Factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection

Abstract

The characterization of the cross-reactive, or heterologous, neutralizing antibody responses developed during human immunodeficiency virus type 1 (HIV-1) infection and the identification of factors associated with their generation are relevant to the development of an HIV vaccine. We report that in healthy HIV-positive, antiretroviral-naïve subjects, the breadth of plasma heterologous neutralizing antibody responses correlates with the time since infection, plasma viremia levels, and the binding avidity of anti-Env antibodies. Anti-CD4-binding site antibodies are responsible for the exceptionally broad cross-neutralizing antibody responses recorded only in rare plasma samples. However, in most cases examined, antibodies to the variable regions and to the CD4-binding site of Env modestly contributed in defining the overall breadth of these responses. Plasmas with broad cross-neutralizing antibody responses were identified that targeted the gp120 subunit, but their precise epitopes mapped outside the variable regions and the CD4-binding site. Finally, although several plasmas were identified with cross-neutralizing antibody responses that were not directed against gp120, only one plasma with a moderate breadth of heterologous neutralizing antibody responses contained cross-reactive neutralizing antibodies against the 4E10 epitope, which is within the gp41 transmembrane subunit. Overall, our study indicates that more than one pathway leads to the development of broad cross-reactive neutralizing antibodies during HIV infection and that the virus continuously escapes their action.

FIG. 1.

Cross-NAb responses in the Vanderbilt cohort. Values represent the plasma dilution at which 50% neutralization was detected. Plasma samples were tested in a dilution range of 1:20 to 1:2,560. Gray-shaded boxes indicate the neutralization of a particular isolate by the indicated plasma sample. (-), 50% neutralization was not recorded at a 1:20 plasma dilution; ETI, estimated time of infection; EY, estimated time (in years) since infection that the plasma sample was obtained and tested. Values listed in the % breadth column are the percentages of the isolates tested that each plasma neutralized (i.e., the breadth of neutralization). Clade B viruses that are part of a panel of viruses created to evaluate the anti-HIV neutralizing responses elicited during infection or during immunization (17) are indicated by an asterisk. Cl. A, clade A.

FIG. 2.

Cross-NAb responses in the UW/CFAR cohort. Values represent the plasma dilution at which 50% neutralization was detected. Plasma samples were tested in a dilution range of 1:20 to 1:2,560. Gray-shaded boxes indicate neutralization of a particular isolate by the indicated plasma sample. (-), 50% neutralization was not recorded at a 1:20 plasma dilution. The times of infection for this cohort are unknown. TO, time point (in years) after enrollment at which the plasmas were collected and tested for neutralizing activity. Values listed in the breadth column are the percentages of the isolates tested that each plasma neutralized (i.e., the breadth of neutralization). Clade B viruses that are part of a panel of viruses created to evaluate the anti-HIV neutralizing responses elicited during infection or during immunization (17) are indicated by an asterisk. Cl. A, clade A.

FIG. 3.

Peptide competition neutralization assays. (A to F) Serially diluted plasmas were preincubated with the consensus clade B peptides corresponding to various regions of the HIV Env before use in neutralization assays against the primary isolate SF162. (G) Competition of plasma VC10028 using the 4E10 and a scrambled 4E10 peptide with the JRFL virus. A reduction in neutralization after preincubation with peptides indicates that the plasma contains NAbs directed at the corresponding regions of the HIV Env. The breadth of neutralization is indicated in parenthesis after each plasma's code. Ab, antibody.

FIG. 4.

(A) Contribution of anti-V1, anti-V2, or anti-V3 antibody to the plasma's neutralizing activity against SF162. (B) Contribution of V3-directed antibodies to the plasma's overall neutralizing activity against six primary HIV-1 isolates. The values indicate the percent reduction of the plasma's neutralizing activity against the indicated isolates following the preincubation of the plasma with the indicated peptides. TSI, the time since infection (in years) that plasmas were collected in the Vanderbilt cohort; (-), no reduction in the plasma's neutralizing activity was recorded; ND, the plasma has no neutralizing activity against the indicated virus.

FIG. 5.

Contribution of anti-CD4-BS antibodies to plasma-neutralizing activities. Plasmas were incubated with beads coated with either WT gp120 or gp120 with a mutated CD4-BS. The flowthrough and the eluted antibodies were tested for neutralizing activity against HIV. (A and B) Sample from patient VC10042; (C and D) sample from patient VC10014; (E and F) sample from patient CC1161; (G and H) sample from VC10002. Flowthroughs (A, C, E, and G) and eluted antibodies (B, D, F and H) are shown. Nondepleted, neutralization curves obtained with unadsorbed plasmas; WT depleted, plasma flowthroughs from WT gp120; BS-mutant depleted, plasma flowthroughs from the CD4-BS mutant gp120.

FIG. 6.

Presence of 2F5- or 4E10-like NAbs in plasma. (A) Values indicate the percent reduction in each plasma's neutralizing activity against SF162 following the preincubation of plasma with either r2F5 or 4E10 peptide. (B) The percent reduction in each plasma's neutralizing activity against six primary isolates following the preincubation of the plasma with the 4E10 peptide. (-), no reduction was recorded; ND, the plasma does not neutralize the indicated virus.

FIG. 7.

Detection of anti-CD4-BS antibodies in HIV⁺ plasmas. (A) Recognition of WT gp120 and CD4-BS mutant gp120 by specific anti-gp120 MAbs. IgG-CD4, chimeric molecule that binds to the CD4-BS of gp120; b12, an antibody that binds to an epitope that overlaps the CD4-BS; 447D, anti-V3 antibody; 2G12, an antibody that binds to a complex conformational epitope on gp120 formed by mannose residues. Closed symbols denote WT gp120 and open symbols denote CD4-BS mutant gp120 as the antigen in the ELISA. (B) Recognition of WT gp120 by the flowthroughs from WT gp120 and the CD4-BS mutant gp120 shown for seven HIV⁺ subjects with distinct breadths of NAb responses. Non Dep, relative end-point ELISA antibody titers in plasma; gp120, flowthrough following plasma's mixing with beads coated with WT gp120; and gp120bs, flowthrough following plasma's mixing with beads coated with CD4-BS mutant gp120. Arrows indicate the relative titers of antibodies binding to the CD4-BS of gp120.