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1.
Arch Virol ; 152(4): 717-25, 2007.
Article in English | MEDLINE | ID: mdl-17171298

ABSTRACT

Equine herpesviruses 1 and 4 (EHV-1 and EHV-4) cause equine respiratory disease worldwide. However, only EHV-1 is a cause of abortion and neurological disease, despite the two viruses having all 76 genes in common. In addition EHV-1 has a broader host range in cell culture than EHV-4, as exemplified by the rabbit kidney (RK) cell line that is permissive for EHV-1, but not for EHV-4. Here we describe that when EHV-4 produced in equine cells was inoculated onto RK cells expressing glycoprotein D of EHV-1 (RKgD1), infection developed as clusters of rounded cells, and this infectivity could be passaged in RKgD1 cells. The progeny virus could also infect single RK cells, consistent with EHV-4 acquiring EHV1 gD from the complementing cell line. No such infection was observed for EHV-4 in RK cells expressing EHV-1 glycoprotein C. The results are consistent with gD homologues being major determinants of host cell tropism and raise the possibility that gD may be a factor in the differential pathogenicity of EHV-1 and EHV-4.


Subject(s)
Herpesvirus 4, Equid/physiology , Viral Envelope Proteins/physiology , Virus Internalization , Animals , Cell Line , Herpesvirus 1, Equid/genetics , Microscopy, Confocal , Rabbits , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/genetics
2.
Vet Immunol Immunopathol ; 105(1-2): 47-57, 2005 May 01.
Article in English | MEDLINE | ID: mdl-15797474

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.


Subject(s)
Herpesviridae Infections/veterinary , Herpesvirus 1, Equid/immunology , Horse Diseases/immunology , Horse Diseases/virology , Immunization/veterinary , Viral Envelope Proteins/immunology , Adjuvants, Immunologic/pharmacology , Animals , Animals, Newborn , Antibodies, Viral/biosynthesis , Antibodies, Viral/blood , Enzyme-Linked Immunosorbent Assay/veterinary , Female , Herpesviridae Infections/immunology , Herpesviridae Infections/prevention & control , Herpesviridae Infections/virology , Horse Diseases/prevention & control , Horses , Neutralization Tests/veterinary , Pregnancy , Recombinant Proteins/immunology , Vaccines, DNA/immunology , Vaccines, Subunit/immunology , Viral Vaccines/immunology
3.
Virus Res ; 79(1-2): 125-35, 2001 Nov 05.
Article in English | MEDLINE | ID: mdl-11551653

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 alpha-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.


Subject(s)
Antigens, Viral/immunology , Herpesviridae Infections/prevention & control , Herpesvirus 1, Equid/immunology , Vaccines, Synthetic/immunology , Viral Envelope Proteins/immunology , Viral Vaccines/immunology , Animals , Antigens, Viral/genetics , Antigens, Viral/isolation & purification , Antigens, Viral/metabolism , Disease Models, Animal , Female , Gene Expression , Glycosylation , Herpesvirus 1, Equid/genetics , Horses/virology , Mice , Mice, Inbred BALB C , Pichia/metabolism , Vaccination , Vaccines, Synthetic/genetics , Vaccines, Synthetic/isolation & purification , Vaccines, Synthetic/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/isolation & purification , Viral Envelope Proteins/metabolism , Viral Vaccines/genetics , Viral Vaccines/isolation & purification , Viral Vaccines/metabolism
4.
Vet Microbiol ; 76(3): 211-20, 2000 Oct 01.
Article in English | MEDLINE | ID: mdl-10973696

ABSTRACT

DNA-mediated immunization was assessed in a murine model of equine herpesvirus 1 (EHV-1) abortion. Whilst there are differences between the model and natural infection in the horse, literature suggests that EHV-1 infection of pregnant mice can be used to assess the potential ability of vaccine candidates to protect against abortion. Female BALB/c mice were inoculated twice, 4 weeks apart, with an expression vector encoding EHV-1 glycoprotein D (gD DNA). They were mated 15 days after the second inoculation, challenged at day 15 of pregnancy and killed 3 days later. The gD DNA-inoculated mice had fewer foetuses which were damaged or had died in utero (6% in gD DNA, 21% vector DNA and 28% in nil inoculated groups challenged with EHV-1), a reduction in the stunting effect of EHV-1 infection on foetuses (gD DNA: 0.40g+/-0.06, vector DNA: 0.34g+/-0.10), reduced placental and herpesvirus-specific lung histopathology and a lower titre of virus (TCID(50)+/-SEM/lung) in maternal lung than control groups (gD DNA 4.7+/-0.3, vector 5.3+/-0.2, nil 5.6+/-0.2). Maternal antibody to EHV-1 gD was demonstrated in pups born to a dam inoculated 123 days earlier with gD DNA. Although protection from abortion was incomplete, immunization of mice with gD DNA demonstrated encouragingly the potential of this vaccine strategy.


Subject(s)
Abortion, Veterinary/prevention & control , Abortion, Veterinary/virology , Herpesviridae Infections/veterinary , Herpesvirus 1, Equid , Vaccines, DNA , Abortion, Veterinary/etiology , Animals , Antibodies, Viral/biosynthesis , DNA, Viral/immunology , Disease Models, Animal , Female , Herpesviridae Infections/complications , Herpesvirus 1, Equid/genetics , Male , Maternal-Fetal Exchange , Mice , Mice, Inbred BALB C , Pregnancy
5.
Vet Microbiol ; 76(2): 117-27, 2000 Sep 25.
Article in English | MEDLINE | ID: mdl-10946142

ABSTRACT

We have shown previously that equine herpesvirus 1 (EHV-1) glycoprotein D (gD) DNA elicited protective immune responses against EHV-1 challenge in murine respiratory and abortion models of EHV-1 disease. In this study, 20 horses, all with pre-existing antibody to EHV-4 and two with pre-existing antibody to EHV-1, were inoculated intramuscularly with three doses each of 50, 200 or 500microg EHV-1 gD DNA or with 500microg vector DNA. In 8 of 15 horses, inoculation with EHV-1 gD DNA led to elevated gD-specific antibody and nine horses exhibited increased virus neutralising (VN) antibody titres compared to those present when first inoculated. A lack of increase in gC-specific antibody during the 66 weeks of the experiment showed that the increase in gD-specific antibodies was not due to a natural infection with either EHV-1 or EHV-4. The increase in EHV-1 gD-specific antibodies was predominantly an IgGa and IgGb antibody response, similar to the isotype profile reported following natural EHV-1 infection.


Subject(s)
Antibodies, Viral/analysis , Hemagglutinins, Viral/immunology , Herpesviridae Infections/veterinary , Herpesvirus 1, Equid/immunology , Herpesvirus 4, Equid/immunology , Horse Diseases/prevention & control , Vaccination/veterinary , Vaccines, DNA , Viral Envelope Proteins/immunology , Animals , Blotting, Western/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Herpesviridae Infections/prevention & control , Horses , Neutralization Tests/veterinary
6.
Vaccine ; 18(14): 1367-73, 2000 Jan 31.
Article in English | MEDLINE | ID: mdl-10618534

ABSTRACT

The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding equine herpesvirus 1 (EHV-1) glycoprotein D (gD) followed by EHV-1 gD expressed by a recombinant baculovirus was assessed in a murine model of EHV-1 respiratory infection. Compared with mice inoculated with DNA or protein only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA and neutralizing antibody titres to EHV-1 and had accelerated clearance of virus from lungs following challenge infection. The enhanced protective effects of this consecutive immunization were also evident in mice which had a previous infection with EHV-1 and had pre-existing antibodies. The T-helper 1 (Th1) type of immune response induced by EHV-1 gD DNA was maintained after the protein boost, despite the gD protein alone appearing to direct a Th2 response.


Subject(s)
Herpesviridae Infections/prevention & control , Herpesvirus 1, Equid/immunology , Immunization, Secondary , Vaccines, DNA/immunology , Viral Envelope Proteins/immunology , Animals , Baculoviridae/genetics , Female , Injections, Intramuscular , Mice , Mice, Inbred BALB C , Vaccines, Combined/immunology , Vaccines, Combined/therapeutic use , Vaccines, DNA/therapeutic use , Vaccines, Synthetic/immunology
7.
Vet Microbiol ; 68(1-2): 35-48, 1999 Aug 16.
Article in English | MEDLINE | ID: mdl-10501160

ABSTRACT

The potential of DNA-mediated immunisation to protect against equine herpesvirus 1 (EHV-1) disease was assessed in a murine model of EHV-1 respiratory infection. Intramuscular injection with DNA encoding the EHV-1 envelope glycoprotein D (gD) in a mammalian expression vector induced a specific antibody response detectable by two weeks and maintained through 23 weeks post injection. Immune responses were proportional to the dose of DNA and a second injection markedly enhanced the antibody response. EHV-1 gD DNA-injected mice developed neutralising antibodies, and a predominance of IgG2a antibodies after the DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunised with 50 microg of EHV-1 gD DNA were able to clear virus more rapidly from lung tissue and showed reduced lung pathology in comparison with control mice. The data indicate that DNA-mediated immunisation may be a useful strategy for vaccination against EHV-1.


Subject(s)
Herpesviridae Infections/veterinary , Herpesvirus 1, Equid/immunology , Horse Diseases/prevention & control , Respiratory Tract Infections/veterinary , Viral Vaccines/standards , Animals , Antibodies, Viral/blood , Blotting, Western/veterinary , DNA, Viral/administration & dosage , Disease Models, Animal , Electrophoresis, Polyacrylamide Gel/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Female , Herpesviridae Infections/immunology , Herpesviridae Infections/prevention & control , Histocytochemistry , Horse Diseases/immunology , Horses , Injections, Intramuscular/veterinary , Lung/pathology , Mice , Mice, Inbred BALB C , Plasmids , Respiratory Tract Infections/immunology , Respiratory Tract Infections/prevention & control , Sensitivity and Specificity , Vaccination/veterinary , Vaccines, DNA/immunology , Vaccines, DNA/standards , Viral Vaccines/immunology
8.
Vaccine ; 17(3): 237-44, 1999 Jan 21.
Article in English | MEDLINE | ID: mdl-9987159

ABSTRACT

DNA-mediated immunization was assessed in a murine model of equine herpesvirus 1 (EHV-1) respiratory infection. A single intramuscular injection with plasmid DNA encoding EHV-1 glycoprotein D (EHV-1 gD), including its predicted C-terminal membrane anchor sequence, induced a specific antibody response detectable by 2 weeks and maintained through 23 weeks post injection. A second injection at 4 weeks markedly enhanced the antibody response and all EHV-1 gD-injected mice developed neutralizing antibodies. A lymphocyte proliferative response to whole EHV-1 was observed and a predominance of IgG2a antibodies after DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunized with EHV-1 gD DNA were able to clear virus significantly more rapidly from lung tissue and showed reduced lung pathology, in comparison to control mice.


Subject(s)
DNA/genetics , Herpesviridae Infections/prevention & control , Herpesvirus 1, Equid/immunology , Immunization , Respiratory Tract Infections/prevention & control , Viral Envelope Proteins/immunology , Animals , Antigen-Antibody Reactions , Disease Models, Animal , Female , Genetic Code , Injections, Intramuscular , Mice , Mice, Inbred BALB C , Plasmids/genetics
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