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Bovine respiratory disease (BRD) remains a major health problem despite extensive use of vaccines during the post-weaning period. Apparent vaccine failure is attributed, in part, to primary vaccination during the period of greatest risk for BRD, providing inadequate time for onset of protective immunity. The current study investigated whether intranasal (IN) vaccination of 3-6 week old calves with a modified-live viral (MLV) vaccine induced sufficient immune memory to prevent respiratory disease and accelerate onset of protective immunity 5 months later. Vaccine groups included naïve controls, a single IN vaccination at 3-6 weeks of age, primary IN vaccination at 6 months, and either an IN or subcutaneous (SC) booster vaccination at 6 months (n = 10/group). All calves were challenged with BHV-1 four days after vaccination at 6 months of age. Primary IN vaccination at 6 months did not significantly reduce clinical disease but significantly (P 0.01) reduced virus shedding. A single IN vaccination at 3-6 weeks of age significantly (P 0.05) reduced weight loss but did not reduce fever or virus shedding. Both IN and SC booster vaccinations, significantly (P 0.01) reduced clinical disease but virus shedding was significantly (P 0.001) reduced only by IN booster vaccination. Reduction in virus shedding was significantly (P 0.01) greater following booster versus primary IN vaccination at 6 months. All vaccination regimes significantly (P 0.01) reduced secondary bacterial pneumonia and altered interferon responses relative to naïve controls. Only IN booster vaccination significantly (P 0.05) increased BHV-1 specific IgA in nasal secretions. These results confirm primary MLV IN vaccination at 3 to 6 weeks of age, when virus neutralizing maternal antibody was present, induced immune memory with a 5 month duration. This immune memory supported rapid onset of protective immunity four days after an IN booster vaccination.

DNA vaccines are capable of inducing humoral and cellular immunity, and are important to control bovine herpesvirus 1 (BoHV-1), an agent of the bovine respiratory disease complex. In previous work, a DNA plasmid that encodes a secreted form of BoHV-1 glycoprotein D (pCIgD) together with commercial adjuvants provided partial protection against viral challenge of bovines. In this work, we evaluate new molecules that could potentiate the DNA vaccine. We show that a plasmid encoding a soluble CD40 ligand (CD40L) and the adjuvant Montanide™ GEL01 (GEL01) activate in vitro bovine afferent lymph dendritic cells (ALDCs). CD40L is a co-stimulating molecule, expressed transiently on activated CD4+ T cells and, to a lesser extent, on activated B cells and platelets. The interaction with its receptor, CD40, exerts effects on the presenting cells, triggering responses in the immune system. GEL01 was designed to improve transfection of DNA vaccines. We vaccinated cattle with: pCIgD; pCIgD-GEL01; pCIgD with GEL01 and CD40L plasmid (named pCIgD-CD40L-GEL01) or with pCIneo vaccines. The results show that CD40L plasmid with GEL01 improved the pCIgD DNA vaccine, increasing anti-BoHV-1 total IgGs, IgG1, IgG2 subclasses, and neutralizing antibodies in serum. After viral challenge, bovines vaccinated with pCIgD-GEL01-CD40L showed a significant decrease in viral excretion and clinical score. On the other hand, 80% of animals in group pCIgD-GEL01-CD40L presented specific anti-BoHV-1 IgG1 antibodies in nasal swabs. In addition, PBMCs from pCIgD-CD40L-GEL01 had the highest percentage of animals with a positive lymphoproliferative response against the virus and significant differences in the secretion of IFNγ and IL-4 by mononuclear cells, indicating the stimulation of the cellular immune response. Overall, the results demonstrate that a plasmid expressing CD40L associated with the adjuvant GEL01 improves the efficacy of a DNA vaccine against BoHV-1.

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle including respiratory, fetal diseases, and reproductive tract infections. Control programs usually include vaccination with a modified live viral (MLV) vaccine. On occasion BoHV-1 strains are isolated from diseased animals or fetuses postvaccination. Currently there are no markers for differentiating MLV strains from field strains of BoHV-1. In this study several BoHV-1 strains were sequenced using whole-genome sequencing technologies and the data analyzed to identify single nucleotide polymorphisms (SNPs). Strains sequenced included the reference BoHV-1 Cooper strain (GenBank Accession JX898220), eight commercial MLV vaccine strains, and 14 field strains from cases presented for diagnosis. Based on SNP analyses, the viruses could be classified into groups having similar SNP patterns. The eight MLV strains could be differentiated from one another although some were closely related to each other. A number of field strains isolated from animals with a history of prior vaccination had SNP patterns similar to specific MLV viruses, while other field isolates were very distinct from all vaccine strains. The results indicate that some BoHV-1 isolates from clinically ill cattle/fetuses can be associated with a prior MLV vaccination history, but more information is needed on the rate of BoHV-1 genome sequence change before irrefutable associations can be drawn.

Increasingly effective vaccination strategies are needed to counteract the high incidence of contagious diseases associated with intensive swine breeding. Recombinant viral vaccines are a promising new avenue in this direction. Key features of viral vectors suitable for immunoprophylaxis are safety, ease of manipulation and the ability to replicate in a variety of hosts. Most of the above requirements are met by bovine herpesvirus 4 (BoHV-4), a non-pathogenic dsDNA virus capable of infecting a broad range of cell types in vitro. Here we report the results of an exploratory study using an engineered BoHV-4 virus (eBoHV-4) expressing two unrelated glycoprotein antigens from bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1), to assess the potential of recombinant BoHV-4 as a self-adjuvanted immunogen in pigs. Free eBoHV-4 virions and virions preloaded into homologous swine adipose-derived stromal cells (SADSC) were tested. Neither virus formulation elicited neutralizing anti-BoHV-4 antibodies, nor any disease symptom, yet both induced specific immune responses against the heterologous antigens. However, a much earlier (18 vs 28 days post-infection) and more robust neutralizing response against BVDV and BoHV-1 viruses was elicited by eBoHV-4-preinfected SADSCs compared to free virions. The data validate BoHV-4 as a safe and effective heterologous antigen carrier/producer and identify SADSCs as helpful tools for the formulation of increasingly efficacious recombinant immunogens for pig vaccination.

Infectious Bovine Rhinothracheitis (IBR) caused by bovine herpesvirus 1 (BoHV-1) infection is distributed worldwide. BoHV-1 either alone or in association with other respiratory cattle pathogens causes significant economic losses to the livestock industry. The aim of this work was to validate a guinea pig model as an alternative method to the current BoHV-1 vaccine potency testing in calves. Guinea pigs were immunized with two doses of vaccine, 21 days apart and sampled at 30 days post vaccination (dpv). BoHV-1 antibody (Ab) response to vaccination in guinea pigs, measured by ELISA and virus neutralization (VN), was statistically compared to the Ab response in cattle. The guinea pig model showed a dose-response relationship to the BoVH-1 antigen concentration in the vaccine and it was able to discriminate among vaccines containing 1log(10) difference in its BoHV-1 concentration with very good repeatability and reproducibility (CV < or = 20%). A regression analysis of the Ab titers obtained in guinea pigs and bovines at 30 and 60dpv, respectively, allowed us to classify vaccines in three potency categories: "very satisfactory", "satisfactory" and "unsatisfactory". Bovines immunized with vaccines corresponding to each of these three categories were experimentally challenged with BoVH-1 virus, the level of protection, as measured by reduction of virus shedding and disease severity, correlated well with the vaccine category used. Data generated by 85 experiments, which included vaccination of calves and guinea pigs with 18 reference vaccines of known potency, 8 placebos and 18 commercial vaccines, was subjected to statistical analysis. Concordance analysis indicated almost perfect agreement between the model and the target species for Ab titers measured by ELISA and almost perfect to substantial agreement when Ab titers were measured by VN. Taken together these results indicate that the developed guinea pig model represents a novel and reliable tool to estimate batch-to-batch vaccine potency and to predict efficacy of killed BoHV-1 veterinary vaccines.

Viral infections are associated with production losses in many animal production industries. Important examples of this are Marek's disease (MD) and bovine respiratory disease (BRD) which are significant issues in the chicken and cattle industries, respectively. Viruses play key roles in MD and BRD development and consequently have also been utilised in vaccination strategies to control these diseases. Despite the widespread availability and use of vaccines to control these diseases both are still major issues for their respective industries. Here the dual role of members of viruses from the family Herpesviridae in causation and control of MD and BRD will be discussed. The technologies that may lead to the development of improved vaccines to provide more sustainable control of MD and BRD will also be identified.

Bovine herpesvirus 1 (BHV-1) causes respiratory and genital infections in cattle. Previously we demonstrated that a DNA vaccine encoding a truncated, secreted form of BHV-1 glycoprotein B (tgB) induces cytotoxic T lymphocyte (CTL) responses in C3H mice. In this study we investigated the potential of interleukin 12 (IL-12) to further enhance the CTL response. C3H mice were immunized with a plasmid encoding tgB or with plasmids encoding tgB and murine IL-12. When the plasmid encoding tgB was delivered intramuscularly or epidermally by a gene gun, co-administration with IL-12 plasmid stimulated the synthesis of more IgG2a, the production of higher levels of IFN-gamma, and more effective killing by CTLs. In contrast, after intradermal delivery no effect of co-administration of IL-12 encoding plasmid was observed. Further investigation suggested that antigen and IL-12 need to be expressed in the draining lymph nodes, where IL-12 can have a direct effect on T cells.

In the present study, cross-protection to bovine herpesvirus type 5 (BHV-5) induced by bovine herpesvirus type 1 (BHV-1) vaccination was examined following inoculation of rabbits and calves with a glycoprotein E (gE)-negative BHV-1 vaccine and subsequent challenge with BHV-5. Rabbits (n=5) and calves (n=8) were vaccinated [five rabbits intranasally (IN), four calves IN and four intramuscularly (IM)] with 7.1 log(10)median tissue culture infective dose (TCID(50)) of the BHV-1 vaccine. Rabbits and calves were challenged IN [rabbits 2 weeks post-vaccination (pv); calves 5 weeks pv] with 9.1log(10)TCID(50) of BHV-5. Two out of five vaccinated rabbits died after challenge with typical BHV-5 disease, as did 3/5 non-vaccinated controls. In calves, 4/8 vaccinated animals displayed mild signs of disease, whereas 6/6 non-vaccinated controls developed signs of disease, so severe that 2/6 had to be killed. Besides, nasal virus shedding post-challenge was not reduced by vaccination. At necropsy, on day 21 post-challenge, typical BHV-5 lesions were evident in brain tissues of both vaccinated and non-vaccinated calves. Dexametasone administration at 180 days post-infection did not reactivate clinical signs despite BHV-5 shedding in nasal secretions of both vaccinated and non-vaccinated calves. These results show that the BHV-1 vaccine evaluated here did not confer protection to BHV-5 in rabbits. In calves, BHV-1 vaccination did confer some protection to BHV-5 induced clinical disease, but it did not prevent infection and had no effect on nasal virus shedding or on the development of encephalitic lesions.

DNA vaccines have frequently been associated with poor efficacy in large animals. In the present study, one administration of an inactivated marker vaccine to cattle considerably boosted both humoral and cellular arms of the immune response primed with Bovine herpesvirus-1 (BoHV-1) DNA vaccines encoding glycoprotein D (gD) or gC+gD. Calves vaccinated according to the DNA prime-inactivated boost also showed significantly enhanced virological protection as compared to controls. The 4-logarithms reduction of virus shedding observed in primed-boosted animals was comparable to the one previously reported in calves immunized twice with marker vaccines. Intradermal immunization of cattle with DNA vaccines promoted a Th2-biased immune response but also primed a cellular component that was further boosted by the inactivated vaccine. Individual IgG2 titers of vaccinated calves were significantly correlated to IFN-gamma production. The immunization protocol described in the present study demonstrates the complementarity between DNA and conventional marker vaccines.

The relative efficacy of four commercially available inactivated bovine herpesvirus-1 (BHV-1) vaccines was directly assessed in controlled studies in 5-6-month-old, BHV-1 naïve calves. The sero-response due to the basic course of two intramuscular vaccinations was closely similar for three of four vaccines whereas one vaccine did not induce seroconversion in five of six calves. At the level of challenge virus shedding in nasal mucus, all four vaccines were significantly protective but to a markedly variable degree. Clinically, however, the relative protection ranking of the vaccines was different to that observed for challenge virus shedding. There was no obvious correlation observed between pre-challenge circulating virus neutralising antibody titre and vaccine-induced protection against virus shedding or clinical signs following the intranasal BHV-1 challenge. Present comparative findings suggest that commercially available BHV-1 vaccines are likely to be of variable potency in the natural host.

The passive immunity transferred to calves from their dams was investigated in a beef herd to determine half-life of antibody, estimated time to seronegative status and effect on immunization. One hundred two beef calves in a commercial ranch under standard management conditions were utilized. Samples were collected at branding (day 0). This was the first possible date to collect samples postcalving. This was approximately 2 months postcalving, and days 95 and 116. The calves were divided into two groups: vaccinates (51) and nonvaccinates (51). The calves were vaccinated with a commercial inactivated viral vaccine containing bovine viral diarrhea virus (BVDV)1a, BVDV2, bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV) on days 0 and 95. Half of the vaccinated and unvaccinated calves also received one dose of an experimental Mannheimia haemolytica and Pasteurella multocida vaccine at day 95. Serums were tested for neutralizing antibody titers to BVDV1a, BVDV1b, BVDV2, BHV-1, PI-3V, and BRSV. Antibodies were detected by ELISA to M. haemolytica whole cell, M. haemolytica leukotoxin, and P. multocida outer membrane protein (OMP). The mean half-life of viral antibodies in nonvaccinated calves to each virus was: BVDV1a, 23.1 days (d); BVDV1b, 22.8 d; BVDV2, 22.9 d; BHV-1, 21.2 d; PI-3V, 30.3 d; and BRSV, 35.9 d. The mean half-life of viral antibodies was greater for vaccinates than for nonvaccinates for all viruses except BRSV. The calculated mean time to seronegative status for nonvaccinates based on titers at day 0 was: BVDV1a, 192.2 d; BVDV1b, 179.1 d; BVDV2, 157.8 d; BHV-1, 122.9 d; PI-3V, 190.6 d; and BRSV, 186.7 d. There was an active immune response after vaccination with two doses to all the viruses, except BRSV. Mean antibody titers of vaccinates at day 116 were statistically higher than nonvaccinates for all viruses except BRSV. However on an individual calf basis there were few seroconversions (four-fold rise or greater to BVDV1a, BVDV1b, BVDV2, PI-3V, or BRSV; or two-fold rise for BHV-1) in the presence of viral antibodies. The predicted time of seronegative status for a group of calves for vaccination programs may not be appropriate as there may be a range of titers for all calves at day 0. In this study the range for BVDV1a was 16-16,384; BVDV1b, 8-8192; BVDV2, 0-8192; BHV-1, 0-935; PI-3V, 8-2048; and BRSV, 8-4096. Using the half-life of 23 d for BVDV1a, the time thereafter for seronegative status would be 46 and 299 d compared to the calculated date of 192.2 d using the mean of estimated time to seronegative status for all the calves. There was an active humoral response in the vaccinated calves to M. haemolytica and P. multocida. Cowherd humoral immunity based on serum antibodies should be monitored as it may relate to transfer of maternal antibodies to calves. Exceptionally high levels of viral antibodies transferred to calves could interfere with the antibody response to vaccination.

Cytokines play a key role as regulators of the immune response. To elucidate whether the efficacy of a live virus vaccine can be improved by co-expression of cytokines, expression cassettes for bovine interleukins (boIL)-2, -4, -6, and -12 and bovine interferon-gamma (boIFN-gamma) were integrated into the glycoprotein E (gE)-locus of the bovine herpesvirus 1 (BHV-1) vaccine virus strain GK/D. Cell culture analyses demonstrated that expression of the cytokines did not impair the replication of the recombinant viruses. To test safety and efficacy, groups of 4-6 months old BHV-1 seronegative calves were vaccinated intranasally with the parental virus strain GK/D or the recombinants, and challenged intranasally 3 weeks later with virulent BHV-1. The animals were monitored for clinical signs, virus excretion and antibody status after vaccination and challenge. All vaccines were well tolerated and protected the immunised calves from clinical disease following challenge, and reduced duration and titres of challenge virus shedding. Calves inoculated with the boIL-6, boIL-12 and boIFN-gamma expressing recombinants showed a significant reduction in vaccine virus shedding but secreted more challenge virus than the other vaccinees. These findings indicate that expression of these cytokines mediates a better control of the vaccine virus replication which, however, interferes with the immunogenicity of the vaccine. In summary, all recombinant viruses were safe and effective, but protection afforded by the recombinants was not improved as compared to vaccination with the parental virus strain GK/D.

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.

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.

Replication-defective human adenoviruses type 5 (HAd5) expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein gC or gD under the control of the human cytomegalovirus immediate-early promoter/enhancer (AdCMVgC or AdCMVgD) or the 5′ regulatory region of the human desmin gene (AdDESMgC or AdDESMgD) were generated. A preliminary experiment performed on rabbits showed that the intranasal administration of AdCMV elicited higher levels of BHV-1 neutralizing antibodies than the intramuscular administration of AdDESM. The obtained results allowed to select the replication-defective AdCMVgC and AdCMVgD for further assessment of their potential as a recombinant vaccine in cattle. Calves were injected intranasally twice 3 weeks apart with either AdCMVgC or AdCMVgD or a combination of these two recombinants or a commercially available live vaccine for comparison. The highest BHV-1 neutralizing antibody titres were obtained with AdCMVgD followed by the live vaccine and to a lower extent with the combination of the two recombinants (AdCMVgC+AdCMVgD). Calves were protected against intranasal BHV-1 challenge performed 3 weeks after the second immunization. In view of the obtained results, recombinant HAd5 may be developed as an intranasal vaccine vector in cattle administrated either alone or sequentially with non-human adenovirus-based vectors.

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle. Control programs in North America incorporate vaccination with modified live viral (MLV) or killed (KV) vaccine. BoHV-1 strains are isolated from diseased animals or fetuses after vaccination. There are markers for differentiating MLV from field strains using whole-genome sequencing and analysis identifying single nucleotide polymorphisms (SNPs). Using multiple primer sets and sequencing of products permits association of BoHV-1 isolates with vaccines. To determine association between vaccine virus and strains isolated from clinical cases following vaccination, we analyzed 12 BoHV-1 isolates from animals with various clinical syndromes; 9 corresponded to BoHV-1.1 respiratory group. The remaining three corresponded to BoHV-1.2b, typically found in genital tracts of cattle. Four BoHV-1 isolates were identical to a vaccine strain; three were from post-vaccination abortion episodes with typical herpetic lesions whose dams had received MLV vaccine during pregnancy, and one from a heifer given a related MLV vaccine; Sequences of two respiratory isolates perfectly matched mutations characterizing RLB106 strain, a temperature sensitive mutant used in intranasal and parenteral vaccines. The last three respiratory strains clearly appeared related to a group of MLV vaccines. Previously the MLV vaccines were grouped into four groups based on SNPs patterns. In contrast with above-mentioned isolates that closely matched SNP patterns of their respective MLV vaccine virus, these 3 strains both lacked some and possessed a number of additional mutations compared to a group of MLV vaccine viral genome. Finding BoHV-1.2b in respiratory cases indicates focus should be given BoHV-1.2b as an emerging virus or a virus not recognized nor fully characterized in BRD.

Bovine herpesvirus 1 (BoHV-1) causes respiratory infections and abortions in cattle, and is an important component of bovine respiratory disease complex, which causes a considerable economic loss worldwide. Several efforts have been made to produce safer and more effective vaccines. One of these vaccines is a glycoprotein E (gE)-deleted marker vaccine which is currently mandated for use in EU countries. In the present study, we have constructed a three-gene-mutated BoHV-1 vaccine virus (UL49.5 luminal domain residues 30–32 and cytoplasmic tail residues 80–96 deleted, gE cytoplasmic tail- and entire Us9-deleted) and compared its protective vaccine efficacy in calves after intranasal vaccination with that of a gE-deleted virus. Following vaccination, both the triple mutant and gE-deleted vaccine virus replicated well in the nasal epithelium of the calves. The vaccinated calves did not show any clinical signs. Four weeks post-vaccination, the animals were challenged intranasally with a virulent BoHV-1 wild-type virus. Based on clinical signs, both the gE-deleted and triple mutant group were protected equally against the virulent BoHV-1 challenge. However, based on the quantity and duration of nasal viral shedding, virus neutralizing antibody and cellular immune responses, the triple mutant virus vaccine induced a significantly better protective immune response than the gE-deleted virus vaccine. Notably, after the virulent BoHV-1 challenge, the triple mutant virus vaccinated group cleared the challenge virus three days earlier than the BoHV-1 gE-deleted virus vaccinated group.

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.

To assess the efficacy of a live glycoprotein E-negative bovine herpesvirus 1 (BHV1) vaccine to reduce transmission of BHV1 in cattle, a randomised, double-blind, placebo-controlled field trial including 84 herds was conducted in the Netherlands. The incidence of BHV1 infections during 17 months was monitored by detecting antibodies against BHV1 glycoprotein E. In the placebo-treated group 214 seroconversions in 3985 paired sera, and in the vaccinated group 67 seroconversions in 3601 paired sera were detected. Based on these data, the transmission ratio R 0 was estimated for each treatment, using the maximum likelihood approach and the martingale approach. In placebo-treated herds R 0 was 2.5 (CI 1.4–3.1) using maximum likelihood and 2.8 (S.E. 0.4) using the martingale approach. In the vaccinated group these estimations were 1.2 (CI 0.5–1.5) and 1.5 (S.E. 0.4) respectively. The vaccinated and placebo-treated group differed significantly in transmission of BHV1. These results suggest that the use of this live gE-negative BHV1 vaccine will reduce the incidence and transmission of BHV1 infections in the field.

The efficacy and safety of a gene-deleted bovine herpesvirus-1 (BHV-1) vaccine was determined in a bovine herpesvirus challenge trial in calves. Three different doses of the vaccine were administered intramuscularly at 10(5), 10(6) and 10(7) PFU/ml and compared to a commercial vaccine and non vaccinated control calves. Challenge was performed by intranasal aerosolization with the Cooper strain of BHV-1 (3 x 10(4) PFU/ml). The non-vaccinated calves shed significantly (P0.05) more virus than all other groups on days 4, 8 and 10 post challenge. By day 14 post challenge, antibody titers for BHV-1 of calves vaccinated with 10(7) PFU/ml were significantly (P0.05) higher than the commercial or non-vaccinated calves. Clinical scores of non-vaccinated calves were significantly (P0.05) higher than all other groups on days 4-14 post challenge. With both radioimmunoprecipitation and competitive enzyme-linked immunosorbent assays (C-ELISA), calves in the gene-deleted vaccine groups mounted comparable specific responses against gB, gC and gD post vaccination as calves in the commercial vaccine group, but in a dose dependent manner. These data suggest that the gene-deleted BHV-1 vaccine tested may be used as an effective vaccine in controlling BHV-1 infections.

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.

An inactivated glycoprotein E-negative vaccine and an experimental glycoprotein D-subunit vaccine against bovine herpesvirus 1 (V1) were examined for their effectiveness in a randomized, double-bline, placebo-controlled field trial comprising 130 dairy farms. The use of these marker vaccines enabled us to monitor the incidence of infections in vaccinated populations. The aims of this trial were to evaluate whether these vaccines: (1) reduce the proportion of outbreaks in dairy herds; and (2) reduced virus transmission within dairy herds and to what extent. Vaccination with either of the two vaccines significantly reduced the proportion of herds wherein an outbreak occurred as well as the virus transmission within herds, as compared to placebo-treated herds. The estimated number of secondary cases caused by one infectious animal, expressed as the reproduction ratio R , was for both vaccines significantly >1. This indicates that when BHV1 is introduced into vaccinated herds, major outbreaks may still occur.

Previous studies have suggested an important role of the cytokine adjuvant IL-6 in the induction of mucosal immune responses in animals, including mice. Here, we report the in vivo ability of bovine adenovirus (BAdV)-3 expressing bovine (Bo) IL-6, to influence the systemic and mucosal immune responses against bovine herpesvirus (BHV)-1 gDt in calves. To co-express both antigen and cytokine, we first constructed a recombinant BAdV-3 expressing chimeric gDt.BoIL-6 protein (BAV326). Secondly, we constructed another recombinant BAdV-3 simultaneously expressing gDt and BoIL-6 using IRES containing a bicistronic cassette gDt-IRES.IL-6, (BAV327). Recombinant proteins expressed by BAV326 and BAV327 retained antigenicity (gDt) and biological activity (BoIL-6). Intranasal immunization of calves with recombinant BAV326, BAV327 or BAV308 (gDt alone) resulted in demonstrable levels of gDt-specific IgG responses in sera and IgA response in nasal secretions, in all animals. In addition, all calves developed complement-independent neutralizing antibody responses against BHV-1. However, no significant difference could be observed in the induction of systemic or mucosal immune response in animals immunized with recombinant BAV326 or BAV327 co-expressing BoIL-6. Moreover, there was no difference in the protection against BHV-1 challenge particularly in the amount of virus excretion in the nasal cavity in calves immunized with BAV326, BAV327 or BAV308. These data suggest that the BoIL-6 had no modulating effect on the induction of gDt specific mucosal and systemic immune responses in calves.

We previously found that aqueous-based spermine-alginate or spermine-chondroitin sulfate microcapsules enhanced rotavirus-specific humoral immune responses after intramuscular inoculation of mice. To extend our observations with whole, infectious rotavirus to vaccine strategies which include inactivated virus and purified proteins, we determined the capacity of aqueous-based microcapsules to enhance virus-specific immune responses to bovine herpes virus type 1 glycoprotein D (BHV-1-gD) or ether-treated influenza virus. We found that spermine-alginate microcapsules decreased the quantity of BHV-1-gD necessary to induce protein-specific antibodies about 5000-fold. However, spermine-alginate microcapsules did not enhance influenza virus-specific antibody responses. Microcapsules composed of spermine-chondroitin sulfate did not enhance either BHV-1-gD or influenza virus-specific immune responses. Possible mechanisms of enhancement of virus-specific antibody responses by microencapsulation are discussed.

We previously reported that the bovine herpesvirus 1 (BHV 1) gene homologous to herpes simplex virus gene UL49 is dispensable; nevertheless, a mutant with the UL49 homolog (UL49h) gene deletion exhibited significantly impaired growth in cell culture. To further evaluate the role of the UL49h in virus infectivity in the natural host of BHV 1, the pathogenesis of the UL49h negative mutant was studied in cattle. An additional mutant with a combined defect in UL49h, UL49.5h and dUTPase genes was also studied in parallel. We found that both mutants were avirulent in cattle inasmuch as intranasal (i.n.) administration of either mutants induced no apparent clinical disease, nor did animals receiving the mutants shed virus. Following i.n. inoculation with the mutants animals developed low levels of serum neutralizing (SN) antibodies, and were partially protected against wild-type BHV 1 challenge. Intramuscular immunizations with either mutant induced good SN titers, and moreover, they induced nearly complete protection against respiratory challenge with wild-type virus. The results from this study establish that BHV 1 UL49h is an important virulence factor, and also suggest that deletion of the nonessential viral genes UL49h, UL49.5h and dUTPase may be useful in developing recombinant BHV 1 vaccines or BHV 1-based vaccine vectors.

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

Live virus vaccines against bovine and porcine diseases were examined for the presence of adventitious pestivirus RNA or pestiviruses by reverse transcription-polymerase chain reaction (PCR). Pestivirus RNA was detected in the live virus vaccines against Akabane disease, Ibaraki disease, infectious bovine rhinotracheitis, porcine parvovirus infection, transmissible gastroenteritis and Japanese encephalitis. Pestivirus RNA or pestivirus in the fetal bovine serum used to grow the host cells used to prepare the bovine and swine viral vaccines is a likely source of the contamination. Nucleotide sequence analysis of the PCR products suggests that modified live virus vaccines being used for immunization of cattle against bovine viral diarrhoea was not responsible for the contamination of the vaccines examined.

Reciprocal competition ELISA (rcELISA) was conducted to map monoclonal antibodies (mAbs) reactive with gI, gIII and gIV glycoproteins of bovine herpesvirus type 1 (BHV-1) into epitope groups. mAbs to glycoproteins gI and gIV were divided into six epitope groups each, while gIII mAbs had been previously divided into four areas. mAbs were chosen from each epitope group to compete in cELISA with bovine sera collected during a typical regimen of vaccination and transportation from farm to auction to feedlot. The immunodominant epitopes were identified for each BHV-1 glycoprotein. With glycoprotein gI, three epitopes defined by mAbs 1F10, D9 and 4807 were the most dominant; with glycoprotein gIII epitopes defined by mAbs G2 and 1507, and with glycoprotein gIV epitopes defined by mAbs 1102, 1106, 3C1, 3402 and 3E7 showed the maximum responses. The overall cELISA responses to each glycoprotein among two vaccination groups were also compared and it was shown that cELISA responses were significantly higher for each glycoprotein in calves receiving two vaccinations, one on the farm of origin and one at auction, than in calves receiving only one vaccination at auction.

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.

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.

A bovine herpesvirus 4 was isolated from the milk cell fraction of a healthy cow and his full genome cloned as a bacterial artificial chromosome. So cloned viral genome was used as a vector platform to deliver in vitro and in vivo an optimized secreted chimeric peptide obtained by the fusion of the bovine viral diarrhoea virus glycoprotein E2 ectodomain with the bovine herpesvirus 1 glycoprotein D ectodomain. Recombinant virus infected cells robustly expressed and secreted the chimeric peptide into the culture medium and inoculated animals with the recombinant virus successfully responded toward antigens, gE2 and gD. Thus, this work has implications for the development of safe and effective polyvalent vaccines.

Summary The objective of this study was to evaluate Th1 promoting strategies for vaccination of neonates against bovine herpesvirus-1 (BHV-1). A plasmid encoding a secreted truncated version of glycoprotein D (tgD) and tgD protein formulated with CpG oligodeoxynucleotide (ODN) effectively primed the immune system of newborn lambs, whereas without CpG ODN the tgD protein was less effective. Furthermore, a heterologous DNA prime–protein/CpG boost induced stronger and more balanced immune responses than either the DNA vaccine or a protein/CpG prime–DNA boost. Three of these strategies were compared as an approach to induce protective immunity in newborn calves with BHV-1-specific maternal antibodies. Whereas the DNA vaccine induced minimal protection, the DNA prime–protein boost resulted in reduced temperature response, weight loss and virus shedding in comparison to the placebo group. Close to complete protection against BHV-1 challenge was elicited in the calves immunized with the protein/CpG formulation, as these animals lost very little weight, had only slightly elevated temperatures and shed almost no virus.

The target animals and vaccination regimes for vaccines against the bovine rhinotracheitis (IBR) and the bovine viral diarrhoea virus (BVDV) are very similar. Therefore, we have compared different schedules for the combined use of a live IBR marker vaccine and an inactivated BVD vaccine. The neutralizing antibody response against BVDV did not reveal any differences between the group vaccinated only with the BVD vaccine and the groups that were vaccinated simultaneously (together in the same syringe) or concurrently (two separate injections) with the IBR marker vaccine at the first or second dose and the third dose of the BVD vaccine. Likewise, the bovine herpesvirus 1 (BHV-1) neutralizing antibody titres did not exhibit any negative effect by the simultaneous or concurrent use of the two products as compared to the single IBR marker vaccination. These results indicate that the two vaccines can be applied at the same day for the first or second dose of the BVD basic vaccination and then at the booster vaccinations (third dose onwards).

The chemical characterization of aqueous extracts (AE) of barks, leaves and branches and the saponin fraction denominated QB-90 obtained from Quillaja brasiliensis, a native species from Southern Brazil, show remarkable similarities to Quillaja saponaria saponins which are known as adjuvants in vaccine formulations. In vivo toxicity assays of AE and QB-90 showed not to be lethal for mice in doses ranging from 50 to 1600 microg and 50-400 microg, respectively. Experimental vaccines prepared with bovine herpesvirus type 1 (BHV-1) antigen and either AE (barks 100 microg, leaves 400 microg, branches 400 microg) or QB-90 (100 microg) were able to enhance the immune responses of mice in a comparable manner to saponins from Q. saponaria (QuilA, 100 microg). BHV-1 specific IgG, IgG1 and IgG2a antibody levels in serum were also significantly enhanced by AE, QB-90 and QuilA compared to control group (p0.05). These results showed that AE and QB-90 from Q. brasiliensis are potential candidates as adjuvants in vaccines.

To optimize the efficiency of a Minimalistic Immunogenically Defined Gene Expression (MIDGE) vector, peptides containing proven (SV40 T-antigen and bovine herpesvirus-1 VP8) or putative (herpes simplex virus-1 VP22) nuclear localization signals (NLSs) were linked to a MIDGE® vector encoding a truncated, secreted form of BHV-1 glycoprotein D (tgD) (MIDGE-tgD). Conjugation of an NLS to the MIDGE-tgD vector improved the tgD expression in vitro and the humoral and cellular immune responses induced in mice in vivo. The NLS from BHV-1 VP8 was most efficient at enhancing the tgD production and tgD-specific immune responses when conjugated to the MIDGE-tgD vector.

The effects of the vaccination of neonatal calves with a glycoprotein E (gE)-negative bovine herpesvirus type 1 (BHV-1) were investigated in naive and passively immunised calves either with the recommended dose or a 5-fold concentrated one. After inoculation (PI), all calves excreted the virus vaccine except three passively immunised calves inoculated with the lower titre. No antibody response could be detected in passively immunised calves, whatever the dose used, and they all became BHV-1 seronegative and remained so after dexamethasone treatment (PDT). Nevertheless, as shown by a gamma-interferon assay, all calves that excreted the vaccine PI developed a cell-mediated immune response and a booster response was observed PDT, suggesting viral reactivation. The vaccine virus was recovered PDT from nasal secretions in two calves and BHV-1 DNA were detected in trigeminal ganglia from five calves belonging to all inoculated groups. The results show that the BHV-1 gE-negative vaccine can establish latency not only in naive but also in passively immunised neonatal calves after a single intranasal inoculation. Moreover, this study shows for the first time that the gE-negative vaccine, when used in passively immunised calves, can lead to seronegative vaccine virus carriers.

We investigated the excretion of either a glycoprotein E (gE)-negative bovine herpesvirus type 1 (BHV1) vaccine strain or a conventional modified-live vaccine strain in both naive and passively immunised calves. The replication of gE-negative strain was considerably reduced in the maternally immunised calves, in comparison with the non-immune calves. On the other hand, the excretion of the gE-positive conventional vaccine strain was not reduced and even seemed to be prolonged in the presence of maternal antibodies. These results suggest that BHV1 gE may play a role in virus survival in the presence of antibodies.

The antibody and cell mediated immune responses induced by BHV-1 were analysed in cattle after vaccination and challenge exposure to the virulent strain LA of BHV-1. Animals were vaccinated intramuscularly (IM) with inactivated virus vaccines against BHV-1 containing either a water in mineral oil adjuvant (W/O), a water in mineral oil adjuvant plus Avridine (W/O+Avridine) or sulfolipo-cyclodextrin in squalane in-water emulsion (SL-CD/S/W). No significant differences were registered in the antibody response induced by the three evaluated vaccines. However, the BHV-1 specific cell-mediated immunite response was stronger and appeared earlier when SL-CD/S/W was included in the formulation. The efficacy of the vaccines was also evaluated after intranasal challenge of the calves with a virulent BHV-1 LA strain. Animals vaccinated with SL-CD/S/W had reduced virus excretion and clinical symptoms compared with the mock-vaccinated animals. Comparison of levels of BHV-1 specific IgG2 and IgG1 with virus shedding revealed that, regardless of the adjuvant administered, animals showing BHV-1 specific IgG2/IgG1 ratios higher than 1 were those with a significant lower number of individuals shedding virus. Additionally, animals vaccinated with SL-CD/S/W presented no post-vaccinal reactions. These factors, combined with the higher efficacy and the ease of manipulation of the biodegradable oil, makes the vaccine formulated with this new adjuvant an important contribution for the veterinary vaccines industry.

Calves were inoculated with the bovine herpes virus 1 (BHV-1) vaccine strain (RLB 106), which is a temperature sensitive mutant. The route of inoculation was intranasal instillation or intramuscular (i.m.) injection (flank or neck). As a control, five calves were given placebo by i.m. injection of the neck. Regardless of the infection route, clinical symptoms did not occur. However, BHV-1 neutralizing antibodies were detected after inoculation demonstrating that sero-conversion occurred. At 60 days post-inoculation, dexamethasone was given by i.m. injection to attempt reactivation of RLB 106. Only those calves inoculated by the intranasal route shed virus leading to an increase in BHV-1 specific antibodies. As expected, viral DNA and the latency related-RNA were detected in trigeminal ganglia (TG) of calves inoculated by the intranasal route. In contrast, viral nucleic acid was not detected in TG of calves inoculated by the i.m. route or in calves inoculated with placebo. In cervical ganglia or sacral dorsal root ganglia, viral nucleic acid was not consistently detected. This study provides evidence that efficient latency and reactivation does not occur following i.m. inoculation. Since serum-neutralizing antibodies were detected in all inoculated calves, i.m. inoculation led to sero-conversion.

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.

Major histocompatibility complex (MHC) class I molecules present endogenously derived viral peptides to CD8+ cytotoxic T-lymphocytes (CTLs). The objective of this study was to identify the H-2Dd- and H-2Kd-restricted CTL epitopes of bovine herpesvirus 1 (BHV-1), based on the allele-specific peptide motifs (ASPMs) of the above class I molecules. Nine sequences conforming to the H-2Dd and H-2Kd ASPMs were identified on BHV-1 proteins, and the respective peptides were synthesized. Five of these peptides exhibited moderate to strong binding to the Dd molecule. CTLs generated by BALB/c mice immunized with BHV-1 proteins emulsified in a suitable adjuvant effectively lysed peptide-pulsed syngeneic targets, indicating that these epitopes were generated in vivo. Mice immunized with these peptides emulsified in a suitable adjuvant also developed anti-BHV-1 CTLs. These CTLs identified three veritable CTL epitopes among the "potential epitopes" synthesized based on the ASPMs. The elucidation of the CTL epitopes of BHV-1 should aid in the development of efficacious vaccines against this virus.

Bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) engenders mucosal and systemic immunity and protects cattle from viral infection. Chimerization of cytokines with gD is being explored to confer intrinsic adjuvanticity on gD. Addition of the appropriate cytokine may convert gD into an antigen that specifically engenders protective mucosal immunity. Here DNA coding for the mature bovine interleukin-6 (IL-6) protein was fused through a synthetic glycine linker to the 3' end of DNA coding for the mature BHV-1 gD (tgD) external domain. It was cloned behind the yeast alpha prepro signal sequence and transfected into Pichia pastoris which secreted the chimeric protein (tgD-IL-6) as a 100 kDa molecule. This chimera combined the immunogenic properties of native gD and the in vitro biological activity of bovine IL-6 based on the following observations. A panel of BHV-1 gD-specific monoclonal antibodies recognizing five neutralizing epitopes on native gD reacted with tgD-IL-6. Sera from yeast tgD-IL-6-immunized mice neutralized BHV-1 infection in vitro. The chimeric protein enhanced total bovine immunoglobulin production 16-fold above tgD alone in pokeweed-stimulated bovine peripheral blood mononuclear cells (P0.05). This chimeric protein may be a potent mucosal immunogen.

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.

Using quantitative polymerase chain reaction (PCR) we have studied the latency established by wildtype (WT) bovine herpesvirus-1 (BHV-1) after challenge of cattle that had been vaccinated with a double deletion (gC-/tk-) mutant BHV-1 vaccine. Fourteen animals were vaccinated intramuscularly with 2 ml containing 10(7.4) CCID50 (cell culture infectious dose 50%) of IBRV (NG) dltkdlgC and challenged, along with six unvaccinated control animals, 30 days later with 10(8.2) CCID50 of WT BHV-1 (Cooper). The ability of this vaccine to prevent acute clinical BHV-1 infection after this challenge has been previously reported. Sixty days after challenge, eight of the vaccinates and the six control animals were euthanitized and the trigeminal ganglia (TG) examined for the amount of WT BHV-1 DNA by an internal standard quantitative PCR. The quantitative protocol that we used is based on co-amplification of BHV-1 gC specific sequences (present in WT BHV-1 but absent in the vaccine strain) and sequences from the bovine growth hormone (BGH) gene, which is used as an internal standard. The TG of the eight vaccinates contained BHV-1 WT DNA, but in a statistically significantly lower amount than the unvaccinated controls. These results are significant from the standpoint that, to our knowledge, this is the first report of a systematic quantitative approach to the study of the effect of BHV-1 vaccines on latency. This technique could be used to measure and compare the efficiency of various BHV-1 vaccines in preventing or diminishing latency, which is a significant factor for the perpetuation of BHV-1 in cattle populations.