Ruolo della proteina Tat nell’infezione da HIV: studi in vitro e immunogenicità in vivo

Role of Tat protein in HIV infection: in vitro studies and in vivo immunogenicity The HIV virus is responsible for the acquired immunodeficiency syndrome, AIDS, and infects CD4+ T cells, with a first acute phase of replication, then entering latency. This mechanism of latency allows the virus to survive in the body for years, in cellular compartments, known as viral reservoirs and recognized as CD4+ T cell memory. The HIV Tat protein, important in the regulation of viral transcription, is known to modulate the transcriptional profile, the activation and the differentiation of T lymphocytes. These capabilities suggest a possible involvement of Tat in the activation of CD4+ T cells, which would lead to an increase of the target of infection, and in the establishment and maintenance of viral reservoirs. In this study, we evaluated the effect of Tat on the activation of CD4+ T cells, in resting and activated (with anti-CD3 / CD28) conditions. We analyzed the release of cytokines, the phenotypic expression of activation markers and cell proliferation in response to the treatment with Tat. The data obtained showed an increased release of IL2 and IFNγ in activated CD4+ T cells treated with Tat, while no effect was observed on cell proliferation and expression of activation markers. We also analysed the transcriptional profile of important genes involved in CD4+ T lymphocytes activation as T-bet, Eomes, Bcl-2 and Blimp-1. We show that Tat up-regulates all genes evaluated on activated CD4+ T cells, while on resting CD4+ T cells there was a decline in the expression of Blimp- and Eomes in response to treatment with Tat. The data obtained confirm the effect of Tat in increasing the activation of activated CD4+ T cells, thus suggesting that Tat favors the spread of virus in the course of HIV infection. Intriguingly, the double effect on the expression of Blimp-1 and Eomes suggests its involvement in viral latency too. The Tat effect on the establishment and maintenance of viral reservoirs was evaluated on total CD4+ T lymphocytes, analyzing their differentiation into memory cells through analysis of phenotypic marker as CCR7 CD62L, CD44, CD127 and CD45RA. By these studies, we observed that Tat increases the expression of markers characterizing a memory phenotype. Then, we used a stimulation model, "unpolarized", to generate and maintain central memory CD4+ T cells obtained from naϊve CD4+ T lymphocytes,in the presence or absence of Tat. By this model, we demonstrated that Tat increases the differentiation of naϊve CD4+ T cells into central memory CD4+ T lymphocytes. Interestingly, we also observed that Tat increases the viability of cells stimulated in the presence of Tat under non-polarized conditions. In conclusion, the analysis of transcription factors, cell viability and phenotype demonstrate that Tat favors the activation of CD4+ T cells, and suggests that it may also have a role on the maintenance of memory lymphocytes, potential reservoir of HIV. Further studies are needed to understand the role of Tat in the differentiation of T cells into memory. Thus, Tat, which is deeply involved in the mechanisms of HIV infection and maintenance, is a good vaccine candidate. Indeed, the use of the Tat protein in vaccines against AIDS showed promising results in primate and human studies. To characterize the impact of the administration route on the induction of humoral responses at systemic and mucosal levels, we compared intradermal, intramuscular and mucosal immunizations with Tat and a Tat-derived peptide. Mice were immunized with the Tat protein by different routes and the titer and isotype of anti-Tat antibodies were assessed in serum and mucosal lavages. Intramuscular and intradermal administrations showed comparable immunogenicity, while the mucosal administration was unable to induce IgM in serum and IgG at mucosal sites but showed superior immunogenicity in terms of IgA induction. Anti-Tat antibodies were also obtained upon vaccination with the immunodominant Tat 1-20 peptide which was, however, less immunogenic than the whole Tat protein.