Background:  A novel anti-gp120 V3 monoclonal antibody, KD-247, which is 1 of the broad neutralizing anti-HIV-1 antibodies, neutralizes around 60% of subtype B isolates. In this study, we induced HIV-1 variants escape from KD-247 in vitro by continuously exposing a primary R5 virus, HIV-1_MOKW. The escape variants had 4 amino acids substitutions in non-V3 regions and 1 in V3-tip. The object of this study is to determine whether the non-V3 mutations are responsible for the reduced sensitivity to KD-247-resistant mutants of R5 primary isolate.

Methods:  To select HIV-1 variants escape from KD-247 in vitro, we exposed PM1-CCR5 cell, which had high expression of CCR5 to HIV-1_MOKW, and the virus was serially passaged in the presence of increasing concentrations of KD-247. The monoclonal antibody concentration was increased by as much as 2000 mg/mL. We determined the amino acid sequences of the gp120-encoding region of the HIV-1_MOKW escape mutants. To confirm that the mutations were associated with the reduced sensitivity of the escape variants, pseudotyped viruses containing the mutations of KD-247 resistance were constructed.

Results:  In first passage (KD-247, 10 mg/mL), 2 amino acid mutations in V2 (8 of 9 clones) and 2 amino acid mutations in C3 region (5 of 9 clones) appeared immediately. These substitutions in the V2 and C3 regions were continued by fifth passages (P-5, 200 mg/mL). However, at passage 9 (P-9, 2000 mg/mL), the C3 mutations had completely disappeared and alternatively V3-tip substitution (P313L) emerged in addition to the mutations in V2 region. To determine whether and how these substitutions were responsible for KD-247 resistance, we constructed luciferase-reporter viruses, which pseudotyped with HIV-1_MOKW envelope of P-5, P-9, and passaged virus without KD-247 and measured the sensitivity to KD-247. The P-9 pseudovirus was completely resistant to KD-247 to as much as 100 µg/mL and P-5 pseudovirus was partially resistant. While P-9 clone was sensitive to CCR5 inhibitors and anti-CCR5 antibody compared with control virus, the P-5 clone was the same as the wild type clone.

Conclusions:  In the present study, upon selection of HIV-1_MOKW in the presence of low concentration of KD-247, the escaped variants appeared with mutations in the V2 and C3 regions, but not in V3. These data suggest that HIV can escape from anti-V3 antibody by acquiring mutations in non-V3 region in an environment of low concentration of the antibody.