Figure 2.

Applied and experimental bacterial vaccines. Today, established bacterial vaccines that are in use are the immunizations with WCA, recombinant proteins including toxoids, polysaccharide/protein conjugates, live attenuated vaccines (LAV), and, since the last few years, also bacterial outer membrane vesicles (OMVs). Vaccination with WCA, bacterial ghosts (BGs), and recombinant proteins/toxoids predominantly results in the processing of protein components for the presentation via MHCII and the activation predominantly of CD4⁺ T cells. In addition, antigen-specific activated B cells produce high-affinity IgG antibodies with the help of CD4⁺ T cells, and a memory response is induced. In the case of polysaccharide/protein conjugates, the carrier protein serves as the protein component that can be recognized by CD4⁺ T cells. This enables B cells to produce high-affinity IgG antibodies against the polysaccharide instead of the production of low-affinity IgM without T cell help. Immunization against intracellular bacterial pathogens requires the activation of cytotoxic CD8⁺ T cells, which is usually not efficiently achieved with these methods. Antigens recognized by CD8⁺ T cells predominantly derive from cytosolic proteins that are degraded by the proteasome and further processed for the presentation via MHCI. A major difficulty of efficient vaccination against intracellular bacterial pathogens lies in the introduction of immunogens into the cytosol of host cells. This can be achieved by the use of OMVs, LAV, viral vectors, bacterial vectors, immunization with DNA or mRNA, and the use of the T3SS translocation system of bacteria such as Salmonella. Immunization with OMVs and LAV results in both antigen presentation by MHCII and MHCI molecules for the activation of CD4⁺ and CD8⁺ T cells, whereby the mechanisms of MHCI presentation in the case of OMV immunization are not well understood and may be a result of cross presentation, either by the release of proteins from the lysosome into the cytosol or by fusion of lysosomes with MHCI-containing vesicles. LAV may release proteins into the cytosol. In addition, surface proteins may be accessible to the proteasome for processing via the MHCI presentation pathway. The most frequently used viral vectors for vaccination are adenoviruses and modified vaccinia virus Ankara (MVA). Adenoviruses translocate their double-stranded (ds) DNA genome into the nucleus of non-dividing cells for replication. Viral mRNA transcription products are exported into the cytosol of the infected cells where ribosomal translation occurs. In contrast, MVA, which also carries a dsDNA genome, has a unique replication cycle that is restricted to the cytosol. In both cases, proteins are expressed in the cytosol of infected cells, which has also been shown for bacterial vectors that carry plasmid DNA with eukaryotic expression cassettes for the expression of immunogens. Cytosolic protein expression is also achieved by the direct transfection of target cells with either DNA or mRNA. While mRNA is transferred directly into the cytosol for protein translation, DNA has to enter the nucleus of the target cell for transcription, which is usually more efficiently achieved with viral vectors. Finally, a rather experimental approach to the introduction of antigens into the cytosol of target cells is the use of recombinant attenuated bacteria such as Salmonella that possess a T3SS translocation system. This system allows active and direct injection of proteins into the cytosol of target cells. In contrast to the use of WCA and LAV, all other methods generally require knowledge of the immunogenic determinants of the pathogen to prepare recombinant vaccines.