Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis

Abstract

T cells are the immunological cornerstone in host defense against infections by intracellular bacterial pathogens, such as virulent Francisella tularensis (Ftt). The general paucity of novel vaccines for Ftt over the past 60 years can, in part, be attributed to the poor understanding of immune parameters required to survive infection. Thus, we developed a strategy utilizing classical immunological tools to elucidate requirements for effective adaptive immune responses directed against Ftt. Following generation of various Francisella strains expressing well-characterized lymphocytic choriomeningitis virus (LCMV) epitopes, we found that survival correlated with persistence of antigen-specific CD4+ T cells. Function of these cells was confirmed in their ability to more effectively control Ftt replication in vitro. The importance of understanding the antigen-specific response was underscored by our observation that inclusion of an epitope which elicits high avidity CD4+ T cells converted a poorly protective vaccine to one that engenders 100% protection. Together, these data suggest that improved efficacy of current tularemia vaccine platforms will require targeting appropriate antigen-specific CD4+ T cell responses and that elucidation of Francisella epitopes that elicit high avidity CD4+ T cell responses, specifically in humans, will be required for successful vaccine development.

Systems Vaccinology for a Live Attenuated Tularemia Vaccine Reveals Unique Transcriptional Signatures That Predict Humoral and Cellular Immune Responses

“…Animal studies have previously shown that B and T cell responses are important in the induction and regulation of an effective immune response to live tularemia vaccines [5,6]; specifically, maintaining either CD4+ or CD8+ T cells in mice appeared to be essential for survival. Animals challenged with virulent Francisella require both T cell subsets for survival [7,8], and in vitro studies of human cells suggest that CD8+ T cell proliferation and cell survival depend on CD4+ proliferation [9]. However, the natural course and efficacy of acquired immunity is not well studied in large human cohorts.…”

Protection induced by a Francisella tularensis subunit vaccine delivered by glucan particles

“…To inform our antigen down selection process, we attempted to assess the in vitro antimicrobial potential of vaccine induced immunity using a functional killing assay based on previously reported methodology [ 35 ]. Splenocytes from immunized mice were stimulated overnight with heat-killed F .…”

“…The macrophage/splenocyte co-culture killing assay was a modification of a method from Roberts et al [ 35 ]. In summary, murine splenocytes were isolated as described above and cultured in L15 medium (Life Sciences, UK) with either medium alone, 10 ng/ml phorbol myristate acetate(PMA; Sigma) + 1ug/ml ionomycin (Sigma) or heat killed F .…”

Development of a novelFrancisella tularensisLive Vaccine Strain expressing ovalbumin provides insight intoFrancisella tularensis-specific CD8+T cell responses

“…Antibody-mediated immunity appears to be a poor correlate of immunity to highly virulent F. tularensis strains; antibody titers do not correlate with protection in humans[15], and the transfer of immune serum fails to protect recipient mice against the challenge with virulent strain of F. tularensis [16–18]. In contrast, both CD4 + and CD8 + T cells are known to be required for protection, as depletion of either subset abolishes protective immunity [12,19,20]. To truly hone in on correlates of protective T cell responses, it is necessary to be able to differentiate cells specifically responding to the pathogen of interest from cells of other specificities [21].…”

“…For instance, a protective vaccine leads to more antigen-specific CD4 + T EM in the mediastinal lymph node (MLN) and spleen, as compared to a non-protective vaccine [20]. Additionally, the tool has been used to study how these cells respond to a prime-boost strategy [13] and has revealed the dramatic influence high avidity CD4 + T cell epitopes have on protection [13,20]. While this tool will undoubtedly yield many more insights into the role of CD4 + T cells in immunity to F. tularensis, no such tool currently exists to characterize CD8 + T cells, which are also required for protection against this predominantly intracellular pathogen [12,19,20].…”