Enhancing the functionality of RHDV VLP

Abstract

Virus-like particles (VLP), such as the rabbit haemorrhagic disease virus (RHDV) VLP, can be utilised as vectors for the delivery of heterologous antigens to the immune system. Model tumour antigens, such as gp33 and ovalbumin have been conjugated to RHDV VLP, allowing them to be cross-presented by antigen presenting cells (APC), leading to the induction of powerful immune responses against tumour challenges. The aim of this research was to enhance the functionality of RHDV VLP as a versatile vaccine vector for the delivery of tumour associated antigens, by increasing antigen loading on each VLP, incorporating sequence specific linkers between antigenic epitopes to enhance processing and targeting receptor mediated internalisation of VLP. In order to determine the effect of increased antigen loading and incorporation of sequence specific linkers on the functionality of VLP in tumour immunotherapy, the N-terminus of the RHDV VLP capsid protein VP60 was genetically manipulated to express the melanoma associated antigen gp10025-33. VLP was modified to express one copy of gp100 (VLP.gp100-1), two copies (VLP.gp100-2), two copies with a linker between copies (VLP.gp100-2L) and three copies with linker sequences (VLP.gp100-3L). Previously used linker sequences were gathered from the literature, and the linker most likely to enhance antigen processing was determined using proteasome and immunoproteasome prediction programs. This linker was determined to be ala-ala-leu. The synthesis of the different VLP.gp100 was confirmed by SDS-PAGE, electron microscopy and mass spectrometry. The ability of these constructs to induce gp100 specific immunity was tested by in vitro T cell proliferation assays, as well as an in vivo cytotoxic assay and a therapeutic tumour trial. Comparison of VLP.gp100-1 to VLP.gp100-2 and VLP.gp100-2L to VLP.gp100-3L suggested that increasing antigen loading on VLP could enhance antigen specific immune responses, but that the addition of too much antigenic material (VLP.gp100-3L) can lead to hindered VLP formation and reduced vaccine efficacy. Furthermore, comparison of VLP.gp100-2 and VLP.gp100-2L indicated that the addition of amino acid linker sequences to target enhanced processing of antigens could further augment immune responses. Overall, VLP.gp100-2L was found to be significantly better than the other VLP.gp100 at generating gp100 specific immune responses, and therapeutic vaccination with this VLP significantly delayed the growth of murine melanoma (B16). Internalization of peptides by APC is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. Therefore, this work aimed to target receptor mediated internalisation of VLP. New schemes for the synthesis of a monomannoside (N-succinimidyl 6-[α-D-mannopyranosyloxy]hexanoate) and a novel dimannoside (N-succinimidyl 6-[2-O-(α-D-mannopyranosyl)-α-D-mannopyranosyloxy]hexanoate) were developed, allowing conjugation of mannose to VLP at neutral pH while retaining VLP structural integrity. The mannosides were successfully conjugated to lysine residues in the RHDV VLP capsid protein (VP60), providing an estimated 270 mannose groups on the surface of each virus particle. Fluorescently labelled VLP conjugated to the mannoside or dimannoside showed significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose-specific uptake, demonstrating that mannosylation of VLP targets mannose receptor based uptake. Analysis of the immune response to mannosylated VLP demonstrated no enhanced activation of dendritic cells. However, partial re-targeting of the intracellular processing of mannosylated RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens. Mannosylation of VLP expressing the model tumour antigen SIINFEKL (VLP.SIIN) induced a delay in the SIINFEKL-specific T cell proliferation in vitro, but this was not matched with a reduction in the ability to mannosylated VLP.SIIN to induce cytotoxic responses in vivo. In the case of VLP expressing the model antigen gp33 (VLP.gp33) and the more relevant TAA gp100 (VLP.gp100-2L), modification with dimannose and to a lesser extent monomannose induced enhanced anti-tumour efficacy. Overall, this demonstrated that the conjugation of mannosides to VLP results in an augmented targeting and delivery system for antigen delivery, facilitating an increase in the efficacy of VLP as vaccination vectors for tumour immunotherapy.