD plasmid DNA induced a long term neutralizing antibody response and led to accelerated clearance of infectious virus from lungs of mice (Ruitenberg et al., 1999a, Ruitenberg et al., 1999b) and afforded partial protection in the murine abortion model of EHV-1 infection (Walker et al., 2000). Recently we reported that i.m. inoculation of horses with EHV-1 gD DNA elevated serum antibody titres to EHV-1 gD against a background of pre-existing antibodies to EHV-1 and/or cross-reacting EHV-4 (Ruitenberg et al., 2000a). The immunogenicity and efficacy of EHV-1 gD DNA in the mouse respiratory model was enhanced by a prime-boost strategy involving a booster inoculation with baculovirus-expressed EHV-1 gD recombinant protein (Ruitenberg et al., 2000b).

An alternate expression system for the production of recombinant protein is the methylotrophic yeast Pichia pastoris which has been used to express other herpesvirus proteins including truncated bovine herpesvirus 1 glycoprotein D (BHV-1 gD) (Zhu et al., 1997), a chimeric BHV-1 gD and bovine IL-6 protein (Zhu et al., 1999) and human cytomegalovirus ppUL44 (Battista et al., 1996). P. pastoris has many of the advantages of higher eukaryotic systems including post-translational modifications, while being easy to manipulate (Cregg et al., 1993, Rosenfeld, 1999, Cereghino and Cregg, 2000). P. pastoris culture and gene expression is faster, simpler and less expensive to use than baculovirus and mammalian expression systems.

Here, we have investigated P. pastoris as a means of producing secreted, glycosylated EHV-1 gD. The EHV-1 gD gene, lacking the native signal sequence and the C-terminal transmembrane region, was cloned into the P. pastoris expression vector pPICZα which uses the methanol inducible alcohol oxidase 1 (AOX1) promoter and directs proteins for secretion with the Saccharomyces cerevisiae α-factor prepro secretion signal. We describe the expression and characterization of Pic-gD and demonstrate its ability to induce protective immune responses in a respiratory mouse model of EHV-1 infection.