Comparative immunological evaluation of recombinant Salmonella typhimurium strains expressing model antigens as live oral vaccines

Abstract

Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines. Here, a series of recombinant Salmonella Typhimurium strains were constructed to express either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. To investigate different delivery and expression methods, antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in the cytoplasm, or the outer membrane. In addition, combinations of two expression strategies were employed to evaluate the efficacy of combined delivery approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities, the immunogenicity of the constructed recombinant Salmonella strains was evaluated and compared in mice. Using soluble model antigen EGFP, my results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, while eukaryotic expression or colonization with good construct stability is critical for T cell responses. For insoluble antigen model HA, the outer membrane strategy induced better B cell and T cell responses than cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited as initially expected. Based on the advantages, deleterious or synergistic effects of different strategies identified in this study, I conclude that different construction strategies of recombinant Salmonella vaccine strains are needed for different forms of antigens (soluble or insoluble antigens). If the antigen (such as EGFP) is soluble and easily expressed in Salmonella, a low-copy plasmid-based strategy should be employed, as it can provoke both strong B cell and T cell responses with better plasmid stability. If a T cell response is preferred, a eukaryotic plasmid, or chromosome-based, cytoplasmic-expression strategy may achieve better results. For heterologous antigens that are likely to be expressed in an insoluble form inside Salmonella (such as HA), an outer membrane-targeting approach is recommended. In addition, I found that the combination of two expression strategies did not enhance the immune response, and hence I caution the use of such an approach.