Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP.

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.

Bluetongue Disabled Infectious Single Animal (DISA) vaccine: Studies on the optimal route and dose in sheep.

Bluetongue (BT) is a disease of ruminants caused by bluetongue virus (BTV) transmitted by biting midges of the Culicoides genus. Outbreaks have been controlled successfully by vaccination, however, currently available BT vaccines have several shortcomings. Recently, we have developed BT Disabled Infectious Single Animal (DISA) vaccines based on live-attenuated BTV without expression of dispensable non-structural NS3/NS3a protein. DISA vaccines are non-pathogenic replicating vaccines, do not cause viremia, enable DIVA and are highly protective. NS3/NS3a protein is involved in virus release, cytopathogenic effect and suppression of Interferon-I induction, suggesting that the vaccination route can be of importance. A standardized dose of DISA vaccine for serotype 8 has successfully been tested by subcutaneous vaccination. We show that 10 and 100times dilutions of this previously tested dose did not reduce the VP7 humoral response. Further, the vaccination route of DISA vaccine strongly determined the induction of VP7 directed antibodies (Abs). Intravenous vaccination induced high and prolonged humoral response but is not practical in field situations. VP7 seroconversion was stronger by intramuscular vaccination than by subcutaneous vaccination. For both vaccination routes and for two different DISA vaccine backbones, IgM Abs were rapidly induced but declined after 14days post vaccination (dpv), whereas the IgG response was slower. Interestingly, intramuscular vaccination resulted in an initial peak followed by a decline up to 21dpv and then increased again. This second increase is a steady and continuous increase of IgG Abs. These results indicate that intramuscular vaccination is the optimal route. The protective dose of DISA vaccine has not been determined yet, but it is expected to be significantly lower than of currently used BT vaccines. Therefore, in addition to the advantages of improved safety and DIVA compatibility, the novel DISA vaccines will be cost-competitive to commercially available live attenuated and inactivated vaccines for Bluetongue.

Development of a competitive ELISA for NS3 antibodies as DIVA test accompanying the novel Disabled Infectious Single Animal (DISA) vaccine for Bluetongue.

Recently, we have developed a novel vaccine for Bluetongue named BT Disabled Infectious Single Animal (DISA) vaccine. Due to the lack of non-essential NS3/NS3a protein, BT DISA vaccine is a replicating vaccine, but without the inherent risks of live-attenuated vaccines, such as residual virulence or reversion to virulence by mutations, reassortment with field virus, horizontal spread by vectors and vertical transmission. The immune response induced by BT DISA vaccines is rapidly induced, highly protective and serotype specific which is dependent on the immunodominant and serotype determining VP2 protein. The BT DISA vaccine platform provides the replacement of exclusively VP2 from different serotypes in order to safely formulate multivalent cocktail vaccines. The lack of NS3/NS3a directed antibodies by BT DISA vaccination enables differentiation of infected from vaccinated animals (DIVA principle). A highly conserved immunogenic site corresponding to the late domain was mapped in the N-terminal region of NS3. We here established an NS3-specific competitive ELISA (NS3 cELISA) as serological DIVA test accompanying BT DISA vaccines. To this end, NS3 protein missing putative transmembrane regions was produced in large amounts in bacteria and used as antigen in the NS3 cELISA which was investigated with a variety of sera. The NS3 cELISA displayed a high sensitivity and specificity similar to the commercially available VP7-specific cELISA. Results of previously performed vaccination-challenge trials with BT DISA vaccines clearly demonstrate the DIVA system based on the NS3 cELISA and BT vaccine free of NS3 protein.

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA.

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA). BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine. Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses. Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.

Vaccination with recombinant modified vaccinia virus Ankara expressing bovine respiratory syncytial virus (bRSV) proteins protects calves against RSV challenge.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and calves. Bovine RSV (bRSV) is a natural pathogen for cattle, and bRSV infection in calves shares many features with the human infection. Thus, bRSV infection in cattle provides the ideal setting to evaluate the safety and efficacy of novel RSV vaccine strategies. Here, we have evaluated the efficacy and safety of modified vaccinia virus Ankara (rMVA)-based vaccine candidates, expressing the bovine RSV-F protein, either or not in combination with the G protein, in colostrums-deprived SPF calves born by caesarean section. Vaccination induced bRSV-specific IgG and CD8 T cell responses. Importantly, no IgE responses were detected. After bRSV challenge, rMVA vaccinated calves experienced less severe symptoms of lower respiratory tract disease compared to the mock-immunized control group. Immunized animals showed reduced pulmonary virus loads, and no eosinophilic infiltration or enhanced respiratory distress. In conclusion, candidate rMVA/bRSV vaccines induced protective and safe immune responses in calves.

Vaccine-induced immunopathology during bovine respiratory syncytial virus infection: exploring the parameters of pathogenesis.

The bovine and human respiratory syncytial viruses cause severe lower respiratory tract infections. Effective vaccines against the respiratory syncytial viruses have been lacking since vaccine failures in the 1960s and 1970s. In this report, we describe a bovine respiratory syncytial virus (bRSV) challenge model in which both classical bRSV respiratory infection and vaccine-enhanced immune pathology were reproduced. The classical, formalin-inactivated (FI) bRSV vaccine that has been associated with vaccine failure was efficient in inducing high antibody titers and reducing viral loads but also primed calves for a far more serious enhanced respiratory disease after a bRSV challenge, thereby mimicking the enhanced clinical situation in FI human RSV (hRSV)-immunized and hRSV-infected infants in the 1960s. We show that immunization with FI-bRSV mainly primes a Th2-like inflammatory response that is characterized by a significant eosinophilic influx in the bronchial alveolar lung fluid and lung tissues and high levels of immunoglobulin E serum antibodies. The current model may be useful in the evaluation of new bRSV candidate vaccines for potency and safety.