Abstract
Immunotherapies have the potential to harness the immune system in order to target and clear cancer cells. One branch of immunotherapeutic research is the development of cancer vaccines. Virus-like particles (VLP) make effective cancer vaccines and are made of the capsid proteins of a virus that spontaneously form an immunogenic shell. VLP made from Rabbit haemorrhagic disease virus (RHDV) have been shown to be a highly immunogenic vaccine. They can act as an adaptable scaffold onto which antigens can be recombinantly expressed or physically coupled. Our group has previously shown that RHDV VLP can be cross-presented by dendritic cells (DCs) inducing a cytotoxic anti-cancer response when used in combination with the adjuvant CpG. The physical coupling of an adjuvant to a vaccine can vastly enhance the immune response by mimicking the stimulation that would occur in a natural viral infection. It has been reported that CpG is a strong B cell stimulus that induces proliferation and increases expression of surface co-stimulatory markers. Alongside the reports of activation it has been shown that B cells are able to cross-present CpG conjugated proteins. This and the fact that B cells are 30 times more common than DCs makes them an attractive APC target for vaccinations.
The overarching aims of this project was to determine if the adjuvant CpG chemically coupled to a VLP would increase the activation, proliferation and cross-presentation of antigen by B cells generating a stronger cytotoxic response against a model antigen.
This study used both RHDV VLP and a modified form that expressed the model antigen SIINFEKL. The thiol modified CpG adjuvant or GpC control sequences were conjugated to the exterior of RHDV VLP using the heterobifuntional linker SMCC producing VLP with CpG or GpC control conjugated (VLP-CpG or VLP-GpC). B cells responded strongly to VLP-CpG but not to VLP-GpC, inducing the upregulation CD86 and MHC-II expression and stimulating proliferation. In order to measure the cross-presentation ability of B cells an antibody was used that identifies SIINFEKL bound to MHC-I. There was no detectable presentation of SIINFEKL on MHC-I using this method. However B cells pulsed with VLP.SIINFEKL could stimulate the proliferation of antigen specific CD8+ T cells. This result shows for the first time B cells have the capacity to cross-present RHDV VLP. B cells also internalised VLP-CpG and VLP-GpC at a significantly higher rate compared with VLP and CpG in solution. Pretreating B cells with GpC impaired this increased internalisation, indicating that this increase is receptor-mediated. The internalisation was explored in subsets of B cells, and marginal zone B cells were found to internalise significantly more VLP than follicular B cells.
Overall these results indicate that the conjugation of CpG to VLP stimulates significantly higher internalisation in a receptor-mediated manner, and triggers both the activation and proliferation of B cells. Furthermore, this is the first report of VLP cross-presentation by B cells.