Bruno Guy

Qualitative and quantitative analysis of human cytotoxic T-lymphocyte responses to HIV-1 proteins.

ObjectiveTo study the degree of immunogenicity of each HIV-1 protein. DesignIn most viral systems, antiviral cytotoxic T-lymphocytes (CTL) from a given donor preferentially recognize only one or a small number of viral proteins. MethodsAnti-HIV CTL were generated by in vitro stimulation of peripheral blood mononuclear cells from seropositive donors and tested against multiple HIV-1 proteins or groups of proteins encoded by seven genes (env, gag, pol, nef, rev, tat and vif). Using autologous target cells infected with recombinant vaccinia viruses expressing one of the HIV-1 LA1 proteins, we compared the cytolytic activities obtained from bulk culture with those found in limiting dilution analysis (LDA). ResultsOur results were noteworthy for the following reasons. (1) Each responding donor reacted simultaneously to multiple proteins; this is very unusual in other viral systems. Anti-Gag CTL were detected in most, and anti-Pol in approximately three-quarters, of the patients, together with very high amounts of the corresponding CTL precursors in LDA. CTL against Env and Nef were found in two-thirds of the patients, while Vif- and Rev-specific CTL were less frequent. Finally, Tat was seldom recognized by CTL, but its antigenicity was revealed in LDA. (2) All responding cells revealed in bulk cultures as well as in LDA were CD8+ T-cells, and their in vitro differentiation did not require the help of CD4+ T-cells. (3) Proteins from the HIV-1LA1 isolate were recognized with high frequency by CTL from seropositive donors, most certainly being infected by other isolates, which suggests that relatively conserved epitopes are predominant targets of CTL. ConclusionTaken together, these results are encouraging for vaccine purposes, since anti-HIV-1 CTL stimulation is thought to be a requirement for such a vaccine.

Different patterns of HIV-1-specific cytotoxic T-lymphocyte activity after primary infection.

Objective: To analyse the HIV‐1‐specific cytotoxic T‐lymphocyte (CTL) responses of nine HIV‐seropositive subjects in relation with primary infection. Methods: Anti‐HIV CTL were generated by in vitro stimulation of peripheral mononuclear cells obtained from HIV‐seropositive donors at various times after primary infection. They were tested against several structural or regulatory HIV‐1 proteins, using autologous target cells infected with recombinant vaccinia viruses expressing one of the HIV‐1LAI proteins. Results: An important CTL activity was found during the first month following seroconversion only in those donors who showed clinical symptoms during primary infection. The temporal evolution of this response differed for each subject; one remained a non‐responder even 30 months after seroconversion. The structural proteins were recognized particularly early, while the antigenicity of regulatory proteins appeared later. Conclusion: Different patterns of HIV‐specific CTL response can be observed after primary infection. The evolution of infection in these different HIV‐seropositive subjects will be particularly interesting to analyse. AIDS 1995, 9:421‐426

Production of a non-functional nef protein in human immunodeficiency virus type 1-infected CEM cells

The nef gene product of the human immunodeficiency virus (HIV) is suggested to be a negative factor involved in down-regulating viral expression by a mechanism in which the correct conformation of the nef protein is essential. The nef protein expressed by vaccinia virus recombinants is phosphorylated by protein kinase C. We investigated the synthesis of the nef protein and its state of phosphorylation during HIV-1 infection of a T4 cell line (CEM cells). Maximum synthesis of viral proteins occurred 3 days after infection, when more than 90% of cells were producing viral proteins. The synthesis of the nef protein was detected in parallel with the env and gag proteins. As expected, the nef protein was myristylated but not phosphorylated, and its half-life was less than 1 h. By the use of the polymerase chain reaction technique, we isolated and sequenced the nef gene of this HIV-1 stock. Two significant mutations were observed. Firstly, threonine, at amino acid number 15, the site of phosphorylation by protein kinase C, was mutated into an alanine, and secondly aspartic acid of the tetrapeptide WRFD, which is probably involved in GTP binding, was mutated into an asparagine. The mutated nef gene was expressed in a vaccinia virus system, in which it was not phosphorylated and its half-life was dramatically reduced compared to the wild-type nef gene product. Furthermore, down-regulation of CD4 cell surface expression was no longer affected by the mutated nef gene. These results emphasize that phosphorylation of the nef protein provides an efficient test to monitor its biological activity.