Bovine Herpesvirus-1 gD Research Articles

A neutralizing monoclonal antibody-based blocking ELISA to detect bovine herpesvirus 1 and vaccination efficacy.

Infections caused by bovine herpesvirus 1 (BoHV-1) remain a serious global issue to the health and welfare of the bovine industry. Monitoring of neutralizing antibodies is essential not only for epidemic diagnosis, but also to assess vaccination efficacy. In this study, we generated a neutralizing monoclonal antibody, termed as 3F8, targeting glycoprotein D (gD) of BoHV-1. This monoclonal antibody could neutralize BoHV-1 with a 50% inhibitory concentration (IC50) of 37.82ng/mL. Furthermore, 3F8 could inhibit BoHV-1 infection and cell-to-cell spread at the prebinding stage. A blocking enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies against BoHV-1 was then developed based on 3F8 and protein gD generated using a baculovirus expression system. The sensitivity and specificity of the test were estimated to be 94.59% and 93.42%, respectively. A significant correlation (R2 = 0.9583, p < 0.01) was observed between the results obtained with the blocking ELISA and a virus neutralization test, which suggested that the blocking ELISA could detect neutralizing antibodies against BoHV-1. A serological survey was carried out in the dairy farms in Beijing district using 3F8-based blocking ELISA to monitor the annual neutralization antibody against BoHV-1 during 2012-2020. It revealed that the dairy farms in Beijing were at high risk of BoHV-1 infection during 2012-2017 but were protected since 2018 upon implementation of an immunization program. Our results demonstrated that this assay is suitable for BoHV-1 surveillance and vaccination efficacy in cattle as a replacement for the virus neutralization test. KEY POINTS: • Prevention of BoHV-1 infection requires the monitoring of neutralizing antibodies. • A blocking ELISA for the neutralizing antibody was developedbased on mAb 3F8 against BoHV-1 gD. • It can replace the labor-intensive and time-consuming viral neutralizing tests.

Generation by self re-fusion of bovine3 × murine2 heterohybridomas secreting virus-neutralizing bovine monoclonal antibodies to bovine herpesvirus 1 glycoproteins gB, gC, and gD

Seventy-eight heterohybridomas (HH) stably secreting bovine monoclonal antibodies (BomAb) to Bovine herpesvirus 1 (BHV1) were produced by fusing lymph node cells from a BHV1 hyperimmunized calf with 3 types of non-secreting fusion partners. Seven were generated through fusion with the murine×murine (murine2) hybridoma SP2/0, 3 through fusion with bovine–murine2 HH previously generated using cells from the same calf, and 68 through fusion with bovine2-murine2 HH previously generated by sequential fusions using cells from the same calf. The chromosome number of example HH increased with increasing numbers of input fusions. A variety of indirect fluorescent antibody assay patterns was observed using the BomAb, suggesting diverse antigen specificity. Three bovine3–murine2 HH secreted IgG1 BomAb neutralizing BHV1 without complement, and were chosen for further characterization. SDS-PAGE of detergent-solubilized BHV1 proteins bound to the 3 neutralizing BomAb demonstrated their individual specificities for BHV1 envelope glycoproteins gB, gC, and gD, the major neutralization targets for BHV1. The 3 HH stably secreted the BomAb in culture for over one year, and pilot-scale production of the BomAb was accomplished by in vivo and in vitro methods. A cocktail of the 3 BomAb was administered intravenously (i.v.) to a 6-month-old calf and its serum neutralization activity decreased with a half-life consistent with non-immune clearance, suggesting that BomAb may be useful for passive immune treatment of disease in cattle. Rabbits were passively protected by i.v. injection with each of the anti-gB and anti-gD BomAb when challenged i.v. with BHV1 24h later. Self re-fusion was shown to be advantageous for efficiently producing HH stably secreting host monoclonal antibodies. The BomAb described should prove useful in studies of the host immune response to BHV1, as reagents, and as sources of bovine immunoglobulin sequences.

Immunization of cattle with recombinant Newcastle disease virus expressing bovine herpesvirus-1 (BHV-1) glycoprotein D induces mucosal and serum antibody responses and provides partial protection against BHV-1

Bovine herpesvirus-1 (BHV-1) is a major cause of respiratory tract diseases in cattle. Vaccination of cattle against BHV-1 is a high priority. A major concern of currently modified live BHV-1 vaccines is their ability to cause latent infection and subsequent reactivation resulting in many outbreaks. Thus, there is a need for alternative strategies. We generated two recombinant Newcastle disease viruses (NDVs) expressing the glycoprotein D (gD) of BHV-1 from an added gene. One recombinant, rLaSota/gDFL, expressed gD without any modification. The other recombinant, rLaSota/gDF, expressed a chimeric gD in which the ectodomain of gD was fused with the transmembrane domain and cytoplasmic tail of the NDV fusion F glycoprotein. Remarkably, the native gD expressed by rLaSota/gDFL virus was incorporated into the NDV virion 2.5-fold more efficiently than the native NDV proteins, whereas the chimeric gD was not detectably incorporated even though it was abundantly expressed on the infected cell surface. The expression of gD did not increase the virulence of the rNDV vectors in chickens. A single intranasal and intratracheal inoculation of calves with either recombinant NDV elicited mucosal and systemic antibodies specific to BHV-1, with the responses to rLaSota/gDFL being higher than those to rLaSota/gDF. Following challenge with BHV-1, calves immunized with the recombinant NDVs had lower titers and earlier clearance of challenge virus compared to the empty vector control, and reduced disease was observed with rLaSota/gDFL. Following challenge, the titers of serum antibodies specific to BHV-1 were higher in the animals immunized with the rNDV vaccines compared to the rNDV parent virus, indicating that the vaccines primed for secondary responses. Our data suggest that NDV can be used as a vaccine vector in bovines and that BHV-1 gD may be useful in mucosal vaccine against BHV-1 infection, but might require augmentation by a second dose or the inclusion of additional BHV-1 antigens.

Caracterización genética de la cepa de vacuna egipcia Abu-Hammad del herpesvirus bovino 1

The Egyptian Abu-Hammad vaccinal strain of bovine herpesvirus-1 (BHV-1) was genetically characterised by comparing HindIII endonuclease genomic fingerprints of the Egyptian BHV-1 with reference strain Cooper 1 of BHV-1 subtype 1 (BHV-1.1). Analyses of nucleotide (nt) and deduced amino acid (aa) sequences and phylogeny of the major viral immunogen, glycoprotein D (gD), were used to compare the Egyptian BHV-1 with related alphaherpesviruses. HindIII restriction digests revealed close identity between the Egyptian BHV-1 and reference BHV-1.1. Both nt and aa sequence alignments revealed variable degrees of sequence similarity with other alphaherpesviruses. Possible mutational frameshifts were observed at nt 509 and 615 of the Egyptian BHV-1 gD. The Egyptian vaccinal BHV-1 was grouped with BHV-1.1 in a distinct branch of the phylogenetic tree. Conservation of five cysteine residues and glycosylation domains emphasised the importance of the amino terminus for immunological and biological function of alphaherpesvirus gD. The most divergent domain of 17 residues at positions 168-184 and an additional cysteine residue at position 178 distinguish the Egyptian BHV-1 from other herpesviruses. This work demonstrated that HindIII genomic fingerprinting and sequencing of the gD gene are useful for genetic characterisation of BHV-1. They may also be applied to epidemiological studies and development of BHV-1 vaccines.

Characterization of caprine herpesvirus 1 glycoprotein D gene and its translation product

Caprine herpesvirus 1 (CpHV-1) is responsible of systemic infection in neonatal kids as well as abortion and fertility disorders in adult goats. This virus is closely related to bovine herpesvirus 1 (BoHV-1) which causes infectious bovine rhinotracheitis. Glycoprotein D (gD) mediates important functions in alphaherpesviruses and is also a main immunogen. The sequence of CpHV-1 gD gene and the biochemical properties of its translation product were analyzed and compared to those of BoHV-1 and other alphaherpesviruses. A relatively high homology was found between CpHV-1 and BoHV-1 glycoproteins D amino acid sequences (similarity of 68.8%). Moreover, six cysteine residues are conserved by CpHV-1 gD and the other studied alphaherpesviruses. CpHV-1 gD has a molecular mass similar to BoHV-1 gD and contains complex N-linked oligosaccharides. In contrast to the BoHV-1 gD, CpHV-1 gD is expressed as a late protein. In spite of the observed differences which could influence its biological functions, CpHV-1 gD shares most characteristics with other alphaherpesviruses and especially BoHV-1.

Expression of the genomic form of the bovine viral diarrhea virus E2 ORF in a bovine herpesvirus-1 vector.

Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of cattle with a world-wide distribution. Recently, the possibility of using recombinant virus vectors to immunize cattle against selected BVDV genes has gained widespread interest. Among the virus vectors tested, bovine herpesvirus-1 (BHV1) provides many unique advantages. However, results of recent studies have raised the possibility that the codon usage pattern required for optimal expression in a BHV1-infected cell may be incompatible with the codon usage pattern of BVDV. If true, use of BHV1 to express BVDV proteins would require construction of synthetic BVDV genes that have been modified to resemble the codon pattern of BHV1. To explore this possibility, we constructed a BHV1 recombinant containing the genomic form of the BVDV (NADL) E2 ORF and compared expression of the E2 protein with that of the endogenous BHV1 gD protein. We observed that E2 was expressed at a significant rate compared to that of the gD protein. We conclude that codon usage problems are unlikely to constitute a serious problem for expression of BVDV proteins in BHV1 vectors.

Induction of cytotoxic T-lymphocytes specific for bovine herpesvirus-1 by DNA immunization

Cytotoxic T-lymphocytes (CTLs) are critical for the defense against herpesvirus infections, in which cell-to-cell spread occurs earlier than the hematogenous spread. The ability of bovine herpesvirus-1 (BHV-1) to undergo latency, to induce apoptosis of CD4 + T-lymphocytes, and to down-regulate the expression of major histocompatibility complex (MHC) class I molecules, necessitates the development of immunization strategies that do not involve the live virus. The objective of this study was to evaluate the feasibility of DNA immunization as a means of induction of CTLs against BHV-1. Mice were injected either by intramuscular (IM) or intradermal (ID) route with a Sindbis virus-based plasmid carrying the gene encoding the glycoprotein D (gD) of BHV-1. Splenocytes from the immunized mice were re-stimulated in vitro with gD-transduced syngeneic fibroblasts. The CTLs generated specifically lysed syngeneic targets, either transduced with gD or infected with BHV-1. IM route of inoculation induced a better CTL response when compared to ID route with respect to onset, magnitude and duration of immunity. These results indicate the feasibility of using a plasmid carrying the gene encoding BHV-1 gD as an immunogen to induce CTLs against BHV-1.

Use of chimeric nectin-1(HveC)-related receptors to demonstrate that ability to bind alphaherpesvirus gD is not necessarily sufficient for viral entry.

Human nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1-124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor.

Heat shock protein-peptide complexes elicit cytotoxic T-lymphocyte and antibody responses specific for bovine herpesvirus 1

Epitope-based vaccines offer a promising alternative to modified live vaccines against viruses such as herpesviruses which give rise to latent infections, and induce immunosuppression. The success of this approach depends on the ability to direct the CTL epitopes to the MHC class I antigen presentation pathway. The objective of this study was to evaluate the potential of the heat shock protein gp96 in this regard. A group of BALB/c mice was injected with three murine CTL epitope peptides of bovine herpesvirus 1 (BHV-1) complexed in vitro with bovine gp96 (gp96-peptides). Three other groups were injected with either the peptides alone, gp96 alone, or the peptides complexed with BSA. CTLs from mice immunized with gp96-peptides specifically lysed the peptide-pulsed syngeneic targets, as well as BHV-1-infected targets. CTLs from the other three groups did not lyse these targets. To further evaluate the utility of this approach, groups of BALB/c mice were immunized with gp96 isolated from a syngeneic cell-line transduced with BHV-1 glycoprotein D (BC-gD). Mice immunized with gp96 from BC-gD developed CTLs, as well as Abs specific for BHV-1 gD. Furthermore, in vitro stimulation of naive bovine PBMCs with gp96 from BC-gD resulted in CTLs specific for BHV-1. These results demonstrate the feasibility of using gp96-peptide complexes isolated from cells expressing BHV-1 proteins to induce CTL and Ab responses against BHV-1, without the prior knowledge of the CTL and Ab epitope sequences.

Biolistic-mediated gene transfer using the bovine herpesvirus-1 glycoprotein D is an effective delivery system to induce neutralizing antibodies in its natural host

A genetic vaccine consisting of the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) gene was constructed and administered to cattle using the biolistic (gene-gun) process. Results were compared to standard intramuscular injection of an inactivated whole BHV-1 commercial vaccine. Cattle genetically immunized by the gene-gun-delivered gD subunit vaccine developed high titers of IgG antibodies specific to gD demonstrating that this immunization method is a potent humoral response inducer. Further, gene-gun vaccinated cattle produced high neutralizing antibody titers to BHV-1 similar to levels induced in the commercial vaccine immunized animals. Additionally, cellular immunity was measured by an increased level of IFN-γ mRNA detected in PBMC of cattle immunized with the gD gene or with the commercial vaccine, whereas augmented levels of IL-4 were not detected following vaccination. Because of its simplicity and effectiveness in inducing an immune response in cattle similar to a commercial vaccine, gene-gun delivery of a subunit BHV-1 gD vaccine would be a viable alternative to current immunization protocols.

Characterization of monoclonal antibodies against bovine herpesvirus 1 gD fusion protein expressed in E. coli

A total of 20 hybridoma cell lines secreting monoclonal antibodies (MAbs) against E. coli expressed bovine herpesvirus-1 (BHV-1) gD fusion protein were produced following the fusion of Sp2/0 myeloma cells with splenocytes from BALB/c mice immunized previously with immunoaffinity purified BHV-1 gD fusion protein. An indirect fluorescent antibody test (IFAT) using BHV-1 infected MDBK cells was used for the selection of positive hybridomas secreting specific antibody. The monoclonal antibody isotypes were 11 IgM, six IgG2b, one IgG1 and two IgG3. All MAbs reacted positively with the E. coli expressed BHV-1 gD fusion protein, BHV-1 infected MDBK cell lysates and PCR BHV-1 gD transcription–translation polypeptide antigens by an ELISA.

Susceptibility of bovine antigen-presenting cells to infection by bovine herpesvirus 1 and in vitro presentation to T cells: two independent events.

The aim of the present study was to develop an in vitro system for presentation of bovine herpesvirus 1 (BHV-1) antigens to bovine T lymphocytes and to characterize the antigen-presenting cells (APC) which efficiently activate CD4(+) T cells. Two approaches were used to monitor the infection of APC by BHV-1 as follows: (i) detection of viral glycoproteins at the cell surface by immunofluorescence staining and (ii) detection of UL26 transcripts by reverse transcription-PCR. The monocytes were infected, while dendritic cells (DC) did not demonstrate any detectable viral expression. These data suggest that monocytes are one site of replication, while DC are not. The capacities of monocytes and DC to present BHV-1 viral antigens in vitro were compared. T lymphocytes (CD2(+) or CD4(+)) from BHV-1 immune cattle were stimulated in the presence of APC previously incubated with live or inactivated wild-type BHV-1. DC stimulated strong proliferation of Ag-specific T cells, while monocytes were poor stimulators of T-cell proliferation. When viral attachment to the surface of the APC was inhibited by virus pretreatment with soluble heparin, T-cell proliferation was dramatically decreased. Unexpectedly, incubation of DC and monocytes with the deletion mutant BHV-1 gD-/-, which displays impaired fusion capacity, resulted in strong activation of T lymphocytes by both APC types. Collectively, these results indicate that presentation of BHV-1 antigens to immune T cells is effective in the absence of productive infection and suggest that BHV-1 gD-/- mutant virus could be used to induce virus-specific immune responses in cattle.

Bovine Herpesvirus 1-Induced Apoptotic Cell Death: Role of Glycoprotein D

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in peripheral blood mononuclear cells and in bovine B lymphoma (BL-3) cells. Attachment but not penetration of BHV-1 is necessary to induce apoptosis in target cells, suggesting that one or more BHV-1 envelope glycoproteins could be involved in the activation of the apoptotic process. In the present study, we demonstrate that, although BHV-1 virions devoid of glycoprotein D (BHV-1 gD−/−) still bind to BL-3 cells, they are no longer able to induce apoptosis. In contrast, virions that contain glycoprotein D (gD) in the viral envelope but do not genetically encode gD (BHV-1 gD−/+) induce a level of apoptosis comparable to that produced by wild-type (wt) BHV-1. In addition, monoclonal antibodies directed against gD, but not against gB or gC, strongly reduced the high levels of apoptosis induced by BHV-1. These observations demonstrate that the induction of apoptosis is directly due to BHV-1 viral particles harboring gD in the viral envelope. Interestingly, binding of affinity-purified gD to BL-3 cells did not induce apoptosis but inhibited the ability of wt BHV-1 to induce apoptosis. Altogether, these results provide evidence for the direct or indirect involvement of gD in the mechanism by which BHV-1 induces apoptosis.

Bovine herpesvirus 1 requires glycoprotein H for infectivity and direct spreading and glycoproteins gH(W450) and gB for glycoprotein D-independent cell-to-cell spread.

By analogy with glycoprotein H (gH) of herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV), gH may also be essential for penetration and cell-to-cell spread of bovine herpes-virus 1 (BHV-1). This was verified with a gH-negative BHV-1 mutant (gH-BHV-1), which replicated normally on gH-expressing cells but was unable to form plaques and infectious progeny on non-complementing cells. The block in entry could be overcome by polyethylene glycol-induced membrane fusion, demonstrating that gH is not essential for egress. Propagation of gH-BHV-1 on cell lines expressing wild-type gH or gH(W450), which complements the function of BHV-1 gD for cell-to-cell spread, indicated that gH(W450) is more efficient than wild-type gH in mediating direct spread of BHV-1. This was supported by the plaque sizes induced by rescued gH-BHV-1 that expressed wild-type gH and gH(W450). Infection of cell lines expressing gH of BHV-1, HSV-1 and PRV with gH-BHV-1, HSV-1 and PRV mutants demonstrated that heterologous gH molecules could not complement gH function in penetration or cell-to-cell spread.

Polynucleotide vaccines in animals: enhancing and modulating responses

We immunized cattle, the natural host for bovine herpesvirus 1 (BHV-1), with a polynucleotide vaccine encoding BHV-1 glycoprotein D. These cattle trials clearly indicate that large species can be immunized with polynucleotide vaccines. Recently, using a murine model, we demonstrated that: the cellular compartment to which the expressed antigen is delivered determines the type of immune response (type 1 or type 2), and that the magnitude and direction of the immune response can be modulated by coadministration of plasmid encoded cytokines and antigen. Finally, we demonstrated that immunization of mice with a polynucleotide vaccine encoding BHV-1 gD could circumvent preexisting passively transferred, gD specific, polyclonal antisera and lead to the development of an active immune response.

Yeast-secreted bovine herpesvirus type 1 glycoprotein D has authentic conformational structure and immunogenicity

Bovine herpesvirus-1 (BHV-1) glycoprotein D (gD), an envelope glycoprotein, engenders mucosal and systemic immunity protecting cattle from viral infection. Production of gD with authentic immunogenicity is required for a subunit vaccine. We placed the truncated BHV-1 gD gene, lacking its putative transmembrane and cytoplasmic domains, under the control of the methanol-inducible AOX1 promoter in the yeast Pichia pastoris. Truncated BHV-1 gD (tgD) was efficiently secreted into the culture medium as a 68 kDa protein using either the yeast alpha prepro or native BHV-1 gD signal sequences. The yeast-secreted tgD had N-linked glycosylation and appears to have authentic conformational structure and immunogenicity based on the following observations. A panel of monoclonal antibodies recognizing five neutralizing epitopes reacted with yeast tgD. Sera from yeast tgD-immunized mice immunoprecipitated native BHV-1 gD and neutralized BHV-1 infection in vitro. Yeast tgD competitively blocked all reaction between native gD and monospecific gD polyclonal sera from cattle. Based on these data, yeast-derived BHV-1 tgD is an excellent candidate for a subunit vaccine.

From essential to beneficial: glycoprotein D loses importance for replication of bovine herpesvirus 1 in cell culture.

Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1) has been shown to be an essential component of virions involved in virus entry. gD expression in infected cells is also required for direct cell-to-cell spread. Therefore, BHV-1 gD functions are identical in these aspects to those of herpes simplex virus 1 (HSV-1) gD. In contrast, the gD homolog of pseudorabies virus (PrV), although essential for penetration, is not necessary for direct cell-to-cell spread. Cocultivation of cells infected with phenotypically gD-complemented gD- mutant BHV-1/80-221 with noncomplementing cells resulted in the isolation of the cell-to-cell-spreading gD-negative mutant ctcs+BHV-1/80-221, which was present in the gD-null BIV-1 stocks. ctcs+BHV-1/80-221 could be propagated only by mixing infected with uninfected cells, and virions released into the culture medium were noninfectious. Marker rescue experiments revealed that a single point mutation in the first position of codon 450 of the glycoprotein H open reading frame, resulting in a glycine-to-tryptophan exchange, enabled complementation of the gD function for cell-to-cell spread. After about 40 continuous passages of ctcs+BHV-1/80-221-infected cells with noninfected cells, the plaque morphology in the cultures started to change from roundish to comet shaped. Cells from such plaques produced infectious gD- virus, named gD-infBHV-1, which entered cells much more slowly than wild-type BHV-1. In contrast, integration of the gD gene into the genomes of gD-infBHV-1 and ctcs+BHV-1/80-221 resulted in recombinants with accelerated penetration in comparison to wild-type virions. In summary, our results demonstrate that under selective conditions, the function of BHV-1 gD for direct cell-to-cell spread and entry into cells can be compensated for by mutations in other viral (glyco)proteins, leading to the hypothesis that gD is involved in formation of penetration-mediating complexes in the viral envelope of which gH is a component. Together with results for PrV, varicella-zoster virus, which lacks a gD homolog, and Marek's disease virus, whose gD homolog is not essential for infectivity, our data may open new insights into the evolution of alphaherpesviruses.

Mucosal and systemic immunity to bovine herpesvirus-1 glycoprotein D confer resistance to viral replication and latency in cattle

Mucosal immunity in the respiratory tract directed against bovine herpesvirus-1 (BHV-1) glycoprotein B forms an effective barrier against BHV-1 replication in cattle. Here we investigated the ability of a second BHV-1 glycoprotein, gD, to engender specific antibodies in nasal secretion and serum and protect against infection. We expected gD to give greater protection than gB because anti-gD antibodies prevent viral penetration into cells at much lower concentrations than anti-gB antibodies. Calves vaccinated once subcutaneously and thrice intranasally with affinity-purified BHV-1 gD had mucosal antibodies and three of five were protected against intranasal challenge by 10 7 p.f.u. of BHV-1. Four of the five vaccinated calves were proven free of BHV-1 latency by the lack of viral shedding following immunosuppression. The putative mucosal adjuvant, cholera toxin B subunit (CTB), did not significantly enhance mucosal immunity or protection against challenge or latency ( P0.5) since only 4 of 6 gD plus CTB immunized calves were completely protected. Taken together, these data suggest that BHV-1 gD may be useful in a mucosal vaccine against BHV-1 infection in cattle but is less than totally effective when used alone.

Immunogenicity of bovine herpesvirus 1 glycoprotein D in mice: effect of antigen form on the induction of cellular and humoral immune responses.

For the development of veterinary subunit vaccines, modifications to the antigen may be needed to make the production of these vaccines cost effective. To investigate the effect of antigen modifications on immune response, we used glycoprotein D, one of the major glycoproteins of bovine herpesvirus-1 (BHV-1), as a model antigen. We developed a mouse model to assess the immune response elicited by immunization with either a recombinant truncated (tgD) or the authentic full-length (gD) form of BHV-1 gD in VSA3, a novel water-in-oil adjuvant. Both forms of BHV-1 gD antigen induced good levels of cell-mediated immunity, as evaluated by antigen-specific proliferative response and cytokine (IFN-gamma and IL-4) production. Following primary immunization, the humoral immune response induced by gD was superior to that elicited by vaccination with tgD. However, after a secondary immunization, a strong and similar antibody response to BHV-1 gD was induced by both forms of the antigen. The difference in immunogenicity between gD and tgD after primary immunization was not due to the loss of immunogenic epitopes in the truncated antigen or the ability to associate with the adjuvant VSA3. Our results indicate that both gD and tgD are capable of efficiently inducing a cell-mediated immune response, and although recombinant tgD is less efficient in inducing a primary humoral immune response when compared to the full-length gD, tgD effectively primed for a secondary antibody response.

Fine mapping of bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) neutralizing epitopes by type-specific monoclonal antibodies and sequence comparison with BHV-5 gD

Overlapping fragments of the bovine herpesvirus-1 (BHV-1) glycoprotein (gD) ORF were expressed as trpE-gD fusionproteins in Escherichia coli to map linear neutralizing epitopes defined by BHV-1-specific MAbs. The MAbs 3402 and R54 reacted with the expressed fragments on Western blots that located the epitopes between the amino acids 52–126 and 165–216, respectively, of gD. Bovine covalescent sera with high neutralizing antibody titers against BHV-1 reacted with these bacterially expressed proteins containing both of the epitopes. Alignment of these sequences from BHV-1 with the corresponding region of the BHV-5 gD ORF sequences (reported here) identified several amino acid mismatches. Since the MAbs 3402 and R54 neutralize the BHV-1 and not BHV-5, it was presumed that these were important amino acids in defining the epitope. To further localize the neutralizing epitopes, synthetic peptides corresponding to these regions in the BHV-1 gD ORF were tested for their capacity to block monoclonal antibody neutralization of BHV-1 infectivity. The peptides encompassing amino acids 92—106 (3402 epitope) and amino acids 202–213 (R54 epitope) of the BHV-1 gD competed with BHV-1 for the binding by MAbs 3402 and R54, respectively, in a dose-dependent manner. Antisera produced in rabbits to these peptides conjugated to a carrier reacted strongly with a 30-kDa protein by Western blotting and had neutralizing antibody titers against BHV-1.