Genetic and neutralization properties of subtype C human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in Southern Africa

Abstract

A standard panel of subtype C human immunodeficiency virus type 1 (HIV-1) Env-pseudotyped viruses was created by cloning, sequencing, and characterizing functional gp160 genes from 18 acute and early heterosexually acquired infections in South Africa and Zambia. In general, the gp120 region of these clones was shorter (most evident in V1 and V4) and less glycosylated compared to newly transmitted subtype B viruses, and it was underglycosylated but no different in length compared to chronic subtype C viruses. The gp120s also exhibited low amino acid sequence variability (12%) in V3 and high variability (39%) immediately downstream of V3, a feature shared with newly transmitted subtype B viruses and chronic viruses of both subtypes. When tested as Env-pseudotyped viruses in a luciferase reporter gene assay, all clones possessed an R5 phenotype and resembled primary isolates in their sensitivity to neutralization by HIV-1-positive plasmas. Results obtained with a multisubtype plasma panel suggested partial subtype preference in the neutralizing antibody response to infection. The clones were typical of subtype C in that all were resistant to 2G12 (associated with loss of N-glycosylation at position 295) and most were resistant to 2F5, but all were sensitive to 4E10 and many were sensitive to immunoglobulin G1b12. Finally, conserved neutralization epitopes in the CD4-induced coreceptor binding domain of gp120 were poorly accessible and were difficult to induce and stabilize with soluble CD4 on Env-pseudotyped viruses. These results illustrate key genetic and antigenic properties of subtype C HIV-1 that might impact the design and testing of candidate vaccines. A subset of these gp160 clones are suitable for use as reference reagents to facilitate standardized assessments of vaccine-elicited neutralizing antibody responses.

FIG. 1.

Phylogenetic relationships of subtype C reference env clones. The newly characterized sequences are indicated by solid circles and shaded diamonds, and the 12 env clones that are recommended as standard reference reagents are represented by the solid circles and are bolded. Horizontal branch lengths are drawn to scale (the scale bar represents 0.01 nucleotide substitutions per site), but vertical separation is for clarity only. Values at nodes indicate the percentage of bootstraps in which the cluster to the right was found; only values of 80% or greater are shown. The phylogenetic tree was rooted with subtype D env sequences (NDK, Z2Z3, and 94UG114).

FIG.2.

Alignment of deduced amino acid sequences from acute/early subtype C HIV-1 env genes. Nucleotide sequences of newly derived env genes were translated, aligned, and compared with a consensus sequence generated by MASE. Numbering of amino acid residues begins with the first residue of gp120 and does not include the signal peptide. Dashes denote sequence identity, while dots represent gaps introduced to optimize alignments. Small letters in the consensus sequence indicate sites at which fewer than 50% of the viruses share the same amino acid residue. Triangles above the consensus sequence denote cysteine residues (solid triangles indicate sequence identity, while open triangles indicate sequence variation). V1, V2, V3, V4, and V5 regions designate hypervariable HIV-1 gp120 domains as previously described. The signal peptide and Env precursor cleavage sites are indicated; msd denotes the membrane-spanning domain in gp41; asterisks mark in-frame stop codons. Open circles highlight altered cysteine residues. Potential N-linked glycosylation sites (NXYX motif, where X is any amino acid other than proline and Y is either serine or threonine) are bolded and underlined. Highly conserved sites of potential N-linked glycosylation are shaded. The solid diamond denotes a highly variable amino acid position in the V3 loop.

FIG.2.

Alignment of deduced amino acid sequences from acute/early subtype C HIV-1 env genes. Nucleotide sequences of newly derived env genes were translated, aligned, and compared with a consensus sequence generated by MASE. Numbering of amino acid residues begins with the first residue of gp120 and does not include the signal peptide. Dashes denote sequence identity, while dots represent gaps introduced to optimize alignments. Small letters in the consensus sequence indicate sites at which fewer than 50% of the viruses share the same amino acid residue. Triangles above the consensus sequence denote cysteine residues (solid triangles indicate sequence identity, while open triangles indicate sequence variation). V1, V2, V3, V4, and V5 regions designate hypervariable HIV-1 gp120 domains as previously described. The signal peptide and Env precursor cleavage sites are indicated; msd denotes the membrane-spanning domain in gp41; asterisks mark in-frame stop codons. Open circles highlight altered cysteine residues. Potential N-linked glycosylation sites (NXYX motif, where X is any amino acid other than proline and Y is either serine or threonine) are bolded and underlined. Highly conserved sites of potential N-linked glycosylation are shaded. The solid diamond denotes a highly variable amino acid position in the V3 loop.

FIG.2.

Alignment of deduced amino acid sequences from acute/early subtype C HIV-1 env genes. Nucleotide sequences of newly derived env genes were translated, aligned, and compared with a consensus sequence generated by MASE. Numbering of amino acid residues begins with the first residue of gp120 and does not include the signal peptide. Dashes denote sequence identity, while dots represent gaps introduced to optimize alignments. Small letters in the consensus sequence indicate sites at which fewer than 50% of the viruses share the same amino acid residue. Triangles above the consensus sequence denote cysteine residues (solid triangles indicate sequence identity, while open triangles indicate sequence variation). V1, V2, V3, V4, and V5 regions designate hypervariable HIV-1 gp120 domains as previously described. The signal peptide and Env precursor cleavage sites are indicated; msd denotes the membrane-spanning domain in gp41; asterisks mark in-frame stop codons. Open circles highlight altered cysteine residues. Potential N-linked glycosylation sites (NXYX motif, where X is any amino acid other than proline and Y is either serine or threonine) are bolded and underlined. Highly conserved sites of potential N-linked glycosylation are shaded. The solid diamond denotes a highly variable amino acid position in the V3 loop.

FIG.2.

Alignment of deduced amino acid sequences from acute/early subtype C HIV-1 env genes. Nucleotide sequences of newly derived env genes were translated, aligned, and compared with a consensus sequence generated by MASE. Numbering of amino acid residues begins with the first residue of gp120 and does not include the signal peptide. Dashes denote sequence identity, while dots represent gaps introduced to optimize alignments. Small letters in the consensus sequence indicate sites at which fewer than 50% of the viruses share the same amino acid residue. Triangles above the consensus sequence denote cysteine residues (solid triangles indicate sequence identity, while open triangles indicate sequence variation). V1, V2, V3, V4, and V5 regions designate hypervariable HIV-1 gp120 domains as previously described. The signal peptide and Env precursor cleavage sites are indicated; msd denotes the membrane-spanning domain in gp41; asterisks mark in-frame stop codons. Open circles highlight altered cysteine residues. Potential N-linked glycosylation sites (NXYX motif, where X is any amino acid other than proline and Y is either serine or threonine) are bolded and underlined. Highly conserved sites of potential N-linked glycosylation are shaded. The solid diamond denotes a highly variable amino acid position in the V3 loop.

FIG. 3.

Neutralization sensitivity of acute/early subtype C and subtype B HIV-1 Env-pseudotyped viruses as determined with plasma samples from HIV-1-infected individuals. Bar height represents the GMT of neutralizing Abs against the indicated Env-pseudotyped viruses. A. GMT for all HIV-1-positive plasma samples shown in Table 4 (BB8, BB12, BB28, BB55, BB70, and BB106 and pool A, pool B, pool C, and pool D). Clones selected as standard subtype C reference strains are marked with an asterisk. B. GMT of neutralizing Abs for the individual subtype C plasma samples BB8, BB12, BB28, BB55, BB70, and BB106 assayed against the 12 subtype C reference strains. C. GMT of neutralizing Abs in the individual subtype C plasma samples BB8, BB12, BB28, BB55, BB70, and BB106 assayed against 12 subtype B reference strains.