To investigate strategies for the preferential induction of specific types of T cell responses utilising vaccinia (VV) and bovine herpes virus (BHV) recombinants expressing respiratory syncytial virus (RSV) glycoproteins. To prepare VV and BHV recombinants expressing viral glycoproteins in native form or modified to remain intracellularly or to be produced as secreted proteins. To produce recombinants co-expressing viral proteins and selected cytokines. To analyse immune responses induced by the recombinant viruses in mice and cattle.
In order to study the influence of the site of viral protein expression in infected cells on the induction of immune responses, recombinant (r)VV expressing membrane-bound, secreted or cytoplasmic versions of the F and G proteins of RSV were prepared and used to vaccinate BALB/c mice. Mice inoculated intraperitoneally (i.p.) with rVV expressing membrane-anchored or soluble forms of the F protein developed high levels of antibody to native F protein, and were protected against RSV infection. In contrast, mice inoculated i.p. with rVV expressing versions of the F protein that were retained within the cytosol of infected cells, failed to develop antibodies to native F protein and were not protected against RSV infection. Although all versions of the F protein primed RSV-specific cytotoxic T cells, there were differences in the levels of cytotoxic activity induced. Thus, membrane bound and mutant F protein in which maturation was arrested before the mid-Golgi, induced high levels of CTL, whereas, secreted F and mutants in which the signal sequence is deleted, induced only low levels of CTL. Analysis of the immune responses induced by rVV expressing membrane-anchored, secreted or cytoplasmic versions of the G protein are in progress.
In order to investigate the effect of the route of vaccination on priming of immune responses, mice were vaccinated i.p. or by scarification with rVV expressing the G protein of RSV. Vaccination of mice by scarification induces a Th2 response in mice which is responsible for a large influx of eosinophils into the lungs after RSV challenge. In contrast, vaccination by the i.p. route does not result in lung eosinophilia after RSV challenge, suggesting that different T cell subsets are primed by the i.p. route when compared with the intradermal route.
The influence of murine cytokines on induction of immune responses to the F protein was studied in mice vaccinated i.p. with recombinant (r)VV co-expressing the F proteins together with murine cytokine genes. rVV co-expressing the F protein and IL-2 or IFNgamma induced a significant reduction in all isotypes of antibody to RSV and a reduction in the RSV-specific CTL response, when compared with control mice vaccinated with rVV-F alone. Despite these reduced responses, which appeared to be due to limited replication of the rVV expressing IL-2 or IFNgamma in mice, animals were protected against RSV infection. Similarly, mice vaccinated with rVV-F co-expressing IL-4, were protected against RSV infection. In these mice, the rVV persisted for longer and grew to higher titres when compared with rVV expressing F alone. Although the antibody response in such mice was biased in favour of IgG1, suggestive of Th2 priming, there was a strong CTL response and immune spleen cells produced high levels of IFNgamma as well as IL-4 after restimulation in vitro with RSV.
The production of BHV recombinants expressing the F and G proteins of RSV was delayed by a failure to obtain expression of viral protein using native viral DNAs. This problem was overcome for the G protein by producing a synthetic gene containing nucleotide substitutions that removed potential splice sites. The immune responses induced by recombinant BHV-1 expressing the G protein was studied in calves after intranasal and intratracheal inoculation. Insertion of the G protein into the gE region of BHV-1 increased the virulence of the virus for the lower respiratory tract of calves. Ther is preliminary evidence that the G protein is incorporated into the envelope of the BHV-1 virions and it may, therefore, facilitate virus attachment and replication in the lower respiratory tract. Although BHV-1/g induced only low levels of antibody to RSV in either the serum or respiratory tract, and failed to prime RSV-specific CTL, there was evidence of T cell priming. Thus, 7 days after RSV challenge there was a RSV-specific lymphocyte proliferative response only in the BHV-1/G-vaccinated calves. Vaccinated calves were not only protected against infection with RSV but were also protected against BHV-1 infection. rBHV-1 expressing secreted or cytoplasmic versions of the G protein have also been constructed and will be used to investigate the influence of the cellular location of the G protein on induction of immune responses in cattle.
Bovine cytokine genes (IL-2, IL-4, IL-10 and IFN-gamma) have been inserted into recombination vectors either alone or in combination with the G protein of BRSV for their expression by BHV-1 and BHV-4. Substantial amounts of IL-2 and IL-4 were produced by these recombinants and analysis of the production of IFN-gamma and IL-10 is in progress. The levels of G protein expression in the various recombinants is under investigation and selected viruses will be used to investigate the effects of cytokines on induction of immune responses in calves.
Since the immune response is influenced by the MHC, the MHC of experimental calves has been analysed and a bank of MHC-typed embryos has been prepared for future experiments. This will enable studies on factors which influence induction of an immune response to be studied independantly of the influence of MHC and facilitate studies on the role of different antigen-presenting cells in the induction of immune responses in cattle. Two populations of dendritic cells have been identified in the peripheral blood and afferent lymph of calves, which differ in their ability to stimulate allogenic T cells and to present RSV antigens to immune T cells. Although BHV-1 infects monocytes in vitro, it does not infect dendritic cells. Nevertheless, both monocytes and dendritic cells are able to present BHV-1 antigens to immune CD4+T cells.
MAJOR SCIENTIFIC BREAKTHROUGHS:
The site of expression of viral proteins within infected cells and the route of vaccination influences the induction of the immune response. Although expression of murine IL-2 or IFN-gamma by rVV-F, reduces the virulence of vaccinia virus for mice and reduces both CTL priming and antibody production, the induction of resistance to RSV infection is not affected. Expression of murine IL-4 increases the virulence of VV for mice, biases the antibody in favour of IgG1 but does not affect induction of CTL or resistance to RSV infection. Vaccination of calves with recombinant BHV-1 expressing the G protein of BRSV does not prime CTL and induces only low levels of antibody. Nevertheless calves are almost completely protected against RSV infection. Bovine cytokine genes IL-2, IL-4, IL-10 and IFN-gamma have been expressed in BHV-1 alone or in combination with BRSV G protein. Bovine IL-2 and IL-4 have been expressed in BHV-4 together with the BRSV G protein. Phenotypic and functional differences in bovine dendritic cells have been identified.
Funding SchemeCSC - Cost-sharing contracts
8200 AB Lelystad
17498 Insel Riems
3508 TD Utrecht