Study on Immune Response to Antigens of Francisella tularensis using Humanized Mice Model

Abstract

Francisella tularensis (FT), a highly infectious pathogen, is considered to be a potential biological weapon because of its low infectious dose, and the ability of aerosol transmission. Although an attenuated FT live vaccine strain (LVS) has been developed, this vaccine exhibits side effects and residual virulence. Therefore, subunit vaccines using parts of pathogens have been proposed as an alternative to live vaccines. Tul4 and FopA are outer membrane proteins of FT, and have been reported to play an important role in the bacteriums immunogenicity, which support the potential of subunit vaccines as candidates against FT. <br/> Although in vitro and mouse models are standard paradigms for vaccine development, these models have limitations including differences in susceptibility to FT between mice and humans. Humanized mice (hu-mice) bearing the human immune system have been developed to study human-specific diseases, and have been reported to produce human immunoglobulins against pathogens. Therefore, hu-mice have the potential to overcome the limitations of animal models used in clinical research. In this study, we reported that a subunit vaccine constructed based upon outer membrane epitopes, Tul4 and FopA, elicited the human-specific immunity in hu-mice model.<br/> The study described in Chapter I reported the in vitro and in vivo immune response of a subunit vaccine comprising the recombinant peptides Tul4 and FopA generated from epitopes on FT outer membrane proteins. Dendritic cells (DCs) stimulated by recombinant peptides with adjuvant CpG oligodeoxynucleotide induced robust immunophenotypic changes in DC maturation and secretion of inflammatory cytokines (interleukin [IL]-6 and IL-12). In addition, the matured DCs enabled ex vivo proliferation of naive splenocytes in mixed lymphocyte reactions. Finally, I investigated in vivo immune responses by assessing antibody production in C57BL/6 mice. Total immunoglobulin (Ig) G was produced after immunization and levels peaked 6 weeks later. Moreover, Tul4-specific IgG was confirmed in mice receiving peptides with or without CpG. Based on these results, I revealed that the recombinant peptides Tul4 and FopA have immunogenicity, and could be a safe subunit vaccine candidate against FT. <br/> Chapter II described the immune response of mice—humanized with human CD34+ cells (hu-mice)—to a cocktail of recombinant Tul4 and FopA (rTul4 and rFopA), which were codon-optimized and expressed in Escherichia coli. Not only did the cocktail-immunized hu-mice produce a significant human immunoglobulin response, they also exhibited prolonged survival against an LVS, as well as human T cells in the spleen. These results suggest that a cocktail of rTul4 and rFopA had successfully induced an immune response in the hu-mice, demonstrating the potential of this mouse model for use in the evaluation of FT vaccine candidates. <br/> In conclusion, the present study demonstrates the efficacy of recombinant Tul4 and FopA vaccine and the value of the hu-mice model for FT vaccine research. Overall, I suggest that such an approach might be widely applicable to vaccine studies of the FT.<br/>