DNA immunization of HLA transgenic mice with a plasmid expressing mycobacterial heat shock protein 65 results in HLA class I- and II-restricted T cell responses that can be augmented by cytokines.

Infection with Mycobacterium tuberculosis (MTB) remains a major cause of morbidity and mortality world-wide. An effective vaccination strategy is the immunization with plasmid DNA (pDNA), expressing an antigen (Ag) from a pathogen in vivo, which results in specific immune response against the encoded protein as well as the pathogen itself or cells infected with it. To test the ability to induce HLA-restricted T cell immune response against a mycobacterial antigen in humans by pDNA vaccination, we have used transgenic mice that express HLA class I (A*0201/Kb) or HLA class II (DRB1*0301) molecules. pDNA immunization with mycobacterial heat shock protein 65 (Mhsp65)-expressing plasmid (P3M.65) resulted in HLA-II-restricted, Ag-specific T cell-mediated immune responses characterized by proliferation and cytokine production. These T cell responses could be further augmented by the coinjection of P3M.65 and plasmid expressing murine GM-CSF. Furthermore, coimmunizing HLA-I transgenic mice with P3M.65 and a plasmid expressing murine IFN-gamma induced a specific cytotoxic T lymphocyte response restricted by HLA-A2. These results represent the first evidence of a concomitant in vivo induction of HLA class I- as well as class II-restricted T cell responses by pDNA immunization, which is induced or augmented by the codelivery of cytokine-expressing plasmids, supporting its potential use in clinical trials.

The identification of a common pathogen-specific HLA class I A*0201-restricted cytotoxic T cell epitope encoded within the heat shock protein 65.

Bacterial antigens recognized by CD8(+) T cells in the context of MHC class I are thought to play a crucial role in protection against pathogenic intracellular bacteria. Here, we demonstrate the induction of HLA-A*0201-restricted CD8(+) T cell responses against six new high-affinity HLA-A*0201-binding CTL epitopes, encoded within an immunodominant and highly conserved antigen of Mycobacteria, the heat shock protein 65 (hsp65). One of these epitopes, Mhsp65(9(369)), is identical in a large number of pathogenic bacteria, and is recognized in a CD8-independent fashion. Mhsp65(9(369)) could be presented by either mycobacterial hsp65-pulsed target cells or BCG-infected macrophages. Interestingly, T cells specific for this epitope did not recognize the corresponding human hsp65 homologue, probably due to structural differences as revealed by modeling studies. Furthermore, in vitro proteasome digestion analyses show that, whereas the mycobacterial hsp65 epitope is efficiently generated, the human hsp65 homologue is not, thus avoiding the induction of autoreactivity. Collectively, these findings describe high-affinity HLA class I-binding epitopes that are naturally processed and are recognized efficiently by MHC class I-restricted CD8(+) T cells, providing a rational basis for the development of subunit vaccine strategies against tuberculosis and other intracellular infectious diseases.