Isolation of a monoclonal antibody that targets the alpha-2 helix of gp120 and represents the initial autologous neutralizing-antibody response in an HIV-1 subtype C-infected individual

Abstract

The C3-V4 region is a major target of autologous neutralizing antibodies in HIV-1 subtype C infection. We previously identified a Center for AIDS Program of Research in South Africa (CAPRISA) participant, CAP88, who developed a potent neutralizing-antibody response within 3 months of infection that targeted an epitope in the C3 region of the HIV-1 envelope (P. L. Moore et al., PLoS Pathog. 5:e1000598, 2009). Here we showed that these type-specific antibodies could be adsorbed using recombinant gp120 from the transmitted/founder virus from CAP88 but not by gp120 made from other isolates. Furthermore, this activity could be depleted using a chimeric gp120 protein that contained only the C3 region from the CAP88 viral envelope engrafted onto the unrelated CAP63 viral envelope (called 63-88C3). On the basis of this, a differential sorting of memory B cells was performed using gp120s made from 63-88C3 and CAP63 labeled with different fluorochromes as positive and negative probes, respectively. This strategy resulted in the isolation of a highly specific monoclonal antibody (MAb), called CAP88-CH06, that neutralized the CAP88 transmitted/founder virus and viruses from acute infection but was unable to neutralize CAP88 viruses isolated at 6 and 12 months postinfection. The latter viruses contained 2 amino acid changes in the alpha-2 helix of C3 that mediated escape from this MAb. One of these changes involved the introduction of an N-linked glycan at position 339 that occluded the epitope, while the other mutation (either E343K or E350K) was a charge change. Our data validate the use of differential sorting to isolate a MAb targeting a specific epitope in the envelope glycoprotein and provided insights into the mechanisms of autologous neutralization escape.

Fig. 1.

Adsorption of autologous neutralizing activity by autologous monomeric gp120. (A) Graph adapted from Moore et al. (19) to show the presence of the anti-C3 and anti-V1V2 neutralizing antibodies in CAP88 plasma samples collected during the course of infection. The black line shows the response against the early CAP88 virus 88.2mo.B5, while the gray area delineates the portion attributed to anti-C3 antibodies and the gray stripped lines delineate the portion attributed to anti-V1V2 antibodies. Plasma samples collected at 26 weeks (B and C) and 54 weeks (D and E) postinfection were adsorbed with CAP88 and CAP63 gp120-coated beads as well as blank beads. The plasma sample collected at 26 weeks was also adsorbed with a chimeric CAP63 gp120 grafted with the C3 region of CAP88 (63-88C3). Absorbed plasma samples were tested for neutralization of the autologous virus 88.2mo.B5 (B and D) and for binding to all the proteins used for adsorptions (C and E). OD, optical density.

Fig. 2.

Epitope-specific memory-B-cell staining. PBMCs from CAP88 at 34 weeks postinfection were stained for flow cytometric analysis and single-cell sorting. Memory B cells were selected as lymphocytes using geometric gates and live cells using a viability marker and then as CD3/CD14/CD16/CD235a⁻, CD19⁺, and surface IgD⁻. Memory B cells positively stained with the chimeric protein 63-88C3-AF647 and negative for CAP63-Pacific Blue (PacBlue; square gate) were sorted individually into a 96-well plate. A population of cells that stained positive for both markers was inadvertently included in the sorted population (lower right edge of gate in the bottom panel). FSC and SSC, forward and side scatter, respectively.

Fig. 3.

Binding and neutralization of the isolated antibodies. The seven isolated V_H and V_L pairs were transiently transfected in 293T cells. Their supernatants were screened for binding to CAP63 and 63-88C3 gp120s by Luminex (A) and tested for neutralization of autologous virus 88.2mo.B5 isolated during acute infection in the TZM-bl assay (B). High background levels in the neutralization screening assay, as seen in the mock transfections, may be due to the transfection reagent or other toxins in these concentrated supernatants.

Fig. 4.

Neutralization escape from CAP88-CH06 MAb. Purified CAP88-CH06 MAb was tested for neutralization of autologous CAP88 clones isolated at 1, 2, 6, and 12 months postinfection in the TZM-bl assay (A), infectious molecular clones of viruses from 1 and 6 months in a PBMC neutralization assay (B), and a clone obtained at 6 months in which the N339I and K350E changes were introduced (C). MFI, mean fluorescence index.

Fig. 5.

Location within the alpha-2 helix of the amino acid residues involved in neutralization escape. The CAP88.2mo.B5 gp120 amino acid sequence was modeled using coordinates from the CAP210 gp120 structure in complex with secretory CD4 and 21c Fab (Protein Data Bank accession no. 3LQA) and the Modeler (version 9.8) program. A ribbon diagram of the alpha-2 helix was used to display the location of the 3 amino acids (in red) in the alpha-2 helix of CAP88 that mediated escape from the CAP88-CH06 MAb. Sequences of this region from 2 representative clones at 6 months compared to the sequence of the clone at 2 months showed that the addition of a glycan at position 339 was common, while charge changes at position 343 or 350 were mutually exclusive, occurring on equal numbers of separate genomes (21). The figure was generated with the PyMOL program (DeLano Scientific LLC, South San Francisco, CA [http://www.pymol.org]).

Fig. 6.

Effect of N-linked glycosylation on neutralization. (A) The I339 residue in the neutralization-sensitive clone 2mo.B5 was replaced by a glutamine (Q) or asparagine (N). Charge change E343K or E350K was introduced with or without the N339 glycan. All mutants were tested for neutralization by the CAP88-CH06 MAb starting at 10 μg/ml. (B) The CAP88 clone 2mo.B5 was grown in the presence of 100 μM kifunensine or in GnTI-deficient 293S cells to determine the effect of the glycans on the neutralization by CAP88-CH06 MAb.

Fig. 7.

CAP88 MAb binding to gp120 made from autologous and mutant viruses. Recombinant gp120s produced in 293T cells from wild-type 88.2mo.B5 and escape mutant 88.2mo.B5 containing two amino acid changes (I339, E343K) were tested in an ELISA for binding to anti-C3 CAP88-CH06 (A) and anti-V3 CAP88-3468L (B) MAbs. CAP63 gp120 was used as a control. Virion-associated gp120s were captured from lysed pseudovirus stocks produced in 293T cells via the D7324 antibody and tested for binding to anti-C3 CAP88-CH06 (C) and anti-V3 CAP88-3468L (D) MAbs.

Fig. 8.

Sequence analyses of the V_H and V_L genes of the CAP88-CH06 MAb. The variable region sequences of CAP88-CH06 were compared to the closest germ line sequence for both the heavy-chain (HC) (A) and light-chain (LC) (B) genes. Light gray and black highlights, nonsynonymous and synonymous changes, respectively; dark gray highlighting, a 6-nucleotide insertion in the heavy-chain complementarity-determining region 2 (CDR2). Framework regions (FR) 1, 2, and 3 and complementarity-determining regions (CDR) 1, 2, and 3 are indicated.