To construct SpyCatcher-mi3 nanoparticle vaccine delivery vectors, evaluate their role in enhancing the immunogenicity of the ovalbumin CD8⁺ T-cell epitope peptide, OVA_257-264, and determine its protective effect in a model which mice were immunized and subcutaneously challenged with E. G7-OVA tumor cells.

SpyCatcher-mi3 proteins were expressed by E.coli and purified by affinity chromatography and anion exchange chromatography sequentially. OVA_257-264-SpyTag peptide was obtained by synthesis. The OVA_257-264-mi3 nanoparticles were produced by the SpyTag/SpyCatcher system. The toxicity of OVA_257-264-mi3 was evaluated using hemolysis assay, CCK-8 assay and mouse experiment. A total of 42 female SPF-grade C57BL/6 mice (6~8 weeks old, 18~20 g) were randomly divided into OVA_257-264-mi3, OVA_257-264, and control groups, with 14 mice in each group. Then the mice in each group were immunized on days 0, 14 and 28. In 14 d after the last immunization, the amounts of spot-forming cells (SFCs, indicating IFN-γ secreting cells in splenic lymphocytes) were determined using ELISpot assay to evaluate their immunogenicity. After the immunized mice were subcutaneously implanted with E.G7-OVA tumor cells, the antitumor effect of the vaccine inprophylactic xenograft tumor model was evaluate by observing tumor volumes with a caliper and tumor growth with MRI.

Both SpyCatcher-mi3 and OVA_257-264-mi3 could be self-assembled to form homogeneous and stable nanoparticles, with an average particle size of about 43.8 and 91.3 nm, respectively. The OVA_257-264-mi3 was safe for in vitro and in vivo toxicity evaluation. The number of IFN-γ secreting cells per 1×10⁶ splenic lymphocytes reached 253 in the OVA_257-264-mi3 group of mice, significantly higher than that in the OVA_257-264 group and the Control group (P<0.05). The tumor volume of mice in the OVA_257-264-mi3 group was about 151.1 mm³ on day 22, which was significantly smaller than that of the OVA_257-264 group and the Control group (P<0.05), and the survival rate during the observation period reached 60%, which was significantly higher than that of the OVA_257-264 groups (P<0.05).

Nanoparticle vaccine OVA_257-264-mi3 is successfully constructed, and it shows enhancing effect on the immunogenicity of the antigen epitope peptide, and exerts protective effect on prophylactic xenograft tumor model, providing a theoretical basis for the research of tumor neoantigen vaccines.