Seroreactivity of Analogous Antigenic Epitopes in Glycoprotein 120 Expressed in HIV-1 Subtypes A, B, C, and D

Abstract

This article describes the impact of sequence variation on the distribution and seroreactivity of linear antigenic epitopes in gp120 encoded in new Ugandan HIV-1 clones from subtypes A, C, and D, and in North American clones from the B subtype. A region of the env gene encoding the C2 to V5 domains was PCR amplified from the lysates of peripheral blood leukocytes or from short-term cultured isolates. Computer-assisted analyses were conducted on the amino acid sequences to determine the distribution of surface structures in gp120. Despite marked sequence diversity, eight analogous epitopes were predicted for all clades of the virus analyzed. Synthetic peptides comprising the putative principal neutralizing determinant E2[V3], and other B cell epitopes E3[V3-V4], E4[V3-V4], E7[C3], and E8[V5], from a seroprevalent Ugandan isolate, AUG06c, were tested in ELISA for antigenicity with sera from Uganda, New York, and Thailand. Variable magnitudes of seroreactivity were observed for all of the peptides tested. However, a significantly higher degree of serum cross-reactivity was detected with the V3 loop peptide. ELISA reactivities of the same serum panel indicated that V3 loop peptides containing the apical residues GPGR (clones AUG06c and BRT3) or GPGQ (CUG045 and DUG044) were more antigenic and display extensive cross-reactivity as compared to analogous peptides comprising GLGQ (DUG23c), GQGQ (DUG042), or GPWG (BRT1). BETATURN analysis of the divergent V3 loop apical residues showed a good correlation of probable beta-turn occurrence with strong seroreactivity. These findings suggest that the major antigenic specificities in the divergent clades of HIV-1 are well conserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Relative reactivity of the V3 loop PND of HIV-1 subtypes A, B, C, D, and F with sera from selected Ugandan localities

“…The consistency and reliability of these programs to predict antigenic residues have been previously documented [8,17,18].…”

“…Peptides predicted to comprise the PND were synthesized using the solid-phase procedure [19] on an automated peptide synthesizer (Synergy, Model 432A, Applied Biosystems) with fmoc protected amino acids, and hydroxymethylphenoacetic resin as described [8]. Post-synthesis cleavage from the resin and protecting groups was conducted using an aqueous solmion containing the scavengers: thioanisole, trifluoroacetic acid, and dithoethane.…”

“…The primers were synthesized based on the ENV sequence in pNL43 [12]. The products were cloned into the pT7 vector (Novagen Inc., WI) [8]. The clones were sequenced using the Sanger dideoxy chain-termination reaction [13] or by automated cycle sequencing (ABI Sequencer Model 373A and the Prism kit, Applied Biosystems, CA) with fluorescent nucleotide dye-terminators and Taq DNA polymerase [8].…”

“…The products were cloned into the pT7 vector (Novagen Inc., WI) [8]. The clones were sequenced using the Sanger dideoxy chain-termination reaction [13] or by automated cycle sequencing (ABI Sequencer Model 373A and the Prism kit, Applied Biosystems, CA) with fluorescent nucleotide dye-terminators and Taq DNA polymerase [8]. All nucleotide sequences have been submitted to GenBank under the accession numbers U 11591 -U 11595 and U 11597.…”

Prediction of exposed domains of envelope glycoprotein in Indian HIV-1 isolates and experimental confirmation of their immunogenicity in humans

“…When the gp120 glycoproteins derived from different HIV-1 isolates were compared, five conserved regions (C1 to C5) and five variable regions (V1 to V5) were identified (8,9). Most of the HIV-neutralizing antibodies fall into two categories: those that recognize a determinant in the V3 loop of gp120 (10,11) and those that block the gp120-CD4 interaction (12) by binding to regions in gp120 conserved between different HIV strains (4,13,14).…”