A sopB Deletion Mutation Enhances the Immunogenicity and Protective Efficacy of a Heterologous Antigen Delivered by Live Attenuated Salmonella enterica Vaccines

Recombinant attenuated Salmonella vaccines (RASVs) have been used to induce mucosal and systemic immunity either against itself or to a vectored heterologous antigen (30, 31). It is clear from previous studies that the mode of attenuation (10), antigen burden (12), and the expression system (11, 19, 21) can have a profound effect on the immune response to a vectored antigen. Many attenuating mutations were found to reduce Salmonella survival due to host-induced stresses and/or reduce colonization of lymphoid effector tissues leading to less than ideal immunogenicity (16, 48). An ideal Salmonella vaccine strain should exhibit wild-type abilities to withstand all stresses (enzymatic, acid, osmotic, ionic, etc.) and host defenses (bile, antibacterial peptides, etc.) encountered following oral or intranasal immunization and should exhibit wild-type abilities to colonize and invade host lymphoid tissues while remaining avirulent. Achieving maximal immune responses to a foreign antigen is directly correlated with the amount of the antigen produced, and thus, it is important that the immunizing strain produce adequate levels of antigen. However, for RASVs, this need must be weighed against the fact that high-level antigen production can be a drain on the energy resources of the cell, leading to reduced growth rates and a compromised ability to colonize and stimulate effector lymphoid tissues (55). We have addressed these problems by exporting the antigen outside of the attenuated Salmonella (19), by delaying expression of the attenuated phenotype (6; unpublished results), and by heterologous antigen synthesis (53) until the vaccine strain has colonized host lymphoid tissues. In addition, we have developed the Asd+-balanced lethal system for plasmid maintenance, obviating the need for antibiotic resistance markers (32). The SopB protein in Salmonella, encoded by SPI-5, is secreted and translocated into the cytosol through the SPI-1-encoded type III secretion system (60). SopB has an inositol phosphate phosphatase activity which affects cytoskeletal rearrangement, host-cell invasion and chloride homeostasis (34). sopB mutants of Salmonella enterica serovar Typhimurium are attenuated in their ability to cause gastroenteritis in calves (59), but not for invasion of host tissues (39). In addition, sopB mutants of both Salmonella enterica serovars Typhimurium and Dublin are similarly attenuated in their ability to induce inflammatory responses and fluid secretion in bovine ileal loops (13, 42). In the context of a vaccine, these features may be beneficial in reducing or eliminating reactogenicity and diarrhea sometimes observed in clinical trial volunteers receiving live attenuated bacterial vaccines (17, 47, 49-51). The SopB protein can also protect infected cells from apoptosis by sustained activation of Akt, which may be helpful for Salmonella survival in host tissues but will necessarily affect host immune responses (22). A recent study showed that introduction of a sopB mutation into several different attenuated Salmonella strains led to enhanced immune responses in mice to a vectored test antigen, β-galactosidase (27). Based on these observations, we evaluated the effects of a sopB deletion mutation on the performance of our Salmonella vaccine strains. Streptococcus pneumoniae is the principle cause of bacterial pneumonia in children and adults and is also a major cause of otitis media, bacterial meningitis, and septicemia (2). Developing an inexpensive, safe, and effective vaccine against this pathogen is still in urgent demand. Pneumococcal surface protein A (PspA) is an important virulence factor found on the surface of all pneumococci (29). It is a protective antigen in animal models and is immunogenic in humans (2, 58). Our previous work has demonstrated that mice immunized with an RASV expressing PspA are protected against challenge with virulent S. pneumoniae WU2 (20). In this work, we evaluated the effects of a sopB mutation on two different RASV strains with different modes of attenuation on their ability to deliver PspA. We analyzed humoral, mucosal, and cellular immune responses against both the bacterial vector and PspA and protective immunity against challenge with S. pneumoniae WU2 in immunized mice.