Identification of overlapping HLA class I-restricted cytotoxic T cell epitopes in a conserved region of the human immunodeficiency virus type 1 envelope glycoprotein: definition of minimum epitopes and analysis of the effects of sequence variation.

Abstract

Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine development. Finally, the identification of minimal CTL epitopes presented by class I HLA molecules should facilitate the definition of allele-specific motifs.

Identification of type-specific cytotoxic T lymphocyte responses to homologous viral proteins in laboratory workers accidentally infected with HIV-1.

“…Five HLA-B14-restricted CD8 ϩ CTL clones isolated from subject LWF at four different time points recognized a previously reported epitope in gp41 (ERYLKDQQL, aa 584-592) (15,44). The gp120 epitope CTNVSTVQC, aa 241-249, was recognized by four CTL clones isolated from subject LWF at two different time points.…”

“…T cell clones were then expanded, screened for cytolytic activity, and maintained as previously described (36). Mapping of CTL epitopes recognized by these clones was performed using autologous B-LCL infected with recombinant vaccinia viruses expressing serial truncations of HIV-1 genes, and then autologous B-LCL pulsed with overlapping synthetic HIV-1 synthetic peptides, as previously described (15). Finally, fine mapping of the epitopes was performed using peptide titration assays as previously described (14).…”

“…Synthetic peptides corresponding to the HIV-1 envelope PV22 sequence were synthesized as previously described (15). These peptides were 25 amino acids (aa), and overlapped adjacent peptide by 8 aa.…”

“…HIV-1 sequence variation in the envelope protein may approach 30% (13), and even single amino acid changes may lead to a loss of CTL recognition of variant epitopes (14,15). Most studies analyzing the CTL response to HIV-1 have relied on the use of target cells expressing viral gene products of laboratory strains of HIV, which may differ substantially from the infecting strain of virus and will favor the detection of group-specific CTL responses.…”

Construction and characterisation of infectious recombinant HIV-1 clones containing CTL epitopes from structural proteins in Nef

“…pEX2.1 subclones were digested with Bpu1102I (MBI Fermentas), dephosphorylated using Calf Intestine Phosphatase (BoehringerMannheim), and purified on agarose gels (DNA extraction kit, Boehringer-Mannheim). The nucleotide sequences encoding the Env 589 -597 (ERYLKDQQL) and RT 244 -252 (IVLPEKDSW) CTL epitopes, which are recognised in the context of HLA-B14 and -B57, respectively (Johnson et al, 1992;Klein et al, 1998), were generated by annealing complementary oligonucleotides (Fig. 1A).…”