Search

Intercellular spread of bovine herpesvirus-1 (BHV-1) VP22 was demonstrated in living COS-7 cells transfected with a plasmid expressing VP22-YFP (yellow fluorescence protein) and CFP (cyan fluorescence protein) bicistronically. The intercellular trafficking property of VP22 was localized to the C-terminal portion of the molecule (amino acids 121-258; VP22-C). Plasmids encoding a truncated form of BHV-1 glycoprotein D (tgD) fused to VP22, VP22-C, or the N-terminal portion of VP22 (amino acids 1-120; VP22-N) were constructed. Mice immunized with plasmid encoding tgD-VP22 or tgD-VP22-C developed stronger immune responses when compared to animals immunized with plasmid encoding tgD or tgD fused to tgD-VP22-N.

The immunogenicity and protective efficacy of a bovine herpesvirus 1 (BHV-1) subunit vaccine formulated with Emulsigen (Em) and a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) was determined in cattle. A truncated, secreted version of BHV-1 glycoprotein D (tgD) formulated with Em and CpG ODN at concentrations of 25, 2.5, or 0.25 mg/dose produced a more balanced immune response, higher levels of virus neutralizing antibodies, and greater protection after BHV-1 challenge compared to tgD adjuvanted with either Em or CpG ODN alone. In contrast, tgD formulated with Em and either 25 mg of a non-CpG ODN or another immunostimulatory compound, dimethyl dioctadecyl ammonium bromide, induced similar immunity and protection compared to tgD formulated with Em alone, a finding which confirms the immunostimulatory effect of ODN to be CpG motif mediated. Our results demonstrate the ability of CpG ODN to induce a strong and balanced immune response in a target species.

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge. Local tissue reactions generated by CpG ODN were very mild and transient, whereas reactions induced by alum or a combination of CpG ODN and alum were moderate in severity and duration. These data demonstrate that CpG ODN causes minimal injection site reactions and yet acts as an effective adjuvant in cattle.

Mucosal surfaces are the primary sites for the transmission of infectious agents including viruses, so effective vaccines generally should induce mucosal immunity. Furthermore, noninvasive delivery is desirable because of the ease of application, the high degree of patient compliance, and the improved safety for patients and clinicians due to the elimination of needles. Unfortunately, most of the conventional vaccines are parenterally administered and result in systemic rather than mucosal immunity. Here we present the first report of mucosal immunity by noninvasive DNA immunization in a target species. As an approach to induce mucosal immunity against bovine herpesvirus-1, cows were immunized intravaginally with suppositories containing plasmid coding for glycoprotein D. Serum IgG, as well as IgA both in the serum and in the nasal fluids, were detected, which supports the contention of a common mucosal immune system. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge, which demonstrates the efficacy of suppository-based administration of DNA vaccines to target species. As this is a very practical method of delivery, it has great potential to be applied as vaccine or therapy in a variety of species., (Copyright 2001 Academic Press.)

Outbreaks of infectious bovine rhinotracheitis (IBR) have recently been observed in vaccinated feedlot calves in Alberta a few months post-arrival. To investigate the cause of these outbreaks, lung and tracheal tissues were collected from calves that died of IBR during a post-arrival outbreak of disease. Bovine herpesvirus-1 (BHV-1), the causative agent of IBR, was isolated from 6 out of 15 tissues. Of these 6 isolates, 5 failed to react with a monoclonal antibody specific for one of the epitopes on glycoprotein D, one of the most important antigens of BHV-1. The ability of one of these mutant BHV-1 isolates to cause disease in calves vaccinated with a modified-live IBR vaccine was assessed in an experimental challenge study. After one vaccination, the majority of the calves developed humoral and cellular immune responses. Secondary vaccination resulted in a substantially enhanced level of immunity in all animals. Three months after the second vaccination, calves were either challenged with one of the mutant isolates or with a conventional challenge strain of BHV-1. Regardless of the type of virus used for challenge, vaccinated calves experienced significantly (P < 0.05) less weight loss and temperature rises, had lower nasal scores, and shed less virus than non-vaccinated animals. The only statistically significant (P < 0.05) difference between the 2 challenge viruses was the amount of virus shed, which was higher in non-vaccinated calves challenged with the mutant virus than in those challenged with the conventional virus. These data show that calves vaccinated with a modified-live IBR vaccine are protected from challenge with either the mutant or the conventional virus.

Replication-competent and replication-defective bovine adenovirus type 3 recombinants expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) were tested for induction of gD specific immune responses in calves using intratracheal (1st and 2nd immunization) and sub-cutaneous (3rd immunization) route of immunization. The replication-defective recombinant BAV501 induced systemic immune responses against gD as low titers of anti gD-IgG were detected in the serum. However, the efficacy of the replication-competent BAV3.E3gD to induce gD-specific antibodies in the serum and the nasal secretions was superior to that of replication-defective BAV501 when both viruses were given at the same dosage. Partial protection from challenge was induced in calves immunized with replication-competent BAV3.E3gD. A dramatic increase in the titers of anti-gD IgG and IgA levels, both in serum and nasal secretions, following BHV-1 challenge (anamnestic response) suggested that the animals immunized with replication-defective BAV501 had been primed for gD-specific antibody responses.

Vaccination by a mucosal route is an excellent approach to the control of mucosally acquired infections. Several reports on rodents suggest that DNA vaccines can be used to achieve mucosal immunity when applied to mucosal tissues. However, with the exception of one study with pigs and another with horses, there is no information on mucosal DNA immunization of the natural host. In this study, the potential of inducing mucosal immunity in cattle by immunization with a DNA vaccine was demonstrated. Cattle were immunized with a plasmid encoding bovine herpesvirus 1 (BHV-1) glycoprotein B, which was delivered with a gene gun either intradermally or intravulvomucosally. Intravulvomucosal DNA immunization induced strong cellular immune responses and primed humoral immune responses. This was evident after BHV-1 challenge when high levels of both immunoglobulin G (IgG) and IgA were detected. Intradermal delivery resulted in lower levels of immunity than mucosal immunization. To determine whether the differences between the immune responses induced by intravulvomucosal and intradermal immunizations might be due to the efficacy of antigen presentation, the distributions of antigen and Langerhans cells in the skin and mucosa were compared. After intravulvomucosal delivery, antigen was expressed early and throughout the mucosa, but after intradermal administration, antigen expression occurred later and superficially in the skin. Furthermore, Langerhans cells were widely distributed in the mucosal epithelium but found primarily in the basal layers of the epidermis of the skin. Collectively, these observations may account for the stronger immune response induced by mucosal administration.

Particle-mediated delivery was used as a method to vaccinate ruminants with a DNA vaccine. The optimal conditions for gene gun-based delivery of gold particles into the epidermal layer of the skin were determined. After delivery of the gold particles, an inflammatory response was observed. This response occurred regardless of the presence of plasmid and therefore was a result of the physical disturbance of the skin by the gold particles. To identify transfected cells, a plasmid expressing a green fluorescent protein was delivered into the skin. Fluorescent cells were located primarily in the outermost layers of the epidermis and outside the core of gold particles deposited by the gene gun. Cattle were immunized by gene gun with a plasmid expressing a truncated, secreted form of bovine herpesvirus-1 glycoprotein D. Serum antibody responses, antigen-specific proliferation, and interferon-gamma secretion by peripheral blood lymphocytes were demonstrated. These immune responses were found to be of long duration and sufficient magnitude to protect cattle against challenge with bovine herpesvirus-1, which demonstrates the efficacy of gene gun-based delivery of DNA vaccines to target species., (Copyright 1999 Academic Press.)

The potential for DNA vaccines encoding mutated versions of the same antigen to modulate immune responses in C3H/HeN mice was investigated. We created expression plasmids that encoded several versions of glycoprotein D (gD) from bovine herpesvirus 1, including authentic membrane-anchored glycoprotein (pSLRSV.AgD), a secreted glycoprotein (pSLRSV.SgD), and an intracellular protein (pSLRSV.CgD). Immunization of an inbred strain of mice with these plasmids resulted in highly efficacious and long-lasting humoral and cell-mediated immunity. We also demonstrated that the cell compartment in which plasmid-encoded gD was expressed caused a deviation in the serum immunoglobulin (Ig) isotype profile as well as the predominant cytokines secreted from the draining lymph node. Immunization of C3H/HeN mice with DNA vaccines encoding cell-associated forms of gD resulted in a predominance of serum IgG2a and gamma interferon-secreting cells within the spleens and draining lymph nodes. In contrast, mice immunized with a secreted form of this same antigen displayed immune responses characterized by greater levels of interleukin 4 in the draining lymph node and IgG1 as the predominant serum isotype. We also showed evidence of compartmentalization of distinct immune responses within different lymphoid organs.

The potential for plasmids encoding a secreted form of bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) to elicit immune responses in passively immune mice following intramuscular immunization was investigated. In these experiments, 6- to 8-week-old female C3H/HeN or C57BL/6 mice were passively immunized with hyperimmune antisera raised against BHV-1 recombinant, truncated (secreted) gD immediately prior to immunization with plasmids. A single immunization of passively immune mice with plasmid encoding the secreted form of BHV-1 gD resulted in rapid development of both cell-mediated immunity and antibody responses. Furthermore, 50% of mice immunized with a suboptimal dose of recombinant gD formulated into an adjuvant developed significant levels of serum antibodies if mice were pre-treated with hyperimmune antisera. The apparent failure of passive polyclonal antisera to suppress the induction of immune responses to pSLRSV may be related to the immunoglobulin subtypes present in the hyperimmune sera.

To determine the potential of replication-competent (E3-deleted) bovine adenovirus-3 (BAV-3) as a delivery system for vaccine antigens in calves, we evaluated the ability of recombinant BAV-3 expressing different forms of of bovine herpesvirus-1 (BHV-1) glycoprotein gD to protect against BHV-1 infection in calves that had pre-existing BAV-3 specific antibodies. Three- to four-month-old calves, vaccinated intranasally with recombinant BAV-3 expressing full-length gD (BAV3.E3gD) or a truncated version of gD (gDt) (BAV3.E3gDt), or with E3-deleted BAV-3 (BAV3.E3d; control), were challenged with BHV-1 strain 108. Vaccination with BAV3.E3gD or BAV3.E3gDt induced gD-specific antibody responses in serum and nasal secretions, and primed calves for gD-specific lymphoproliferative responses. In addition, all calves developed complement-independent neutralizing antibodies against BHV-1. Protection against viral challenge was observed in calves vaccinated with recombinant BAV3.E3gD or BAV3.E3gDt as shown by a significant reduction in body temperature and clinical disease, and a partial reduction in the amount and duration of virus excretion in nasal secretions. These results indicate that replication-competent BAV-3-based vectors can induce protective immune responses in calves (the natural host) that have pre-existing BAV-3-specific antibodies.

Nine 2-year-old heifers having BAd3-neutralizing antibody titers between 1:120 and 1:1080 were individually exposed intranasally to an aerosol of 10(8) pfu of wild type (wt) bovine adenovirus type 3 (BAd3). Four animals were kept as non-inoculated controls. The heifers were examined daily for rectal temperature, weight gain/loss, nasal and ocular discharges, and other clinical signs for 10 d post-inoculation. None of the animals showed any sign of clinical disease. Virus excretion was observed in one animal only on Day 3 post-inoculation. All BAd3-inoculated heifers demonstrated a significant (P < 0.005, paired t-test) rise in BAd3-specific serum IgG, IgG1, or IgG2 ELISA titers and virus-neutralizing antibody titers compared to the titers before inoculation. All virus-inoculated animals demonstrated increased levels of BAd3-specific IgA ELISA titers in nasal secretions. These results suggest that in the presence of circulating BAd3-neutralizing antibodies, intranasal inoculation of cattle with wt BAd3 would result in inapparent infection.

We investigated whether pre-existing adenovirus-specific immunity influenced the development of immunity to a foreign antigen expressed by recombinant adenovirus. Active adenovirus-specific immunity was induced in cotton rats by i.n. administration of wild type human adenovirus type 5 (HAd5) two weeks before immunisation with a HAd5 vector expressing the glycoprotein D (gD) of bovine herpesvirus type 1 (gD-dE3 recombinant adenovirus). Active adenovirus-specific immunity inhibited gD-specific immune responses, following either i.n. or gastrointestinal immunisation with gD-dE3. An inhibitory effect was present even if infection with HAd5 and immunisation with gD-dE3 were 13 weeks apart. Passive transfer of adenovirus specific antibodies to cotton rats one day before immunisation, however, did not significantly inhibit gD-specific immune responses induced by i.n. immunisation with gD-dE3. Repeated administration of an adenovirus vector, therefore, may have a limited ability to deliver antigen, while passive immunity to adenovirus may not interfere with the success of immunisation.

One anticipated advantage of DNA immunization is the potential to create multivalent vaccines. We have examined the effects of mixing plasmids into single formulations using plasmids expressing four different membrane bound glycoproteins from bovine herpesvirus-1 (BHV-1), bovine parainfluenzavirus-3 (bPI3) and human influenza virus (HINF). Plasmids were delivered by intradermal injection into the tails of mice and the types of responses generated were clearly affected by the expressed antigen. Plasmids expressing glycoproteins B and D of BHV-1, and the haemagglutinin/ neuraminidase of bPI3, generated responses with a predominance of IgG1, suggestive of a Th2 type of response. In contrast, the plasmid expressing HINF haemagglutinin induced an antibody response biased towards IgG2a, indicating a Th1 type of response. In most instances the mixing of plasmids had only slight effects on the magnitude or bias of the responses to the individual components. However, under certain conditions we found that addition of a second plasmid converted an IgG2a biased response to a response with primarily IgG1 antibody. The reverse situation (i.e. an IgG1 conversion to IgG2a), however, was not found. These findings have important implications for the development of multivalent vaccines.

Using the homologous recombination machinery of E. coli, a 1.245-kb deletion was introduced in the E3 region of bovine adenovirus 3 (BAV3) genomic DNA cloned in a plasmid. Transfection of the restriction enzyme-excised, linear E3-deleted BAV3 genomic DNA into primary fetal bovine retina cells produced infectious virus (BAV3. E3d), suggesting that all the E3-specific open reading frames are nonessential for virus replication in vitro. Using a similar approach, we constructed replication-competent (BAV3.E3gD and BAV3. E3gDt) BAV3 recombinant expressing full-length (gD) or truncated (gDt) glycoprotein of bovine herpes virus 1. Recombinant gD and gDt proteins expressed by BAV3.E3gD and BAV3.E3gDt, respectively, were recognized by gD-specific monoclonal antibodies directed against conformational epitopes, suggesting that antigenicity of recombinant gD and gDt was similar to that of the native gD expressed in bovine herpes virus 1-infected cells. Intranasal immunization of cotton rats induced strong gD- and BAV3-specific IgA and IgG immune responses. These results suggest that replication-competent bovine adenovirus 3-based vectors have potential for the delivery of vaccine antigens to the mucosal surfaces of animals., (Copyright 1998 Academic Press.)

Although intramuscular (i.m.) injection of DNA encoding glycoprotein D (gD) of bovine herpesvirus-1 (BHV-1) induces immune responses in cattle, this route of delivery is inefficient. Here we assessed three parameters that may enhance the efficacy of a gD DNA vaccine in cattle. First, the immune response generated by i.m. injected plasmid expressing a secreted form of gD (tgD) was determined and found to be very similar in magnitude to the response induced by gD-expressing plasmid. Secondly, gD- and tgD-expressing plasmids were administered by intradermal (i.d.) immunization, which resulted in a superior immune response to the secreted form, but no improvement in the response to the membrane-associated form. However, the form of gD used for immunization did not influence the immunoglobulin subtype, the ratio of antigen-specific IgG1 to IgG2 being approximately 4:1. Finally, the effect of promoter strength was assessed by replacing the Rous sarcoma virus (RSV) promoter, which was used in the original experiments, with the human cytomegalovirus immediate early promoter and first intron A (HCMV/IA). Although upon transfection in vitro the HCMV/IA promoter appeared to be stronger than the RSV promoter, there was only a 2-fold higher antibody response in vivo upon i.d. injection of cattle. Protection against virus challenge was obtained in the calves immunized i.d. with tgD-encoding plasmid, as shown by a significant reduction in weight loss, virus excretion, temperature response and clinical disease. No significant protection was observed in the animals vaccinated i.d. with the gD-expressing plasmid, which correlates with the lower level of immunity pre-challenge.

To facilitate the evaluation of vaccines against bovine herpesvirus type 1 (BHV-1), a cotton rat model of intranasal (i.n.) BHV-1 infection was established. Cotton rat lung cells were similar to bovine cells in their ability to support BHV-1 replication in vitro. Furthermore, i.n. inoculation of cotton rats with BHV-1 resulted in pulmonary lesions comparable to BHV-1 infection in cattle. Using this model, the potential of i.n. and gastrointestinal (g.i.) immunization was examined with recombinant human adenoviruses expressing glycoprotein D (gD) of BHV-1 to induce protective immunity against BHV-1. The replication-competent virus (gD-dE3) was more efficient than the replication-defective virus (gD-dE1E3) in inducing gD-specific antibody in the serum and in the respiratory tract. Furthermore, i.n. immunization with gD-dE3 stimulated antigen-specific antibody-secreting cells in the lung 12 weeks following immunization. Protection against BHV-1 challenge correlated with gD-specific antibody levels such that i.n. immunization with gD-dE3 conferred complete protection, while g.i. immunization conferred only partial protection of the lungs of most animals against BHV-1 challenge. In comparison, immunization with gD-dE1E3 by either route resulted in only a partial reduction of BHV-1 titre in the respiratory tract. The results obtained demonstrate that mucosal immunization with replication-competent recombinant adenovirus expressing gD of BHV-1 can induce immunity and protection against BHV-1 challenge.

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.

The efficacy of two experimental subunit gD vaccines and two commercial whole virus vaccines was determined in a bovine herpesvirus-1 (BHV1) challenge trial. Full-length gD and a truncated, secreted form of gD (tgD) were produced using a vaccinia virus expression system and purified by affinity chromatography. Comparison of these forms of gD did not reveal significant structural or antigenic differences. Calves immunized with gD or tgD in avridine developed significantly (P < 0.05) higher neutralizing antibody titers in the serum and nasal mucosa than animals vaccinated with killed virus (KV) or modified live virus (MLV). Following challenge with BHV1, all vaccinated calves had significantly (P < 0.05) lower rectal temperatures and clinical scores than those in the placebo group. In contrast to the KV-, MLV- and placebo-vaccinated calves, the gD and tgD-immunized animals experienced minimal weight loss and virus shedding post-challenge. Glycoprotein B-specific antibodies were detected in KV- and MLV-vaccinated calves, but not in gD- or tgD-immunized animals. These data suggest that full-length or truncated gD, when formulated in an appropriate adjuvant, is more effective than two KV and MLV vaccines and may be used as a marker vaccine for concurrent vaccination and eradication programs of BHV1.

Immunization with naked plasmid DNA effectively induces both humoral and cell-mediated immunity to vaccine antigens and can confer protection against numerous infectious diseases. To explore the potential use of DNA immunization to induce rotavirus-specific immune responses, we used plasmid DNA encoding the VP4 gene of bovine rotavirus (BRV). Intrasmuscular injection of the plasmid encoding the VP4 gene into C57BI/6 mice induced cell-mediated immunity as measured by cytokine production. Although DNA immunization did not induce a detectable BRV-specific antibody response, DNA-immunized animals were primed for antibody production and a cellular immune response. Following viral inoculation, the immunized animals displayed an enhanced number of BRV-specific antibody-secreting cells and cytotoxic activity. The immune response induced by DNA immunization alone or followed by viral inoculation was biased toward IFN-gamma production (Th1-like). CD4+ lymphocytes were the major source of IFN-gamma production in the spleen following DNA immunization. In contrast, a balanced cytokine production was observed in the spleens of animals receiving whole virus. These experiments showed that DNA immunization with a gene encoding the VP4 protein of BRV stimulated a Th1-like immune response in mice, and this bias in the immune response persisted following exposures to whole virus.

Recent advances in our understanding of virulence factors of viruses and the proteins or glycoproteins involved in inducing neutralizing antibodies or cell mediated immunity are forming the foundation for the development of a new generation of viral vaccines. Using bovine herpesvirus as an example, we have identified glycoproteins gB, gC, and gD as important targets for inducing neutralizing antibody responses, with gD being able to induce the highest neutralizing and cellular responses. For subunit vaccine development, the glycoproteins were produced in both prokaryotic and eukaryotic expression systems. Glycoproteins produced in eukaryotic systems were very effective in stimulating a broad range of immune responses in cattle. These glycoproteins were then formulated into effective vaccines that prevented both virus shedding and clinical disease. Herpesviruses also served as an excellent model for the identification and deletion of specific genes which lead to attenuation. In herpesviruses, two major classes of genes can be deleted. Class I includes glycoprotein genes that are nonessential for virus replication in vitro, and Class II includes genes involved in nucleic acid metabolism. these gene deleted regions can then be replaced with genes coding for protective antigens of other pathogens to develop multivalent vaccines in a single vector. Similar approaches are being used for other viruses including vaccinia virus and adenovirus. Finally, we introduced plasmids coding for protective antigens, gB, gC, and gD, into animals and developed immunity to these antigens. This approach has the potential to revolutionize vaccination regimes of the future.

The histopathology of adenovirus pneumonia in cotton rats (Sigmodon hispidus) due to bovine adenovirus type 3-luciferase recombinant virus (BAd3-Luc), which has a 0.7 kb deletion from the early region 3 (E3) replaced with the firefly luciferase gene, was compared with that produced by the parental wild-type (wt) bovine adenovirus type 3 (BAd3). After intranasal inoculation of cotton rats with 3 x 10(7) p.f.u. of BAd3-Luc, the infectious virus titres in the lungs at various times post-infection were similar to those of animals infected with the parental virus. Quantitative analysis of histopathological changes and immunohistochemical staining showed that the character and severity of the lesions were indistinguishable in the two infections. Luciferase activity was detected in the lungs of BAd3-Luc-inoculated animals until 4 days post-infection (p.i.). Antibodies to both BAd3 and luciferase were detected in sera collected from BAd3-Luc-infected animals until at least 6 weeks p.i. These results show that Bad3-Luc produces pulmonary lesions in cotton rats similar to those of wt BAd3 and suggest that BAd3-based vectors may be suitable for the development of live recombinant virus vaccines.

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.

A truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein IV (tgIV) was produced in a novel, non-destructive expression system based upon regulation of gene expression by the bovine heat-shock protein 70A (hsp70) gene promoter in Madin Darby bovine kidney (MDBK) cells. In this system, up to 20 micrograms ml-1 of secreted tgIV, which is equivalent to the yield from 4 x 10(6) cells, was produced daily over a period of up to 18 days. Different doses of tgIV were injected intramuscularly into seronegative calves. Virus-neutralizing antibodies were induced by all doses of tgIV, both in the serum and in the nasal superficial mucosa. However, the low dose (2.3 micrograms) induced significantly (p < 0.05) lower antibody titres than the medium (7 micrograms) and high (21 micrograms) doses. The medium and high doses of tgIV conferred protection from BHV-1 infection, as demonstrated by a significant (p < 0.05) reduction in clinical signs of respiratory disease and virus shedding in the nasal secretions postchallenge. However, the 2.3 micrograms group, although partially protected, was not significantly (p > 0.05) different from the placebo group. This study demonstrated the potential of an intramuscularly administered tgIV subunit vaccine to induce mucosal immunity to BHV-1 using an economic protein production system and an acceptable vaccine formulation. In addition, a strong correlation was observed between neutralizing antibodies in the serum and nasal superficial mucosa, virus shedding and clinical disease. Thus, serum neutralizing antibody levels in tgIV-immunized animals may be a good prognosticator of protection from BHV-1 infection and disease.

Vaccination has been important in controlling a wide variety of viral and bacterial infections of man and animals. Vaccines to herpesvirus infection of cattle are no exception. The present review describes the different types of conventional vaccines that have been used to date and furthermore describes the novel approaches which are presently being implemented to develop more effective vaccines. These include subunit vaccines as well as genetically engineered modified live deletion mutants. Both these novel vaccine approaches appear to be more efficacious than conventional vaccines. Furthermore, these vaccines provide an additional dimension for control and eradication of infection by providing an opportunity to develop companion diagnostic tests to differentiate infected animals from vaccinated animals. This review summarizes these developments as well as present knowledge regarding the important host defence mechanisms required for preventing infection and aiding recovery from infection.

Mice and cattle injected with plasmids encoding bovine herpesvirus 1 (BHV-1) glycoproteins developed gene-specific antibody responses capable of neutralizing BHV-1. The ability of animals to respond serologically to DNA injections was in part dependent on the quantity of DNA injected and was also negatively affected by carrier DNA. Calves injected with a plasmid encoding BHV-1 gIV developed significant antibody titers to gIV and shed less virus than did the control calf after challenge. This report indicates the potential of DNA injection as a method of vaccination.

Rotavirus structural proteins VP4, VP6 and VP7 from Bovine Rotavirus Strain C486 were cloned and expressed in a baculovirus expression system. Combinations of the proteins were assembled into a series of virus-like particles, and a murine model was used to determine the capacity of the recombinant proteins and particles to induce protective immunity. All of the proteins induced humoral immunity as measured by an ELISA against whole virus. However, only the antisera from animals immunized with VP4 neutralized virus and inhibited haemagglutination. Challenge of neonates born to animals immunized with VP4 protein on assembled particles or in cell lysates showed protection against challenge with both homologous (bovine C486) and heterologous (SA-11) strains of rotavirus. In contrast, the offspring of mice immunized with VP6 were only partially protected. Neonates of animals immunized with virus-like particles composed of VP7 assembled on VP6 spherical particles were protected against challenge with the homotypic virus and significantly protected from a heterotypic challenge whereas unassembled VP7 protein provided only partial protection against challenge.

Interleukin-2 has been described as an effective adjuvant for a number of antigens in different host species. Previously, we demonstrated the adjuvant activity of recombinant bovine IL-2 with a glycoprotein IV (gIV) subunit vaccine from bovine herpesvirus type-1 (BHV-1). In the present study, primary antibody responses were assessed in cattle immunized with either 2 or 50 micrograms of gIV, and treated with multiple doses of IL-2 or combinations of IL-2 and IFN-alpha or IL-2 and IFN-gamma. IL-2 was able to augment significantly antibody responses detected by either ELISA or virus neutralization. More significantly, IL-2 was able to enhance antibody titres in animals immunized with only 2 micrograms gIV to levels similar to those immunized with 50 micrograms gIV in the absence of IL-2. For optimal stimulation, multiple injections of IL-2 and Avridine had to be used in the formulation; other oil adjuvants or IL-2 alone could not induce a primary serum antibody response. Addition of IFN-alpha or IFN-gamma to the IL-2/gIV/Avridine formulation did not affect any of the immune parameters tested. As IFN-alpha is an effective immunoprophylactic agent for infectious bovine rhinotracheitis (IBR), combination vaccine-immunoprophylaxis may become feasible using IL-2 as a co-adjuvant. Thus, extremely low doses of antigen and only one immunization may be an effective vaccine given in combination with interferon prophylactic treatment.

Cattle were immunized with glycoprotein IV (gIV) from bovine herpes virus-1 (BHV-1). Groups of five animals were then given either 2, 3, 4, or 5 doses of interleukin-2 (IL-2) (0.5 microgram/kg) at 12-hr intervals. Animals that received no IL-2 exhibited specific immune responses that are typical for BHV-1 infection, i.e. enhanced specific cytotoxicity, lymphocyte proliferative responses to gIV, and increased gIV-specific (ELISA) and virus-neutralizing antibodies. Treatment of animals with five doses of IL-2 significantly augmented all of these responses except serum neutralization (P less than 0.05). Furthermore, the dose of IL-2 that was selected did not induce any non-specific responses, i.e. hypergamma-globulinaemia, changes in blood chemistry, increased lymphokine-activated killer (LAK) cell activity, changes in mitogen responsiveness or alterations in the phenotypic profile of circulating lymphocytes. Nor were there any clinical changes associated with IL-2 therapy (e.g. depression, pyrexia, diarrhea). Animals that were treated with less than five doses of IL-2 also exhibited elevated immune responses, but they were not significantly different from untreated immunized controls. Interestingly, animals given five doses of IL-2 responded to minor contaminants present in the gIV preparation. This allows speculation that this dose regimen of IL-2 is not only a potent adjuvant for conventional vaccine immunizing doses, but will also allow the use of minute quantities of antigen for immunization.

Latent infections by a temperature-sensitive (ts) infectious bovine rhinotracheitis virus vaccines was produced as frequently as by non-ts vaccine virus. Thus virus could be reactivated in seven of eight ts vaccinates and six of eight non-ts vaccinates after dexamethasone treatment. Virus excretion could be detectable for 1 to 8 days at a level of 2 x 10(6) to 3 x 10(8) plaque-forming units per ml of nasal secretions. The reactivated virus was shown to be the same as the original virus used for vaccination by its inability to grow at the restrictive temperature (39 degrees C) as well as by its restriction endonuclease cleavage pattern.

The onset of protection offered by intranasal vaccination with attenuated bovine herpesvirus-1 (BHV-1) was studied in 18 calves given a virulent BHV-1 aerosol challenge inoculum and an aerosol challenge exposure to Pasteurella haemolytica. Calves challenge exposed with virus 3, 7, 11, 15, or 19 days after vaccination and challenge exposed 4 days later with Pasteurella haemolytica did not develop viral-bacterial pneumonia, whereas 2 of 3 control calves died of fibrinous bronchopneumonia 40 and 60 hours after the bacterial aerosol and the 3rd control calf had similar lesions. All vaccinated and control calves had detectable amounts of interferon at the time of viral challenge exposure. Protection was observed before detection of neutralizing antibodies to BHV-1 in nasal secretions or in serum. Protection was therefore present from day 3 through day 19 after vaccination, but the mechanism could not be explained completely by neutralizing antibody or interferon.

Three experiments were conducted with calves in which, following intramuscular or intranasal vaccination with virulent or attenuated bovine herpesvirus 1, calves were protected against bovine herpesvirus 1 -- Pasteurella haemolytica challenge. Calves receiving low doses of vaccine had lower levels of antibody and greater evidence of virus replication upon challenge than those receiving higher doses. In contrast 11/13 unvaccinated controls had fibrino-purulent pneumonia following challenge. The immune response developed later in younger calves and those given low doses of vaccine. Neutralizing antibodies to bovine herpes-virus 1 were not found in nasal secretions, but were present in serum seven days after vaccination. Bovine herpesvirus 1 was isolated before challenge from nasal secretions of calves vaccinated intranasally or intramuscularly with virulent virus but not those vaccinated intramuscularly with vaccine virus. It was concluded that both routes of vaccination with either virulent or attenuated bovine herpesvirus 1 provided protection from challenge with homologous or heterologous bovine herpesvirus 1 and that live vaccines should contain at least 10(3) plaque forming units/dose for effective immunization.

The effect of levamisole on bovine immune responses to infectious bovine rhinotracheitis virus was assessed under laboratory and commercial feedlot situations. In all instances, levamisole appeared to have a beneficial effect on antibody responses of the cattle after vaccination. In the smaller scale pilot trials, levamisole appeared to be more efficacious when given 7 days after vaccination, presumably when a large amount of viral antigens was present as a result of viral replication. In the larger feedlot trial, however, response to administration of levamisole at the time of vaccination appeared to be slightly better than if given 7 days later. In all instances that animals had an antibody response before they were challenge-exposed with virulent virus, rectal temperature responses remained below 40 C, indicating that a threshold level of immunity may be acquired after the vaccination and that elevation of this threshold level does not necessarily alter the clinical disease. However, the amount of virus replication and shedding after challenge exposure seemed to be correlated with the level of immunity. These results are discussed in relationship to the role of immunity levels to spread of virus within a feedlot.

Levamisole was shown to enhance in vitro blastogenic responses of bovine lymphocytes to nonspecific mitogens (phytohemagglutinin and pokeweed mitogen) as well as to infectious bovine rhinotracheitis virus and purified protein derivative. Greatest enhancement was observed at suboptimal concentrations of viral antigen. In addition to enhancing lymphocyte reactivity levamisole also affected macrophage activity as determined by increased Fc receptor activity and [3H]glucosamine incorporation. Levamisole (5-50 micrograms/mL) enhanced type II immune (or gamma) interferon production by macrophage-lymphocyte cultures. Administration of levamisole and attenuated infectious bovine rhinotracheitis vaccine virus in vivo did not elevate cellular or humoral responses.

Previous experiments demonstrated that an antigenic site responsible for virus neutralization and cell attachment was located on a 14,000-molecular-weight fragment of the major bovine rotavirus (BRV) glycoprotein (M. Sabara, J. E. Gilchrist, G. R. Hudson, and L. A. Babiuk, J. Virol. 53:58-66, 1985). However, it was necessary to investigate whether this fragment also had the ability to induce the production of neutralizing antibodies. Upon immunization of mice, the bovine serum albumin-conjugated 14,000-molecular-weight fragment, the unconjugated 14,000-molecular-weight fragment, and the native glycoprotein all induced a similar neutralizing antibody response, albeit to a lesser extent than did the infectious, whole virus. In addition, immuno-blot enzyme-linked immunosorbent assay analysis of the reactivity of anti-peptide serum versus anti-glycoprotein serum with the glycoprotein was very comparable. These results suggest that the 14,000-molecular-weight fragment may represent not only a biologically active region but also an immunodominant area of the glycoprotein.

The major glycoproteins gI, gIII, and gIV of bovine herpesvirus-1 (BHV-1) were found to induce high levels of antibody in cattle which could neutralize virus and participate in antibody-dependent cell cytotoxicity of BHV-1-infected cells. Immunized animals were fully protected from disease, using a BHV-1/Pasteurella haemolytica aerosol challenge model but not from infection with the virus. Thus, virus could still replicate in the nasal passages of immunized animals, although to a lesser extent than in placebo-treated animals or animals immunized with a commercial killed whole virus vaccine. Systemic spread of the virus in immunized animals did not appear to occur since there was not a dramatic alteration of leukocyte function following challenge. These results suggest that any one of the three major BHV-1 glycoproteins may be useful as a subunit vaccine either individually or in combination.