Vaccines containing the HIV tat protein as an adjuvant for the enhancement of cytotoxic T-cell responses

The HIV-1 Tat modifies the catalytic subunit composition and activity of immunoproteasomes in B and T cells which either express Tat, or have been treated with biologically active exogenous Tat protein. This results in modulation of the in vitro CTL epitope hierarchy. In particular, both intracellularly expressed and exogenous native Tat protein increase the major proteolytic activities of the proteasome by up-regulating LMP7 and MECL1 subunits and by down-modulating the LMP2 subunit. This results in a more efficient generation and presentation of subdominant CTL epitopes (Gavioli et al. J. Immunol, 2004, 173:3838-3843 and Patent No. 0323840.9). Since the amount of MHC-I/epitope complexes is crucial in determining the presence and the strength of epitope-specific CTL responses, we set out to verify the biological relevance of these findings for vaccination strategies by evaluating epitope-specific CTL responses against ovalbumin in mice vaccinated with ovalbunin alone, or with ovalbumin in combination with Tat. We found that Tat slightly decreases CTL responses directed to the immunodominant epitope, while it induces those directed to subdominant and cryptic T-cell epitopes which were absent in mice vaccinated with ovalbumin alone (Patent No. 0323840.9). This finding suggests that Tat favours the generation of CTL responses directed to “weak” CTL epitopes and could therefore be used as a tool to increase CTL responses to heterologous antigens. In addition, we found that a mutated form of the HIV-1 Tat protein, carrying glycine instead of cysteine 22 (Tatcys22), like wild-type Tat, modifies the subunit composition of proteasomes. Tatcys22 mutant, in contrast to wild-type Tat, has no effect on the transactivation of the HIV-1 LTR, and does not induce reactivation of latent infection. We also demonstrated that peptide 47-86, derived from the wild-type Tat protein, is sufficient to down-modulate the LMP2 subunit. Therefore, mutated forms of Tat, or Tat-derived peptides, may represent an important alternative to the use of wild-type Tat in vaccination strategies aimed at increasing epitope-specific T cell responses directed to heterologous antigens. We exploited the effect in vivo of wild-type Tat protein and mutant Tatcys22 protein on T cell responses against structural HIV gene products. To this end, Balb/C mice were immunized with HIV-1 Gag or Env protein antigens, either alone or in combination with wild-type Tat or mutant Tatcys22. We found that both wild-type Tat and mutated Tatcys22 increase the number of epitope-specific T cell responses against Gag and Env antigens. In particular, we demonstrated that mice vaccinated with Gag, in combination with wild-type Tat or with the mutant Tatcys22, responded to 7 or 11 T cell Gag-derived epitopes respectively, in contrast to mice vaccinated with Gag alone, which responded to 4 T cell Gag-derived epitopes. Similarly, mice vaccinated with Env, in combination with wild-type Tat or with the mutant Tatcys22 responded to 12 Env-derived pools of epitopes, in contrast to mice vaccinated with Env alone, which responded to 8 T cell Env-derived peptide pools. These observations, together with our previous findings, suggest that Tat is not only an antigen but also a novel adjuvant capable of increasing T cell responses against heterologous antigens. Therefore, the Tat protein, as well as mutant Tatcys22, may represent an important tool in HIV-1 vaccine strategies aimed at broadening the spectrum of the epitopes recognized by T cells.