Additive effects characterize the interaction of antibodies involved in neutralization of the primary dualtropic human immunodeficiency virus type 1 isolate 89.6

Abstract

Human immunodeficiency virus-type I (HIV-1) infection elicits antibodies (Abs) directed against several regions of the gp120 and gp41 envelope glycoproteins. Many of these Abs are able to neutralize T-cell-line-adapted strains (TCLA) of HIV-1, but only a few effectively neutralize primary HIV-1 isolates. The nature of HIV-1 neutralization has been carefully studied using human monoclonal Abs (MAbs), and the ability of such MAbs to act in synergy to neutralize HIV-1 has also been extensively studied. However, most synergy studies have been conducted using TCLA strains. To determine the nature of Ab interaction in HIV-1 primary isolate neutralization, a panel of 12 anti-HIV-1 human immunoglobulin G (IgG) MAbs, specific for epitopes in gp120 and gp41, were used. Initial tests showed that six of these MAbs, as well as sCD4, used individually, were able to neutralize the dualtropic primary isolate HIV-1(89.6); MAbs giving significant neutralization at 2 to 10 microg/ml included 2F5 (anti-gp41), 50-69 (anti-gp41), IgG1b12 (anti-gp120(CD4bd)), 447-52D (anti-gp120(V3)), 2G12 (anti-gp120), and 670-D (anti-gp120(C5)). For studies of reagent interaction, 16 binary combinations of reagents were tested for their ability to neutralize HIV-1(89.6). Reagent combinations tested included one neutralizing MAb with sCD4, six pairs consisting of two neutralizing MAbs, and nine pairs consisting of one neutralizing MAb with another non-neutralizing MAb. To assess the interaction of the latter type of combination, a new mathematical treatment of reagent interaction was developed since previously used methods could be used only when both reagents neutralize. Synergy was noted between sCD4 and a neutralizing anti-gp120(V3) MAb. Antagonism was noted between two pairs of anti-gp41 MAbs (one neutralizing and one non-neutralizing). All of the other 13 pairs of MAbs tested displayed only additive effects. These studies suggest that Abs rarely act in synergy to neutralize primary isolate HIV-1(89.6); many anti-HIV-1 Abs act additively to mediate this biological function.

FIG. 1

Neutralization of HIV-1_89.6 by anti-gp120 MAbs. The percent neutralization of HIV-1_89.6, shown on the y axis, was determined with increasing amounts of anti-gp120 MAbs (x axis) or with an anti-parvovirus MAb, 860-55D, used as a negative control. The GHOST cell neutralization assay was used (18), and the results are represented as the mean of two or three separate experiments for each MAb. Significant neutralization was defined by the 95% confidence limit (shown as the shaded area in gray), established on the basis of 24 assays run with MAb 860-55D and defined in Materials and Methods.

FIG. 2

Neutralization of HIV-1_89.6 by anti-gp41 MAbs. The percent neutralization of HIV-1_89.6, shown on the y axis, was determined with increasing amounts of anti-gp41 MAbs (x axis) or with an anti-parvovirus MAb, 860-55D, used as a negative control. The GHOST cell neutralization assay was used (18), and the results are represented as the mean of two or three separate experiments for each MAb. Significant neutralization was defined by the 95% confidence limit (shown as the shaded area in gray), as described in the legend of Fig. 1.

FIG. 3

Neutralization of HIV-1_89.6 by sCD4. The percentage of neutralization of HIV-1_89.6, shown on the y axis, was determined with increasing amounts of sCD4 (x axis). The GHOST cell neutralization assay was used (18), and the results are represented as the mean of two separate experiments.

FIG. 4

Experimental effects of pairs of MAbs against gp120 and/or gp41 on neutralization of HIV-1_89.6 compared to the predicted additive effects. The percent neutralization of HIV-1_89.6 observed (obs, ▾), as well as the predicted additive effects (pred, ○) are shown on the y axis for 16 binary combinations of reagents, as the means of 2 or 4 independent experiments. The total amount of the combined MAbs used in the virus-MAb mixture for the neutralization assay is shown on the x axes.