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
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)
- et al.
Serum antibody responses to equine 330 herpesvirus 1 glycoprotein D in horses, pregnant mares and young foals
Vet. Immunol. Immunopathol.
(2005) - et al.
Refolding, purification, and characterization of human erythropoietin binding protein produced in Escherichia coli
Protein Exp. Purif.
(1996) - et al.
Characterization of the glycoprotein-D gene products of equine herpesvirus-1 using a prokaryotic cell expression vector
Vet. Microbiol.
(1992) - et al.
DNA-mediated immunization with glycoprotein D of equine herpesvirus 1 (EHV-1) in a murine model of EHV-1 respiratory infection
Vaccine
(1999) - et al.
Potential of DNA-mediated vaccination for equine herpesvirus 1
Vet. Microbiol.
(1999) - et al.
Equine herpesvirus 1 (EHV-1) glycoprotein D DNA inoculation in horses with pre-existing EHV-1/EHV-4 antibody
Vet. Microbiol.
(2000) - et al.
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
(2000) - et al.
Equine herpesvirus 1 glycoprotein D expressed in Pichia pastoris is 360 hyperglycosylated and elicits a protective immune response in the mouse model of EHV-1 disease
Virus Res.
(2001) - et al.
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 Res.
(1997) - et al.
The DNA sequence of equine herpesvirus 1
Virology
(1992)
Comparison of the pathogenesis of acute equine herpesvirus 1 (EHV-1) infection in the horse and the mouse 380 model: a review
Vet. Microbiol.
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
Vet. Microbiol.
Protective immunity against equine herpesvirus type-1 (EHV-1) 400 infection in mice induced by recombinant EHV-1 gD
Virus Res.
Quantitation of virus-specific classes of antibodies following immunization of mice with attenuated equine herpesvirus 1 and viral glycoprotein D
Virology
Advances in understanding of the pathogenesis, epidemiology and immunological control of equine herpesvirus abortion
Cited by (8)
Use of Biologics in the Prevention of Infectious Diseases
2019, Large Animal Internal MedicineImmunoprotective response induced by recombinant glycoprotein D in the BALB/c respiratory mouse model of Equid alphaherpesvirus 1 infection
2019, Revista Argentina de MicrobiologiaCitation Excerpt :Most of the studies on vaccine development against EHV-1 with gD have been performed in mice by using systemic routes, and, although these vaccines stimulate the specific immune response, they do not completely prevent infection25,29,30,37. In horses, few studies on EHV-1 gD immunogenicity have been reported10,23,28,39, and all of them have shown specific serum antibody response. Recently, Liu et al.20 have evaluated the use of the live-attenuated Herpes Simplex Virus 1 vaccine strain VC2 expressing EHV-1 gD and have shown that it can efficiently infect equine cells and generate strong and protective anti-EHV-1 immune responses in mice.
Recombinant Glycoprotein B of Equine herpesvirus Type 1 Elicits Protective Immune Response against Challenge in BALB/ c Mouse Model
2023, Indian Journal of Animal ResearchExpression and characterization of equid herpesvirus 1 glycoprotein D in Pichia pastoris
2020, Arquivo Brasileiro de Medicina Veterinaria e Zootecnia