Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies

Abstract

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.

FIG. 1.

Generation of pseudovirions containing cloned Env from primary PBMC-cultured isolates. Primary isolates of HIV-1, obtained by standard PBMC coculture, were used to infect fresh cultures of PHA-stimulated PBMC. DNA from the infected PBMC was used for the PCR amplification and cloning of rev/env cassettes that contained full-length gp160 genes. The virus-containing culture medium was made cell free by 0.45-μm filtration and stored in aliquots at −80°C as a reference stock of the immediate uncloned parental virus. Cloned gp160 genes were used to produce Env-pseudotyped virus in 293T cells by cotransfection with the pSG3ΔEnv backbone vector. Neutralization phenotypes of the parental PBMC viruses and pseudovirions generated by cotransfection with the cloned primary Envs were compared in JC53-BL cells.

FIG. 2.

Phylogenetic relationships of newly derived HIV-1 env sequences with subtype B reference strains. The newly characterized sequences are indicated by boxes, and the 12 env clones that are recommended as standardized reference reagents are highlighted by shaded boxes. Horizontal branch lengths are drawn to scale (the scale bar represents 0.01 nucleotide substitution 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. 3.

Alignment of deduced amino acid sequences from acute/early 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 indicate sequence identity, while dots represent gaps introduced to optimize alignments. Lowercase 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 indicate 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, membrane-spanning domain in gp41; asterisks, in-frame stop codons; open circles, altered cysteine residues in the extracellular portions of gp120 and gp41. Potential N-linked glycosylation sites (NXYX motif, where X is any amino acid other than proline and Y is either serine or threonine) are in boldface and underlined.

FIG. 3.

Alignment of deduced amino acid sequences from acute/early 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 indicate sequence identity, while dots represent gaps introduced to optimize alignments. Lowercase 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 indicate 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, membrane-spanning domain in gp41; asterisks, in-frame stop codons; open circles, altered cysteine residues in the extracellular portions of gp120 and gp41. Potential N-linked glycosylation sites (NXYX motif, where X is any amino acid other than proline and Y is either serine or threonine) are in boldface and underlined.

FIG. 3.

Alignment of deduced amino acid sequences from acute/early 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 indicate sequence identity, while dots represent gaps introduced to optimize alignments. Lowercase 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 indicate 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, membrane-spanning domain in gp41; asterisks, in-frame stop codons; open circles, altered cysteine residues in the extracellular portions of gp120 and gp41. Potential N-linked glycosylation sites (NXYX motif, where X is any amino acid other than proline and Y is either serine or threonine) are in boldface and underlined.

FIG. 4.

Rank order of the neutralization sensitivities of acute/early HIV-1 Env clones as determined with serum and plasma samples from HIV-1-infected individuals. The bar height represents the GMT of the HIV-1-positive serum and plasma samples shown in Table 4 (samples DUMC-1, DUMC-2, DUMC-3, LW.0013, and TH.10.3 and pool A, pool B, pool C, and pool D). Clones selected as standard reference strains are marked with an asterisk.

FIG. 5.

Neutralization phenotypes of acute/early HIV-1 Env clones compared to the phenotypes of the immediate uncloned parental viruses. Uncloned parental PBMC-grown viruses (gray bars) and the corresponding Env-pseudotyped viruses produced in 293T cells (black bars) were assayed with HIV-1-positive serum samples, sCD4, and human MAbs in JC53-BL cells.