AttenuatedYersinia pseudotuberculosisCarrier Vaccine for Simultaneous Antigen-Specific CD4 and CD8 T-Cell Induction

Abstract

Yersinia pseudotuberculosis employs a type III secretion system for targeting of several virulence factors directly to the cytosol of eukaryotic cells. This protein translocation mechanism mediates the ability of Yersinia to resist phagocytosis and is required for sustained extracellular bacterial replication. In the present study, the Yersinia outer protein E (YopE) was used as a carrier molecule for type III-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. In comparison to wild-type Yersinia, an attenuated Y. pseudotuberculosis yopK-null mutant strain hypertranslocates chimeric YopE/LLO into the cytosol of macrophages, resulting in enhanced major histocompatibility complex (MHC) class I-restricted antigen presentation of an LLO-derived CD8 T-cell epitope. Remarkably, T-cell activation assays also revealed a superior ability of translocated over secreted LLO to induce MHC class II-restricted antigen presentation. These in vitro observations were confirmed after immunization of mice with a single dose of the yopK-null mutant strain. Animals orally inoculated with recombinant Yersinia expressing translocated chimeric YopE/LLO revealed high numbers of gamma interferon-producing LLO-specific CD4 and CD8 T cells. For the first time, it is shown that cytosolic antigen display mediated by an extracellular bacterial carrier vaccine results in simultaneous CD4 and CD8 T-cell priming, conferring protection against an intracellular pathogen.

Yersinia Outer Protein P Inhibits CD8 T Cell Priming in the Mouse Infection Model

“…The development of an immune response depends on the amount of Ag that is translocated into the cytosol of APCs and it has been shown that hypertranslocation of heterologous Ag by a Y. pseudotuberculosis yopK knockout mutant results in a superior immune response (61). To exclude the possibility that the CD8 T cell response induced by yopP-deficient mutants could be the result of compensatory hypertranslocation of YopE/LLO/M45, we demonstrated by Western blot analysis that the deletion of yopP does not influence the amount of YopE/LLO/M45 that is translocated into DCs (data not shown).…”

“…The frequency of LLO 91-99 -specific CD8 T lymphocytes in mice immunized with the Y. enterocolitica mutants was determined by an IFN-␥-specific ELISPOT assay as previously described (61). Assays were performed with nitrocellulose-backed 96-well microtiter plates (Nunc) that were coated with rat anti-mouse IFN-␥ mAb (BioSource International).…”

“…We used the previously described construct pHR430 (61), which harbors the genetic information for secretion and translocation of YopE (amino acids 1-138) fused to amino acids 51-363 of LLO. This fragment of LLO harbors the immunodominant H-2K d -restricted CD8 T cell epitope LLO 91-99 .…”

LcrV Delivered via Type III Secretion System of Live Attenuated Yersinia pseudotuberculosis Enhances Immunogenicity against Pneumonic Plague

“…Previous studies suggest that Y. pseudotuberculosis is a promising candidate for an oral live carrier vaccine, capable of stimulating antigen specific CD8 ϩ T-cell responses (40,41). Additionally, delivery of heterologous antigens by the type III secretion system (T3SS) in Y. pseudotuberculosis and Salmonella stimulated antigen-specific cytotoxic T-cell responses, antigen-specific CD8 ϩ memory T cells, and protection against challenge with different pathogens (40,42,43).…”

“…Additionally, delivery of heterologous antigens by the type III secretion system (T3SS) in Y. pseudotuberculosis and Salmonella stimulated antigen-specific cytotoxic T-cell responses, antigen-specific CD8 ϩ memory T cells, and protection against challenge with different pathogens (40,42,43). Studies have indicated that both humoral immunity and cellular immunity contribute to vaccine efficacy against plague (21,24,(44)(45)(46)(47).…”