Surface display of two neoantigens on Lactiplantibacillus plantarum

Lactic acid bacteria, such as Lactiplantibacillus plantarum, are becoming increasingly popular hosts for combining production and delivery of therapeutic proteins to immune cells. Soluble antigens are susceptible to rapid proteolysis, hence anchoring of antigens to bacterial cells, which likely protects the antigen, is a preferred delivery strategy that may increase immune responses. In cancer research, personalized immunotherapy has high potential and, in this respect, the so-called neoantigens that accumulate in tumor cells are promising tumor specific targets. Here, we demonstrate that, when using the inducible pSIP expression system, L. plantarum can produce and surface display two neoantigens, NAG1 and ETV6. The antigens could be targeted to both the cell membrane and the cell wall, utilizing four different anchoring methods. The production level and the degree of surface exposure of the antigens varied among the anchors. Flow cytometry analysis showed that antigens anchored to the cell wall were more exposed than those anchored to the cell membrane. To our knowledge these are the first reports of neoantigens being produced and surface-displayed in Lactobacillales.