Analysis of genetic variability within the immunodominant epitopes of envelope gp41 from human immunodeficiency virus type 1 (HIV-1) group M and its impact on HIV-1 antibody detection

Abstract

The serodiagnosis of human immunodeficiency virus type 1 (HIV-1) infection primarily relies on the detection of antibodies, most of which are directed against the immunodominant regions (IDR) of HIV-1 structural proteins. Among these, the N-terminal region of gp41 contains cluster I (amino acids [aa] 580 to 623), comprising the cytotoxic T-lymphocyte epitope (AVERYLKDQQLL) and the cysteine loop (CSGKLIC), and cluster II (aa 646 to 682), comprising an ectodomain region (ELDKWA). To delineate the epitope diversity within clusters I and II and to determine whether the diversity affects serologic detection by U.S. Food and Drug Administration (FDA)-licensed enzyme immunoassay (EIA) kits, gp41 Env sequences from 247 seropositive persons infected with HIV-1 group M, subtypes A (n = 42), B (n = 62), B' (n = 13), C (n = 38), D (n = 41), E (n = 18), F (n = 27), and G (n = 6), and 6 HIV-1-infected but persistently seronegative (HIPS) persons were analyzed. While all IDR were highly conserved among both seropositive and HIPS persons, minor amino acid substitutions (<20% for any one residue, mostly conservative) were observed for all subtypes, except for B', in comparison with the consensus sequence for each subtype. Most importantly, none of the observed substitutions among the group M plasma specimens affected antibody detection, since all specimens (n = 152) tested positive with all five FDA-licensed EIA kits. Furthermore, all specimens reacted with a group M consensus gp41 peptide (WGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASW), and high degrees of cross-reactivity (>80%) were observed with an HIV-1 group N peptide, an HIV-1 group O peptide, and a peptide derived from the homologous region of gp41 from simian immunodeficiency virus from chimpanzee (SIVcpz). Taken together, these data indicate that the minor substitutions observed within the IDR of gp41 of HIV-1 group M subtypes do not affect antibody recognition and that all HIV-1-seropositive specimens containing the observed substitutions react with the FDA-licensed EIA kits regardless of viral genotype and geographic origin.

FIG. 1

Analysis of sequence divergence in gp41 cluster I (aa 580 to 623) from 247 HIV-1 group M subtype A to G specimens. The CTL epitope (aa 591 to 602), the cysteine loop (aa 607 to 613), and the glycosylation site (aa 620 to 622) are shaded. The left column shows the number of specimens for each subtype examined. The numbers of specimens with amino acid substitutions are shown for each subtype; dashes represent conserved amino acids in all specimens. ∗, specimens from six HIPS AIDS patients infected with subtype B virus.

FIG. 2

Analysis of sequence divergence in gp41 cluster II (aa 646 to 682) from 247 HIV-1 group M subtype A to G specimens. The glycosylation site (aa 646 to 648) and the ectodomain (aa 671 to 676) are shaded. The left column shows the number of specimens for each subtype examined. The numbers of specimens with amino acid substitutions are shown for each subtype; dashes represent conserved amino acids in all specimens. ∗, specimens from six HIPS AIDS patients infected with subtype B virus.

FIG. 3

Reactivity of HIV-1 group M plasma specimens with five FDA-licensed EIA kits (last five columns, from left to right: Abbott HIVAB HIV-1 EIA, Abbott HIVAB HIV-1/HIV-2, Genetic Systems LAV EIA, Genetic Systems HIV-1/HIV-2 Peptide EIA, and Organon Teknika Vironostika HIV Microelisa System). Representative specimens (n = 152) with and without amino acid substitutions in the cluster I and cluster II regions were tested with the EIA kits. Specific mutations within the CTL epitope, the cysteine loop, and the ectodomain are shown for each subtype. nt, not tested.

FIG. 3

Reactivity of HIV-1 group M plasma specimens with five FDA-licensed EIA kits (last five columns, from left to right: Abbott HIVAB HIV-1 EIA, Abbott HIVAB HIV-1/HIV-2, Genetic Systems LAV EIA, Genetic Systems HIV-1/HIV-2 Peptide EIA, and Organon Teknika Vironostika HIV Microelisa System). Representative specimens (n = 152) with and without amino acid substitutions in the cluster I and cluster II regions were tested with the EIA kits. Specific mutations within the CTL epitope, the cysteine loop, and the ectodomain are shown for each subtype. nt, not tested.

FIG. 4

Detection of gp41-specific antibodies using synthetic peptides comprising cluster I sequences (aa 580 to 623). The percent reactivities of HIV-1 group M plasma specimens are shown for consensus group M peptide (■) and group O peptide (▨) or homologous regions of group N peptide (░⃞) and SIVcpz peptide (▩). The 44-mer peptides representing consensus sequences include group M (WGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASW), group O (WGIRQLRARLLALETLIQNQQLLNLWGCKGKLVCYTSVKWNRTW), group N (WGIKQLQAKVLAIERYLRDQQILGSLGCSGKTICYTTVPWNETW), and SIVcpz (WGVKQLQARLLAVERYLQDQQILGLWGCSGKAVCYTTVPWNNSW). All 131 samples, regardless of substitution in gp41 regions, reacted with the gp41 group M peptide-based EIA, and a high degree of cross-reactivity to HIV-1 group O and N peptides and in SIVcpz peptide was observed.