No abstract

Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp10025–33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN-γ) or the linker at the N-terminus (0.8 ng/ml IFN-γ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN-γ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy.

Conjugation of CpG to an antigen induces a stronger immune response compared to that of the mixture. This study compares the in vitro immunostimulatory activity of CpG conjugated via either its 5′ or 3′ end to the model antigen ovalbumin (OVA). CpG modified with an amine at either the 5′ or 3′ end was conjugated to OVA via a stable bis-aryl hydrazone bond. Similar levels of CpG conjugation to OVA were observed for both conjugates on the basis of the absorbance at 360 nm for the formation of the bis-aryl hydrazone bond, which determined 2.8 ± 0.3 CpGs linked per OVA. Both the 5′ and 3′ CpG–OVA conjugates had similar size-exclusion chromatography elution profiles. The immunostimulatory properties of the conjugates were determined by dendritic cells (DCs) and T-cells isolated from mice. The activation of DCs was determined by the upregulation of activation markers CD86 and CD40. T-cells were co-cultured with stimulated DCs, and the immunogenicity was determined by measuring T-cell proliferation and interferon γ production. Both the CpG 5′- and 3′-linked conjugates induced the same level (p > 0.5) of DC activation markers, which were significantly higher than those of the untreated control. Similarly, T-cell assays showed no significant difference (p > 0.5) between the 5′ and 3′ conjugates with respect to T-cell proliferation and interferon γ production. The 5′ and 3′ conjugates induced T-cell activation significantly higher than the mixture of CpG and OVA. This study showed that the end at which CpG is conjugated to an antigen has no influence on the generation of a T-cell-based immune response in vitro.

No abstract

The dendritic cell (DC) is the foremost antigen-presenting cell (APC) for ex vivo expansion of tumour-specific patient T cells. Despite marked responses in some patients following reinfusion of DC-activated autologous or HLA-matched donor T cells, overall response rates remain modest in solid tumours. Furthermore, most studies aim to generate immune responses against defined tumour-associated antigens (TAA), however, meta-analysis reveals that those approaches have less clinical success than those using whole tumour cells or their components. Tumour lysate (TL) is used as a source of tumour antigen in clinical trials and potentially represents the full range of TAAs in an undefined state. Little is known about how different APCs cooperate to present TL antigens. We examined the effect of oxidised whole-cell lysate (ox-L) versus soluble fraction freeze–thaw lysate (s-L) on bone marrow-derived DCs and macrophages, and magnetic bead-isolated splenic B cells. The APCs were used individually, or in combination, to prime T cells. CD8+ T cells produced interferon (IFN)-γ in response to both s-L and ox-L, but only proliferated in response to ox-L. IFN-γ production and proliferation was enhanced by priming with the DC+B cell combination. Compared to DC alone, a trend toward greater interleukin (IL)-12 production was observed when DC+B cell were loaded with s-L and ox-L antigens. CD8+ T-cell specific lysis in vivo was greatest in ox-L-primed groups and DC+B cell priming significantly increased in vivo cytotoxicity compared to DC alone. These improved T-cell responses with two APCs and stressed cell lysate has implications for APC-based adoptive cell therapies.