In vivo gp41 antibodies targeting the 2F5 monoclonal antibody epitope mediate human immunodeficiency virus type 1 neutralization breadth

Abstract

The broadly neutralizing human monoclonal antibodies (MAbs) 2F5 and 4E10, both targeting the highly conserved human immunodeficiency virus type 1 (HIV-1) envelope membrane proximal external region (MPER), are among the MAbs with the broadest heterologous neutralizing activity and are of considerable interest for HIV-1 vaccine development. We have identified serum antibodies from an HIV-infected subject that both were broadly neutralizing and specifically targeted MPER epitopes that overlap the 2F5 epitope. These MPER-specific antibodies were made 15 to 20 months following transmission and concomitantly with the development of autoantibodies. Our findings suggest that multiple events (i.e., genetic predisposition and HIV-1 immune dysregulation) may be required for induction of broadly reactive gp41 MPER antibodies in natural infection.

FIG. 1.

2F5 MAb competitive ELISA screening of 2F5-like activity in HIV-1⁺ patients. Serum samples were tested for their potency in competing with biotinylated 2F5 MAb in binding to HIV-1 JRFL gp140. The 2F5-equivalent concentrations (2F5 Eq) were calculated from a 2F5 MAb standard curve.

FIG. 2.

Neutralization of TND_669S and TND_669L Env pseudoviruses by HIV-1⁺ sera or antibodies against HIV-1. TND_669S and TND_669L are two HIV-1 isolates that vary by one amino acid in their envelope sequences. TND_669S is 279-fold more sensitive to 2F5 MAb neutralization (black and gray bars, plotted on the secondary y axis) and 275-fold more sensitive to 4E10 MAb neutralization, whereas its sensitivities to various other neutralizing antibodies targeting gp120 and to virus entry inhibitor T20 are not significantly higher than those of TND_669L (data not shown). SC44 neutralized TND_669S 11-fold more potently than it did TND_669L, while another broadly neutralizing serum sample, IBB JT, neutralized TND_669S only 2-fold more potently than it did TND_669L. Values shown above each bar are either the serum IC₅₀s (in cases of serum samples) or the MAb IC₅₀s (in cases of 2F5 MAb).

FIG. 3.

Specificity of neutralizing activity for the 2F5 epitope. (A) 2F5 MAb positive-control data for the peptide absorption neutralization assay. The 2F5 peptide alone did not affect virus entry (infectivity of 2.58 × 10⁵ RLU with peptide and 2.53 × 10⁵ RLU with virus only). On the basis of this dose response, 3 μM of 2F5 peptide was used in subsequent screening assays of patient sera. (B) 2F5 and 4E10 MAb controls for the K665N reduction assay. The K665N mutant was resistant to 2F5 MAb neutralization but remained sensitive to 4E10 MAb neutralization. (C) Absorption of the neutralization activity of SC44 27-month-postenrollment serum by the 2F5 peptide. The percentages above the bars are the relative levels of 2F5-like neutralizing activity, calculated as follows: (IC₅₀ without peptide − IC₅₀ with peptide)/IC₅₀ without peptide. SC03 represents a control HIV⁺ serum sample that was identified to contain nonneutralizing antibodies which bind to the 2F5 peptide. (D) Differential neutralization of the 2F5-sensitive and 2F5-resistant viruses by SC44 27-month-postenrollment serum, in contrast to that of the control serum (from SC03). Bars show the neutralization titers (IC₅₀s) of the serum against the 2F5-sensitive virus (TND_669S, in black) and the 2F5-resistant virus (TND_669S/K665N, in gray). The percentages indicate the relative levels of the 2F5-like neutralizing activity, calculated as follows: (IC₅₀ for TND_669S − IC₅₀ for TND_669S/K665N)/IC₅₀ for TND_669S.

FIG. 4.

Characterization of SC44 serum/plasma antibodies purified by affinity columns coupled with the 2F5 peptide. (A) Tests for neutralizing activities. The 27-month-postenrollment plasma sample (7.5 ml) was purified with an affinity column conjugated with the MPER peptide (QQEKNEQELLELDKWASLWN, containing the 2F5 binding site), and the purified antibody (SC44 puri) was concentrated to ∼500 μl. The total protein concentration in the purified fraction was measured to be 1.36 mg/ml. The SC44 purified fraction (in red) was then tested for neutralization of TND_669S (solid lines), absorption of neutralization activity by the MPER peptide (dotted lines), and abrogation of neutralization of the TND_669S/K665N pseudotyped virus (dashed lines), side by side with 2F5 MAb neutralization controls (in blue). The IC₅₀s of the SC44 puri antibody and the 2F5 MAb were 1.09 and 0.027 μg/ml, respectively. (B) 2F5 competitive ELISA. Approximately 6 ml of SC44 serum (around 27 months postenrollment) was pooled and purified through a 2F5 peptide affinity column, and the purified antibody was concentrated to 350 μl. The purified antibody fraction was then tested in a 2F5 competitive ELISA. The black line shows the standard curve generated with unlabeled 2F5 MAb. The gray drop line indicates the optical density (OD) of the purified SC44 antibody at a 1:50 dilution.

FIG. 5.

Ontogeny of the 2F5-like-antibody activity in patient SC44. The kinetics of the 2F5-like neutralizing activity in SC44 over the course of HIV-1 infection was assessed by a K665N reduction assay (A) and a 2F5 binding ELISA (B) of longitudinal serum samples of SC44. (A) Neutralization titers for 2F5-sensitive virus TND_669S (dark blue) and 2F5-resistent virus TND_669S/K665N (light blue) are shown with the bars and plotted on the primary y axis. The corresponding viral loads over the infection course are shown with the gray line and plotted on the secondary y axis. The first viral load measurement shown is below the limit of detection (<100 copies/ml). The relative levels of 2F5-like activity, as indicated by K665N reduction levels in this assay (calculated as described for Fig. 4D), are shown above each group of bars. Neutralizing anti-MPER antibodies developed after 12 months from the time of enrolment into the acute infection cohort, as indicated by a 41% difference in the results for the reduction neutralization assay, which then peaked at a 65 to 70% difference by 27 months. (B) Direct 2F5 peptide binding ELISA was performed as previously described (1). Multiple serum samples (from both acute and chronic stages of infection) from each patient were diluted 1:50 and tested in the 2F5 peptide binding ELISA.

FIG. 6.

Detection of autoantibodies in patient SC44. Results are shown for detection of anti-dsDNA (A) and anti-Jo-1 (B) antibody in patient SC44 sera. The units are AtheNA U/ml, with 120 U/ml as the positivity cutoff.

FIG. 7.

Detection of anti-cardiolipin antibodies in SC44 serum. Anti-cardiolipin IgG, IgA, and IgM concentrations were measured for SC44 sera from the 7-month-, 15-month-, and 33-month-postenrollment time points. The cutoffs for different antibody species are listed in the table in panel B. ** indicates positive values.

FIG. 8.

Alignment of the 2F5 MAb epitope region of SC44 SGA sequences. SGA of envelope sequences from HIV-1 viruses was performed as previously described (15, 24). Early sequences were derived from plasma samples at enrollment, and late sequences were derived from 45-month-postenrollment plasma samples.