Immunogenicity of

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have emerged as a novel and flexible vaccine platform. OMVs can be decorated with foreign antigens and carry potent immunostimulatory components. Therefore, after their purification from the culture supernatant, they are ready to be formulated for vaccine use. It has been extensively demonstrated that immunization with engineered OMVs can elicit excellent antibody responses against the heterologous antigens. However, the definition of the conditions necessary to reach the optimal antibody titers still needs to be investigated. Here, we defined the protein concentrations required to induce antigen-specific antibodies, and the amount of antigen and OMVs necessary and sufficient to elicit saturating levels of antigen-specific antibodies. Since not all antigens can be expressed in OMVs, we also investigated the effectiveness of vaccines in which OMVs and purified antigens are mixed together without using any procedure for their physical association. Our data show that in most of the cases OMV-antigen mixtures are very effective in eliciting antigen-specific antibodies. This is probably due to the capacity of OMVs to "absorb" antigens, establishing sufficiently stable interactions that allow antigen-OMV co-presentation to the same antigen presenting cell. In those cases when antigen-OMV interaction is not sufficiently stable, the addition of alum to the formulation guarantees the elicitation of high titers of antigen-specific antibodies.