Investigating Cytotoxic T cell Responses to RHDV Virus-like Particles

Abstract

Immunotherapies prime cells of the immune system to generate an appropriate response against a specific target. One area of Immunotherapeutic research is the development of cancer vaccines such as the vaccines based on Virus-like particles (VLP). VLP derived from Rabbit Hemorrhagic Disease Virus (RHDV) have been shown to be effective in the induction of the cytotoxic responses to specific tumour antigens. The cytotoxic response of CD8+ T cells generated in response to a tumour is important for the clearance of tumour cells and the generation of memory T cells. Previous studies have shown that RHDV VLP were taken up by dendritic cells (DC) and cross presented, utilising the MHC-I recycling pathway but the cytotoxic response generated was not 100% effective against tumour challenge, in particular where the specific activation of CD4+ T cells was not activated. The aims of this research were firstly to produce RHDV VLP incorporating tumour epitopes and to determine the dendritic cell subset with the capacity to cross-present VLP to induce cytotoxic responses in vivo. Following on from this was an investigation into the role of CD4+ help to optimise cytotoxic responses and determine when is help required in relation to CD8+ activation. The addition of adjuvant was investigated in its capacity to improve the cytotoxic responses seen in vivo. Finally the production of antibody against VLP protein VP60 was investigated and the impact this antibody has on VLP uptake. To carry out these aims recombinant VLP expressing different immunogenic antigens; gp33 derived from LCMV and OTI, an ovalbumin derived MHC-I peptide, were generated using a baculovirus expression system (VLP.gp33 and VLP.OTI). Some VLP were also chemically coupled with ovalbumin derived MHC-II peptide (VLP.OTII and VLP.OTI.OTII). The importance of DC subsets expressing langerin on cross-presentation for the generation of cytotoxicity was investigated by depleting langerin positive DC using LangDTREGFP mice and vaccinating with VLP.gp33. The requirement for CD4 cells in the induction of in vivo cytotoxicity was assessed by determining the impact of vaccinating mice with VLP.OTI following the adoptive transfer of antigen specific OTI CD4 T cells. The timing of CD4 activation and whether CpG adjuvant could improve in vivo cytotoxic responses were carried out utilising the different VLP previously generated. The presence of antibody was assessed from the serum of animals that had previously been vaccinated with VLP.OTI and the impact on VLP uptake was determined utilising an in vitro uptake assay. These experiments showed that when depletion of the langerin+ DC was maintained, the in vivo specific lysis of gp33-labelled target cells was significantly reduced. The adoptive transfer of OTII CD4 T cells showed that when the transferred cells were activated by the presence of OTII peptide the cytotoxic responses were improved. The improved cytotoxic response was also observed when animals were vaccinated with VLP that were able to activate both CD4 and CD8 T cells (VLP.OTI.OTII). Regardless of when CD4 help was given in relation to CD8 activation there was no significant improvement to the cytotoxic response. Experiments with the addition of adjuvant to the VLP vaccination were able to show an improved cytotoxic response indicating the requirement of an adjuvant. Finally antibody was detected in high quantities against the VP60 protein and the presence of these antibodies was able to improve the uptake of VLP early on during the uptake process. Overall these results indicate that cross-presentation of peptides by langerin+ DC is important for the generation of cytotoxic responses of CD8 T cells and are improved when both CD4 and CD8 T cells are activated.