Abstract
Virus-like particles (VLP) have been shown to be useful nanoscale platforms in a diverse range of applications including their use as vaccines. They can act as vehicles for the delivery of heterologous antigens to the immune system thereby initiating strong anti-tumor responses. Model tumor antigens chemically coupled to rabbit hemorrhagic disease virus VLP (RHDV VLP) have been shown to delay or prevent the development of tumors in vivo using an aggressive model of murine melanoma. RHDV VLP are amenable to conjugation with a range of peptide antigens, single proteins or complex tumor lysates, all of which are relevant for studying immune responses to tumors in mouse models or with human systems in vitro.
Dendritic cells (DC) play a key role in the development of anti-tumor T cell responses due to their ability to take up antigens, process them into peptides and present them to CD8+ T cells on MHC-I by cross-presentation. Inhibitor studies showed RHDV VLP are rapidly taken up by both mouse- and human-derived DC by macropinocytosis and phagocytosis and are subsequently prepared for presentation on MHC-I that has recycled from the cell surface within acidified lysosomal compartments. Additionally, the ability of murine DC to cross-present VLP does not seem to be confined to specific subsets as DC expressing CD8α were able to cross-present VLP-SIINFEKL in vivo, to equivalent levels as other populations of DC.
RHDV VLP conjugated to tumor peptides or lysates do not stimulate phenotypic maturation of the DC required for the cross-priming of naïve T cell responses. The addition of an adjuvant, either OK432 or oncolytic reovirus, to DC pulsed with VLP-MART1 or VLP-Lysates, resulted in phenotypic maturation of DC and enabled the priming of naïve T cells able to produce IFN-γ and directly lyse specific target cells. Furthermore, MART-1 specific MHC-I pentamer staining demonstrated the expansion of CD8+ T cells possessing a TCR specific for MART-1 while the T cells took on a more activated, effector memory phenotype through diminished expression of CD45RA and increased expression of CD45RO and CCR7. Importantly, T cell responses primed using VLP-MART1 or VLP-Lysate with either adjuvant were more pronounced than where MART1 peptide or Lysate was used with that same adjuvant indicating a significant benefit in delivery of antigen on VLP. Collectively, these results indicate that VLP-conjugates, together with adjuvant, have the ability to stimulate cytotoxic T cell responses specifically against tumor cells.
Production of a VLP able to induce DC maturation was attempted by conjugation of melanoma lysates infected previously with oncolytic reovirus to the VLP (VLP-ReoLysate). Results indicate that these VLP-ReoLysate are able to induce activation of innate NK and NKT cells as well as DC. Furthermore, DC pulsed with VLP-ReoLysate resulted in priming of naïve T cells with cytotoxic capabilities against melanoma cells in vitro, however minimal differences were seen where ReoLysates were delivered alone or conjugated to VLP.
Collectively these results demonstrate that the delivery of antigens coupled to VLP, along with an adjuvant, to DC are able to induce strong and highly specific cytotoxic T cell responses against tumor cells to a greater degree than antigens delivered alone. The potential to further modify the VLP to incorporate an adjuvant would further enhance the appeal of VLP as a clinical agent to target tumors.