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Bovine herpesvirus 1 (BHV-1) infection may lead to conjunctivitis, upper respiratory tract problems, pneumonia, genital disorders and abortion. BHV-1 is able to spread quickly in a plaque-wise manner and invade by breaching the basement membrane (BM) barrier in the respiratory mucosa. BHV-1 Us3, a serine/threonine kinase, induces a dramatic cytoskeletal reorganization and BHV-1 Us9, a tail-anchored membrane protein, is required for axonal transport of viruses in neurons. In this study, we investigated the role of Us3 and Us9 during BHV-1 infection in the respiratory mucosa. First, we constructed and characterized BHV-1 Us3 null, Us9 null and revertant viruses. Then, we analysed the viral replication and plaque size (latitude) in Madin–Darby bovine kidney (MDBK) cells and the respiratory mucosa as well as viral penetration depth underneath the BM of the respiratory mucosa when inoculated with these recombinant viruses. Knockout of Us3 resulted in a 1 log10 reduction in viral titre and plaque size (latitude) in MDBK cells and the trachea mucosa. There were no defects in the cell-to-cell spread observed for BHV-1 Us9 null virus. Both BHV-1 Us3 null and Us9 null viruses showed a significant reduction of plaque penetration underneath the BM; however, penetration was not completely inhibited. In conclusion, the current findings demonstrated that Us3 and Us9 play an important role in the invasion of BHV-1 through the BM of the respiratory mucosa, which shows the way forward for research-based attenuation of viruses in order to make safer and better-performing vaccines.

Respiratory syncytial virus (RSV) is one of the primary causative agents of upper and lower respiratory tract infections in young children, in particular infants. Recently, we reported the protective efficacy of a RSV vaccine formulation consisting of a truncated version of the fusion (F) protein formulated with a Toll-like receptor (TLR) agonist and an immunostimulatory peptide in a carrier system (ΔF/TriAdj). To evaluate the duration of immunity induced by this vaccine candidate, we carried out long-term trials. The ΔF was formulated with triple adjuvant (TriAdj) containing either polyinosinic : polycytidylic acid (polyI : C) or cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) and administered intranasally to mice. One year after the second vaccination all mice were challenged with RSV. Both ΔF/TriAdj formulations mediated the induction of high levels of IgG1, IgG2a and virus-neutralizing antibodies, and IgA in the lungs. Based on the numbers of IFN-γ- and IL-5-secreting cells in the spleen, the immune response was slightly T-helper cell type 1 (Th1)-biased. This was confirmed by the presence of F85–93-specific CD8+ effector T cells in the lungs of both ΔF/TriAdj(polyI : C)- and ΔF/TriAdj(CpG)-immunized mice. Both ΔF/TriAdj formulations induced RSV-specific CD8+ T cells. However, ΔF/TriAdj(polyI : C) generated significantly higher IgG affinity maturation and higher numbers of RSV-specific CD8+ effector memory T cells in lungs and CD8+ central memory T cells in spleen and lymph nodes than ΔF/TriAdj(CpG). After RSV challenge, no virus replication and no evidence of vaccine-induced pathology were detected in mice immunized with either of the ΔF/TriAdj formulations, demonstrating that the duration of immunity induced with these vaccines is at least one year.

The UL47 gene product, VP8, is one of the major tegument proteins of bovine herpesvirus 1 (BoHV-1) and is subject to phosphorylation. Analysis of protein bands co-immunoprecipitated with VP8 from BoHV-1-infected cells by mass spectroscopy suggested that VP8 interacts with two protein kinases: cellular CK2 and viral US3. CK2 is a highly conserved cellular protein, expressed ubiquitously and known to phosphorylate numerous proteins. The US3 gene product is one of the viral kinases produced by BoHV-1 during infection. Interactions of CK2 and US3 with VP8 were confirmed outside the context of infection when FLAG–VP8 was expressed alone or co-expressed with US3–haemagglutinin tag in Cos-7 cells. Furthermore, VP8 and US3 were found to co-localize in the nucleus during viral infection. To explore the significance of these interactions, an in vitro kinase assay was performed, which demonstrated that VP8 is heavily phosphorylated by CK2. In the presence of the highly specific CK2 kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), phosphorylation of VP8 was significantly reduced. Phosphorylation of VP8 was also inhibited by the presence of kenpaullone, a less specific CK2 inhibitor, but not by protein kinase CK1 or protein kinase C inhibitors. When VP8 and US3 were both included in the kinase assay in the presence of DMAT, phosphorylation of VP8 was again observed. Autophosphorylation of US3 was also detected and was not inhibited by DMAT. Based on these results, it is proposed that VP8 interacts with cellular CK2 and viral US3 in BoHV-1-infected cells, and is in turn subject to kinase activities associated with both of these proteins.

At present, infections with bovine viral diarrhea virus (BVDV) type 2 occur nearly as frequently as those with BVDV type 1, so development of vaccines that protect cattle from both type 1 and type 2 BVDV has become critical. In this study, we compared various DNA prime–protein boost vaccination strategies to protect cattle from challenge with BVDV-2 using the major protective antigen of BVDV, glycoprotein E2. Calves were immunized with a plasmid encoding either type 1 E2 (E2.1) or type 2 E2 (E2.2) or with both plasmids (E2.1+E2.2). This was followed by a heterologous boost with E2.1, E2.2 or E2.1 and E2.2 protein formulated with Emulsigen and a CpG oligodeoxynucleotide. Subsequently, the calves were challenged with BVDV-2 strain 1373. All vaccinated calves developed both humoral and cell-mediated immune responses, including virus-neutralizing antibodies and IFN-γ-secreting cells in the peripheral blood. Depletion studies showed that CD4+ T cells were responsible for IFN-γ production. Furthermore, the calves vaccinated with either the E2.2 or the E2.1+E2.2 vaccines were very well protected from challenge with BVDV-2, having little leukopenia and showing no weight loss or temperature response. In addition, the animals vaccinated with the E2.1 vaccine were partially protected, so there was a certain level of cross-protection. These data demonstrate that a vaccination strategy consisting of priming with E2.2 or E2.1+E2.2 DNA and boosting with E2.2 or E2.1+E2.2 protein fully protects cattle from BVDV-2 challenge.

The objective of this study was to develop an optimal vaccination strategy for Bovine viral diarrhea virus (BVDV). The E2 protein of BVDV plays a major protective role against BVDV infection. In order to be able to compare DNA, protein and DNA prime–protein boost regimens, a plasmid was constructed encoding a secreted form of the NADL strain E2 protein (pMASIA-tPAsΔE2). Furthermore, a pure secreted recombinant ΔE2 (rΔE2) protein was produced. The rΔE2 protein was formulated with a combination of Emulsigen and CpG oligodeoxynucleotide. Groups of calves were immunized with pMASIA-tPAsΔE2 or with rΔE2, or first with pMASIA-tPAsΔE2 and then with rΔE2. To evaluate the protection against BVDV, calves were challenged with BVDV strain NY-1 after the last immunization. Although all immunized calves developed humoral and cellular immune responses, the antibody responses in the DNA prime–protein boost group were stronger than those elicited by either the DNA vaccine or the protein vaccine. In particular, E2-specific antibody titres were enhanced significantly after boosting the ΔE2 DNA-primed calves with rΔE2 protein. Moreover, protection against BVDV challenge was obtained in the calves treated with the DNA prime–protein boost vaccination regimen, as shown by a significant reduction in weight loss, viral excretion and lymphopenia, compared with the unvaccinated calves and the animals immunized with the DNA or protein only. These results demonstrate the advantage of a DNA prime–protein boost vaccination approach in an outbred species.

Virus-specific cytotoxic T lymphocytes (CTLs) are considered to be important in protection against and recovery from viral infections. In this study, several approaches to induce cytotoxicity against bovine herpesvirus 1 (BHV-1) were evaluated. Vaccination of C57BL/6 mice with BHV-1 induced a strong humoral, but no CTL, response, which may be due to downregulation of major histocompatibility complex class I molecules. In contrast, vaccinia virus expressing glycoprotein B (gB) elicited a weaker antibody response, but strong cytotoxicity, in mice. As an approach to inducing both strong humoral and cellular immune responses, a plasmid vector was then used to express gB. Both antibody and CTL responses were induced by the plasmid encoding gB in C57BL/6 and C3H mice, regardless of the type of vector backbone. This demonstrated that DNA immunization induces a broad-based immune response to BHV-1 gB. Interestingly, removal of the membrane anchor, which resulted in secretion of gB from transfected cells, did not result in reduced cytotoxicity. Here, it is shown that, compared with the cell-associated counterpart, plasmid-encoded secreted protein may induce enhanced immune responses in cattle. Therefore, calves were immunized intradermally with pMASIAtgB, a plasmid encoding the secreted form of gB (tgB), using a needle-free injection system. This demonstrated that pMASIAtgB elicited both humoral responses and activated gamma interferon-secreting CD8+ CTLs, suggesting that a DNA vaccine expressing tgB induces a CTL response in the natural host of BHV-1.

Cell-mediated immune responses to hepatitis C virus (HCV) proteins play a key role in recovery from infection. The NS3 protein of HCV is of special interest, since it is one of the most conserved proteins and NS3-specific immune responses are stronger and more frequently observed in patients resolving the infection than in chronically infected patients. Since these characteristics make NS3 an attractive vaccine candidate, the objective of this study was to optimize NS3-specific immune responses. Results from this group first demonstrated that a plasmid enriched with 24 CpG motifs (pBISIA24-NS3) tends to induce the strongest and most consistent Th1-biased immune response. Subsequently, it was shown that NS3 formulated with CpG oligodeoxynucleotide and Quil A (rNS3+CpG+Quil A) adjuvants induces a balanced immune response in mice, whereas rNS3 combined with either CpG or Quil A elicits a Th2-biased response. To further enhance NS3-specific cell-mediated immune responses, a vaccination regime consisting of priming with pBISIA24-NS3, followed by boosting with rNS3+CpG+Quil A, was explored in mice and pigs. When compared to immunization with rNS3+CpG+Quil A, this regime shifted the immune response to a Th1-type response and, accordingly, enhanced MHC I-restricted killing by cytotoxic T lymphocytes in mice. Although immunization with pBISIA24-NS3 also induced a Th1-biased response, including cytotoxicity in the mice, the humoral response was significantly lower than that induced by the DNA prime–protein boost regime. These results demonstrate the advantage of a DNA prime–protein boost approach in inducing a strong NS3-specific cell-mediated, as well as humoral, immune response, in both inbred laboratory and outbred large animal species.

The potential of CpG-enhanced plasmid DNA vectors encoding a truncated secreted form of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD) to induce enhanced immune responses in cattle was investigated. We created tgD expression plasmids containing 0, 40 or 88 copies of the hexamer 5′ GTCGTT 3′, a known pan-activating CpG motif in several species. The total tgD-specific IgG titre of calves immunized with these plasmids did not correlate with the CpG content of the plasmid backbone. However, the pBISIA88-tgD-vaccinated group showed a significantly lower IgG1:IgG2 ratio than calves immunized with pBISIA40-tgD or pMASIA-tgD, which has no CpG motifs inserted. Antigen-specific lymphocyte proliferation and IFN-γ secretion by peripheral blood mononuclear cells correlated positively with the CpG content of the vectors. In contrast, calves that received a killed BHV-1 vaccine had an IgG1-predominant isotype and low lymphocyte proliferation and IFN-γ levels. Following challenge, the pBISIA88-tgD-immunized group developed the greatest anamnestic response, the highest BHV-1 neutralization titres in serum and a significantly lower level of virus shedding than the saline control group. However, there were no significant differences in clinical symptoms of infection between the DNA-immunized groups and the saline control group. These data indicate that CpG-enhanced plasmids induce augmented immune responses and could be used to vaccinate against pathogens requiring a strong cellular response for protection.

DNA vaccines have several advantages over conventional vaccines. One of the most important characteristics is the presentation of antigen via both MHC class I and class II receptors. Although this generally results in strong T-cell responses, antibody production and protection achieved by DNA immunization are unfortunately not always adequate. In contrast, modified live virus (MLV) vaccines usually induce adequate antibody and moderate cellular responses, whereas killed vaccines tend to elicit weak immune responses in general. A DNA prime–MLV boost regimen should result in enhanced cellular immunity and possibly improved antibody production. To test this hypothesis, plasmids encoding bovine herpesvirus-1 (BHV-1) glycoproteins B and D were delivered by gene gun to the genital mucosa of cattle prior to immunization with modified live BHV-1 vaccine. The immune responses induced were compared to those of an MLV-vaccinated group and a negative control group. Although significantly enhanced T-cell responses were induced by priming with the DNA vaccine, there was no increase in antibody titres. Similar levels of protection were induced by the MLV vaccine alone and the DNA prime and MLV boost regimen, which suggests that there is no correlation between the induction of T-cell responses and protection from BHV-1 challenge.

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.

The glycoproteins of bovine herpesvirus 1 (BHV-1) play important roles in the interactions between virions and target cells. A 108 kDa glycoprotein, designated gII or gp 108, has been identified by two different panels of monoclonal antibodies. The gII- and gp 108-specific monoclonal antibodies were shown to react with the same protein, which was identified by N-terminal sequencing as the homologue of herpes simplex virus type 1 (HSV-1) gH. When BHV-1 gH was purified by immunoadsorbent chromatography, gL was co-purified. The gH-gL complex induced the production of antibodies that neutralized virus infectivity and inhibited virus penetration. Affinity-purified gH-gL did prevent penetration, but not attachment of BHV-1, which suggests that the gH-gL complex is essential for penetration of BHV-1 into susceptible cells.