Cross-clade neutralizing activity of human anti-V3 monoclonal antibodies derived from the cells of individuals infected with non-B clades of human immunodeficiency virus type 1

Abstract

The majority of global human immunodeficiency virus infections are caused by viruses characterized by a GPGQ motif at the tip of the V3 loop. Characterization of anti-V3 monoclonal antibodies (MAbs) that neutralize isolates with the GPGQ V3 motif is an important step in designing vaccines that will induce such Abs. Consequently, seven human anti-V3 MAbs derived from the cells of individuals infected with non-B-subtype viruses (anti-V3(non-B) MAbs) were generated from the cells of individuals from Africa infected with circulating recombinant forms CRF02_AG, CRF09_cpx, and CRF13_cpx, each of which contains a subtype A env gene. Sequence analysis of plasma viruses revealed a GPGQ motif at the apex of the V3 loop from six of the seven subjects and a GPGR motif from one subject. The MAbs were selected with fusion proteins (FP) containing V3(92UG037.8) or V3(JR-CSF) from subtype A or B, respectively. In virus binding assays, five of the seven (71%) anti-V3(non-B) MAbs bound to V3-FPs from both subtype A and subtype B, while only four of the nine (44%) anti-V3(B) MAbs recognized both V3-FPs. Using two neutralization assays, both the anti-V3(non-B) and the anti-V3(B) MAbs neutralized subtype B viruses with similar activities, while the anti-V3(non-B) MAbs exhibited a tendency toward both increased potency and breadth of neutralization against non-B viruses compared to anti-V3(B) MAbs. Statistical significance was not achieved, due in large measure to the sizes of the MAb panels, but the overall pattern of data strongly suggests that viruses with the GPGQ motif at the tip of the V3 loop induce anti-V3 Abs with broader cross-neutralizing activity than do viruses with the GPGR motif.

FIG. 1.

V3 sequences of HIV-1 isolates obtained from donors of the anti-V3_non-B MAbs. The amino acids were aligned with their CRF02_AG consensus sequence or with individual sequences of viruses from CRF09_cpx and CRF13_cpx (shown in bold) obtained from the Los Alamos Database. Dashes represent identity with the reference sequences.

FIG. 2.

Binding patterns of V3-fusion proteins with anti-V3 MAbs derived from individuals infected with non-B-subtype (panels A to D) and subtype B (panels E to H) viruses. Seven human anti-V3_non-B MAbs generated from the cells of individuals infected with non-B-subtype viruses (A to D) and nine human anti-V3_B MAbs from the cells of subtype B virus-infected subjects (E to H) were tested for their abilities to bind to V3-fusion proteins containing a subtype A V3 sequence (V3_A-FP) (A, C, E, G) and to a subtype B V3 sequence (V3_B-FP) (B, D, F, H). Human anti-parvovirus B19 MAb 1418 was used as negative control. The curves represent the mean binding activities from three separate experiments, and error bars indicate the standard deviations. The binding curves for anti-V3_non-B MAbs are shown in red, with the exception of the curves generated with MAb 2182, which are shown in black (C and D); curves generated with anti-V3_B MAbs are shown in blue.

FIG. 3.

Neutralization patterns of pseudotype viruses with anti-V3 MAbs derived from subjects infected with non-B-subtype (panels A and C) and subtype B (panels B and D) viruses. The neutralization capacities of seven anti-V3_non-B MAbs and seven anti-V3_B MAbs with psSF162 (bearing the GPGR motif) and psMW965 (bearing the GPGQ motif) were tested in a single-cycle infectivity assay. Human anti-parvovirus B19 MAb 1418 was used as a negative control. The neutralizing curves represent the means from three experiments, and error bars indicate standard deviations. The curves representing neutralizing activities for anti-V3_non-B MAbs are shown in red, and those for anti-V3_B MAbs are shown in blue. Data for the negative control MAb 1418 are shown in green, and data for the MAb 2182 are shown in black.

FIG. 4.

Neutralization of primary isolates by anti-V3 MAbs derived from individuals infected with non-B- and B-subtype viruses. Neutralization of primary isolates was performed in the GHOST cell neutralization assay, with MAbs at a final concentration of 25 μg/ml. The cutoff value of 29% is based on the 95% confidence level obtained with 60 experiments using the nonneutralizing human anti-C2 MAb 847. The percentages represent the means from three separate experiments. Light gray cells indicate neutralization in excess of the cutoff value; dark gray cells indicate virus/MAb combinations giving >50% neutralization. nt, not tested.