Dissecting the neutralizing antibody specificities of broadly neutralizing sera from human immunodeficiency virus type 1-infected donors

Abstract

Attempts to elicit broadly neutralizing antibody responses by human immunodeficiency virus type 1 (HIV-1) vaccine antigens have been met with limited success. To better understand the requirements for cross-neutralization of HIV-1, we have characterized the neutralizing antibody specificities present in the sera of three asymptomatic individuals exhibiting broad neutralization. Two individuals were infected with clade B viruses and the third with a clade A virus. The broadly neutralizing activity could be exclusively assigned to the protein A-reactive immunoglobulin G (IgG) fraction of all three donor sera. Neutralization inhibition assays performed with a panel of linear peptides corresponding to the third hypervariable (V3) loop of gp120 failed to inhibit serum neutralization of a panel of HIV-1 viruses. The sera also failed to neutralize chimeric simian immunodeficiency virus (SIV) and HIV-2 viruses displaying highly conserved gp41-neutralizing epitopes, suggesting that antibodies directed against these epitopes likely do not account for the broad neutralizing activity observed. Polyclonal IgG was fractionated on recombinant monomeric clade B gp120, and the neutralization capacities of the gp120-depleted samples were compared to that of the original polyclonal IgG. We found that the gp120-binding antibody population mediated neutralization of some isolates, but not all. Overall, the data suggest that broad neutralization results from more than one specificity in the sera but that the number of these specificities is likely small. The most likely epitope recognized by the monomeric gp120 binding neutralizing fraction is the CD4 binding site, although other epitopes, such as the glycan shield, cannot be excluded.

FIG. 1.

Neutralization curves of IgG fractions purified from sera on protein A. The neutralization capacity of purified polyclonal IgG (•) is compared with those of the column flowthrough (▴) and original serum (▪). To allow comparison of all samples, the purified IgG and column flowthrough were concentrated to volumes equal to that of the original volume of serum run over the column. Neutralization of FDA2, LT2, and ITM1 column fractions was compared for the subtype B isolates JR-FL and ADA, the subtype A isolate 92RW020.5, and the subtype C isolate 92BR025.9.

FIG. 2.

Neutralization of JR-FL by HIV-infected donor sera and MAb controls in the presence or absence of competing V3 peptides. Peptides representing portions of immunodominant variable loops of clade B isolates (Table 2) were tested for their ability to inhibit serum neutralization of four primary clade B isolates. Representative neutralization curves are shown for JR-FL, YU2, R2, and L36 V3 peptides tested for their ability to inhibit serum neutralization of JR-FL. Serum or IgG neutralization of JR-FL is shown by the closed symbols, and neutralization in the presence of the competitor peptides is shown by the open symbols. Anti-V3 MAb 447-52D (tested with L36-V3) or F425 B4e8 (tested with JR-FL, YU2, and R2 V3) was used as a positive control for neutralization inhibition in the presence of the V3 peptides, and MAb b12 was tested as a negative control.

FIG. 3.

Neutralization of HIV-2_7312A and chimeric HIV-2_7312A containing the complete HIV-1_YU2 MPER. The presence of NAbs specific for the MPER region of gp41 was evaluated by comparing the abilities of sera (top panels) to neutralize a chimeric HIV-2_7312A virus with the full-length HIV-1_YU2 MPER grafted into an appropriate location and the parental HIV-2_7312A virus. The neutralization specificities were assessed using MAbs 4E10 and 2F5 (bottom panels).

FIG. 4.

Antigenic profiles of recombinant gp120 monomers used in serum depletion experiments. MAb and polyclonal IgG binding to gp120_JR-FL, gp120_JR-CSF, and core gp120_JR-CSF were analyzed.

FIG. 5.

ELISA binding curves of polyclonal ITM1 IgG depleted on gp120-coupled beads. Polyclonal ITM1 IgG was incubated with gp120_JR-FL-, gp120_JR-CSF-, and core gp120_JR-CSF-coupled beads. The initial and depleted IgG fractions were titered on wild-type or core gp120 and gp41 to assess the removal of antigen-specific antibodies in each depletion experiment. wt, wild-type.

FIG. 6.

Neutralization of polyclonal LT2 IgG depleted on gp120-coupled beads. Polyclonal LT2 IgG was incubated with beads coupled with gp120_JR-FL, core gp120_JR-CSF, or gp120_JR-CSF. The neutralization capacities of the initial and depleted fractions were tested against clade A isolate 92RW020.5, clade C isolate 92BR025.9, and clade B isolates JR-FL, ADA, and 6535.3. Error bars represent results from two independent experiments.

FIG. 7.

Neutralization results of polyclonal ITM1 IgG depleted on gp120-coupled beads. Polyclonal ITM1 IgG was incubated with gp120_JR-FL-, core gp120_JR-CSF-, and gp120_JR-CSF-coupled beads. The neutralization capacities of the initial and depleted fractions were tested against clade A isolate 92RW020.5, clade C isolate 92BR025.9, and clade B isolates JRFL and 6535.3. Representative results from two independent experiments are shown for IgG depleted against core gp120_JR-CSF-coupled beads and tested for neutralization against 92BR025.9.

FIG. 8.

Neutralization results of polyclonal FDA2 IgG depleted on gp120-coupled beads. Polyclonal FDA2 IgG was incubated with gp120_JR-FL-, core gp120_JR-CSF-, and gp120_JR-CSF-coupled beads. The neutralization capacities of the initial and depleted fractions were tested against clade A isolate 92RW020.5, clade C isolate 92BR025.9, and clade B isolate JR-FL. Representative results from two independent experiments are shown for IgG depleted against gp120_JR-FL-coupled beads and tested for neutralization against JR-FL and for IgG depleted against core gp120_JR-CSF-coupled beads and tested for neutralization against 92BR025.9.