Identification of HIV-1 protease epitopes target of CD4+ T cell responses in HIV-1 infected patients

Author(s)

Natalie Guida Muller

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2009-12-18

Examining board members

Edecio Cunha Neto; Luís Fernando de Macedo Brigido; Cristina Maria Kokron

• Advisor: Edecio Cunha Neto

Abstract

Introduction: A significant proportion of protease inhibitor (PI)-treated HIV-1 infected (HIV-1+) patients develop resistance mutations. Recent studies have shown that CD8+ T cells from HIV-1 patients can recognize antiretroviral drug-induced mutant Pol epitopes. No HIV-1 protease CD4 epitopes are described in the Los Alamos database. Aims: Given that the protease of HIV-1 is a target of antiretroviral therapy and this pressure may lead to the selection of mutations, we investigated whether PI-induced mutations affect the recognition of HIV-1 protease epitopes by CD4 + T cells in PI-treated patients. We investigated the recognition of three protease regions predicted to harbor CD4+ T cell epitopes as well as PI-induced mutations by CD4+ T cells of PI-treated HIV-1+ patients. Methods: Forty PI-treated HIV-1+ patients were included (30 undergoing Lopinavir/ritonavir, 9 undergoing Atazanavir/ritonavir and 1 undergoing exclusively Atazanavir treatment). For each patients, the endogenous HIV-1 protease sequence, viral genotype and HLA class II typing were determined. We used the TEPITOPE algorithm to select promiscuous, multiple HLA-DR-binding peptides encoding 3 regions of HIV-1 HXB2 strain protease (HXB2 4-23, 45-64, and 76-95) and 32 additional peptides contained in the same regions, but encompassing the most frequent PI-induced mutations in Brazil. The 35 peptides were thus synthesized. Proliferative responses of CD4+ and CD8+ T cells against peptides were determined by the CFSE dilution assay. HLA class II binding assays were made to confirm the promiscuity of these peptides and evaluate their ability to bind the HLA molecules carried by each patient. Results: All tested peptides were recognized by at least one patient and proliferative responses of CD4+ and CD8+ T cells against at least one HIV-1 protease peptide were found in 78% and 75% patients, respectively. The third region (Protease 76-95) was the most frequently recognized. By comparing T-cell responses to HIV-1 endogenous protease sequences, we found that most patients failed to recognize identical peptides of those sequences, but recognized different variant peptides of the same region. Only seven patients responded to endogenous sequences. We found that several endogenous peptides that failed to be recognized showed no binding to the HLA alleles carried by that given patient, suggesting that mutations selected by immune pressure have led to escape of antigen presentation, as well as direct escape of the CD4+ T cell response. Alternatively, it could have been due to the presence of a different replicating virus in the plasma-since we only obtained proviral sequences. Conclusion: Wild-type and mutant HIV-1 protease epitopes recognized by CD4+ T cells were identified. We also found that most patients failed to recognize their endogenous protease sequences, while they recognized variant sequences. The recognition of non-endogenous sequences could hypothetically be a consequence of targeting a minor HIV-1 population; HERV protease, that contains regions of similarity with HIV-1 protease; or HIV-1 sequences present only in viremic partners. The failure to recognize endogenous sequences is most likely due to immune escape, either at the level of presentation or direct T cell recognition. This may have a pathophysiological consequence on evasion of T cell responses against protease and the fact that it has been considered traditionally a poorly antigenic HIV-1 protein. (AU)