Analysis and optimization of immune responses elicited by Outer Membrane Vesicles (OMVs)

Outer membrane vesicles (OMVs) are spheroidal particles released from all gram-negative bacteria and exert different biological functions. OMVs carry many microbe-associated molecular patterns (MAMPs) such as peptidoglycan, lipopolysaccharide (LPS), and lipoproteins and can be decorated with foreign antigens. Thanks to these properties OMVs are becoming an attractive vaccine platform. However, a systematic investigation on the potentiality of OMVs to design vaccines to elicit protective antigen-specific T and B cell responses is missing. In this study we systematically investigated the antigen-specific B and T cell responses elicited by OMVs decorated with heterologous antigens. Regarding the B cell induction by OMVs, we selected and purified 16 OMV endogenous proteins, studying aspects of their immunogenicity and their concentration within OMVs. We also used these proteins to analyze other aspects of the immunogenicity of OMVs such as the minimum amount of protein, decorated to OMVs, sufficient to induce a protein-specific antibody response or the minimum amount of protein to achieve saturating protein-specific antibody titers. We also analysed the influence of physical association of antigens to OMVs in antigen-specific antibody titers, comparing the immunogenicity of OMVs expressing B cell antigens on their surface with the immunogenicity of OMVs absorbed with the same antigens. We also investigated the immunogenicity of OMVs decorated with T cell epitopes in inducing epitope-specific IFNγ+ CD4+ and CD8+ T cell responses. In particular, we analysed important aspects such as the amounts of OMVs and heterologous antigen necessary and sufficient to reach the plateau of antigen-specific T cell responses. We studied the effect of immunization schedule and route of immunization on antigen-specific T cell response and the influence of physical association of peptide epitopes to OMVs in epitope-specific T cell frequencies.