Human immunodeficiency virus type 1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction, and potential for regulation in acute infection

Abstract

Two human monoclonal antibodies (MAbs) (2F5 and 4E10) against the human immunodeficiency virus type 1 (HIV-1) envelope g41 cluster II membrane proximal external region (MPER) broadly neutralize HIV-1 primary isolates. However, these antibody specificities are rare, are not induced by Env immunization or HIV-1 infection, and are polyspecific and also react with lipids such as cardiolipin or phosphatidylserine. To probe MPER anti-gp41 antibodies that are produced in HIV-1 infection, we have made two novel murine MAbs, 5A9 and 13H11, against HIV-1 gp41 envelope that partially cross-blocked 2F5 MAb binding to Env but did not neutralize HIV-1 primary isolates or bind host lipids. Competitive inhibition assays using labeled 13H11 MAb and HIV-1-positive patient plasma samples demonstrated that cluster II 13H11-blocking plasma antibodies were made in 83% of chronically HIV-1 infected patients and were acquired between 5 to 10 weeks after acute HIV-1 infection. Both the mouse 13H11 MAb and the three prototypic cluster II human MAbs (98-6, 126-6, and 167-D) blocked 2F5 binding to gp41 epitopes to variable degrees; the combination of 98-6 and 13H11 completely blocked 2F5 binding. These data provide support for the hypothesis that in some patients, B cells make nonneutralizing cluster II antibodies that may mask or otherwise down-modulate B-cell responses to immunogenic regions of gp41 that could be recognized by B cells capable of producing antibodies like 2F5.

FIG. 1.

Induction of antibodies to the HIV-1 envelope MPER 2F5 epitope in mice. Mice were immunized with HIV-1 Env CON-S formulated in either Ribi or CpG ODN (oCpG) on weeks 0, 2, and 4. Serum samples were collected as indicated and assayed against HIV-1 Env 2F5 epitope peptide in ELISA. Mean values of the absorbance at 405 nm of serum (1:25 dilution) binding to 2F5 epitope peptides are plotted in the y axis (n = 5).

FIG. 2.

(A) Binding of MAbs 2F5, 13H11, and 5A9 to HIV-1 MPER peptides. Biotinylated full-length HR-2 peptide, SP62, or 8926 was immobilized at 200 to 300 RU on a streptavidin (SA) sensor chip. Biotin-DP107 was used as a control to subtract nonspecific binding. Representative overlay of specific (control subtracted) binding curves show binding of each of the MAbs to HR-2 (solid line), SP62 (dotted line), or 8926 (broken line) peptides. (B) K_d and rate constants of each of the indicated MAbs binding to HIV-1 MPER peptides. Rate constants were calculated using the bivalent analyte model to account for the avidity of the bivalent MAb. k_a, association constant.

FIG. 3.

Cross-blocking of 2F5 and 13H11 MAb binding to HR-2 peptide. Biotin-HR-2 (about 200 RU) peptide was anchored on a streptavidin chip (Biacore SA chip) and either blocking antibody 13H11 (A) or 2F5 (B) was injected until the peptide surface was near saturation. Following the blocking step, either 2F5 (A) or 13H11 (B) was injected, and binding was monitored for MAb blocking. Binding of each MAb in the absence of blocking antibody (solid line) or in the presence of blocking antibody (dotted line) is shown. Arrows indicate the injection point for each MAb.

FIG. 4.

Binding of human cluster II MAbs to MPER peptides. Binding of Cluster II MAbs to HR-2 peptide was monitored on a streptavidin chip loaded with HR-2 peptides as described above in the legend of Fig. 2. An overlay of binding of each MAb to HR-2 peptide (DP178; solid line), SP62 (dotted line), and 8926 (dashed line) is shown.

FIG. 5.

Reactivity of HIV-1.Env gp41 cluster II MAbs with the surface of HIV-1-infected T cells. Shown are histograms of viable H9 HIV-1-infected T cells reacted with the indicated MAbs in indirect immunofluorescence assays. In all panels, unstained cells are shown by a shaded curve, and antibody-labeled cells are shown by a black line. Control antibodies (not shown) gave curves overlapping with the unstained cells. The anti-gp120 V3 loop antibody F39F shows a positive signal as do the MPER antibodies 2F5 and 4E10. The cluster II MAbs 98-6, 126-6, and 167-D also reacted with HIV-1-infected T cells. The MAbs 5A9 and 13H11 show no binding to HIV-1 infected T cells. FITC, fluorescein isothiocyanate.

FIG. 6.

(A) Ability of plasma from 75 chronically HIV-1-positive patients (CD4 counts of >400; no antiretroviral therapy) and HIV-1-negative control plasma from 25 healthy donors to inhibit either the biotinylated MAb 2F5 or MAb 13H11. Twenty of 50 plasma samples from the chronically HIV-1-positive patients inhibited MAb 2F5 binding at ≥20% levels, while 62 of 75 plasma samples from the chronically HIV-1-positive patients inhibited MAb 13H11 binding at ≥20% levels. No significant inhibition of either MAb 2F5 or 13H11 binding by HIV-1-negative control plasma (n = 50) was observed. (B) Ability of plasma from 20 acutely HIV-1-infected patients and HIV-1-negative control plasma from 26 healthy donors to inhibit either the biotinylated MAb 2F5 or MAb 13H11. Two of 20 plasma samples from the acutely HIV-1-infected patients inhibited MAb 2F5 binding at ≥20% levels, while 19 of 20 plasma samples from the acutely HIV-1-infected patients inhibited MAb 13H11 binding at ≥20% levels. No significant inhibition of either MAb 2F5 or 13H11 binding by HIV-1-negative control plasma (n = 26) was observed. (C) Ability of plasma from 75 chronically HIV-1-positive patients (CD4 counts of >400; no antiretroviral therapy) to bind either the 2F5 epitope peptide SP62 or the 4E10 epitope peptide 4E10P in direct ELISA. A total of 23% of plasma samples from chronically HIV-1-positive patients were reactive with SP62 2F5 peptide while 8% were reactive with the 4E10 gp41 epitope peptide. Plasma samples were considered positive in peptide reactivity if the OD value was ≥0.500.

FIG. 7.

Antibody responses to HIV-1 Env MPER epitopes in acutely HIV-1-infected patients. Serial plasma samples were collected from 12 individuals from the clade C CAPRISA cohort from the initial study point through approximately 70 weeks, as shown on the x axis, and assayed for their ability to block the binding of biotinylated MAbs 2F5 or 13H11 to HIV-1 JRFL gp140 Env in ELISA. Mean values (n = 12) of microgram equivalents of either 2F5 or 13H11 antibody in plasma are shown on the y axis.