Identification and characterization of monoclonal antibodies specific for polymorphic antigenic determinants within the V2 region of the human immunodeficiency virus type 1 envelope glycoprotein

Abstract

We have identified six monoclonal antibodies (MAbs) mapping to both linear and conformation-dependent epitopes within the V2 region of the human immunodeficiency virus type 1 clone HXB10. Three of the MAbs (12b, 66c, and 66a) were able to neutralize the molecular clones HXB10 and HXB2, with titers in the range of 9.5 to 20.0 g/ml. MAbs mapping to the crown of the V2 loop (12b, 60b, and 74) bound poorly to cell surface-expressed oligomeric gp120, suggesting an explanation for the poor or negligible neutralizing activity of MAbs to this region. In contrast, MAbs 12b and 60b demonstrated good reactivity with recombinant gp120 in an enzyme-linked immunosorbent assay format, suggesting differential epitope exposure between the recombinant and native forms of gp120. Cross-competition analysis of these MAbs and additional V1V2 MAbs for gp120 binding enabled us to assign the MAbs to six groups (A to F). Selection of neutralization escape mutants with MAbs 10/76b and 11/68b, belonging to nonoverlapping competition groups, identified amino acid changes at residues 165 (I to T) and 185 (D to N), respectively. Interestingly, these escape variants remained sensitive to neutralization by the nonselecting V2 MAbs. All MAbs demonstrated good recognition of IIIB viral gp120 yet failed to neutralize nonclonal stocks of IIIB. In addition, MAbs 12b and 62c bound MN and RF viral gp120, respectively, yet failed to neutralize the respective isolates. Cloning and expression of a library of gp120 and V1V2 fragments from IIIB-, MN-, and RF-infected H9 cultures identified a number of polymorphic sites, resulting in antigenic variation and subsequent loss of V2 MAb recognition. In contrast, the V3 region from the clones of the same isolates showed no amino acid changes, suggesting that the V2 region is polymorphic in long-term-passaged laboratory isolates and may account for the reduced antibody recognition observed.* Corresponding author. 222 MATERIALS AND METHODS Sources of reagents.The following gp120 MAbs were used for neutralization and/or gp120-binding studies: 11/65a and 33/79, specific for amino acids (aa) 102 to 121 within C1; 11/4c, 11/41e, and 10/76b, specific for aa 162 to 171 (22); 11/68b, CRA-3, CRA-4, and CRA-5, specific for conserved conformation-dependent epitopes within V2 (22); 39.13g, specific for a conserved conformation-dependent epitope involved in CD4 binding (4, 23); 38.1a, specific for aa 430 to 447 (4, 21); 41.1, specific for a conformation-dependent epitope within V3 (19); and MAb 11/85b, mapping to aa 311 to 321 in V3 (19).Recombinant CHO-expressed gp120 and peptides 740.13 (GEIKNCSFNIST SIRGKVQK), 740.14 (STSIRGKVQKEYAFFYKLDI), and 740.15 (EYAFFY KLDIIPIDNDTTSY) are members of the full set of gp120 overlapping peptides, which were obtained from the Medical Research Council AIDS Directed Programme. The control Raf-c peptide (IVQQFGYQRRASDDGKLTD) was a gift from C. Marshall, Institute of Cancer Research, London, United Kingdom. IIIB, MN, and RF viral stocks were obtained from the Medical Research Council AIDS Dir...

The Human Immunodeficiency Virus Type 1 gp120 V2 Domain Mediates gp41-Independent Intersubunit Contacts

“…An involvement of the V2 loop in the gp120 intersubunit contact site is also consistent with a number of reports suggesting that epitopes within this domain are less accessible to MAb binding to cell surface-expressed versus soluble purified gp120 (34,37,39). Moreover, it has also been shown that sera from infected individuals have few antibodies against V1/V2 epitopes (25).…”

“…Subunit interactions have been inferred from the differential reactivity of epitopes in cell surface-expressed env versus purified soluble gp120. Reduced exposure of epitopes within the V2, C1, C4, and C5 domains of cell surface-expressed gp120 has been reported (24,34,37,39). While it is likely that reduced exposure of the C1 and C5 epitopes is due at least in part to the involvement of these domains in association between gp120 and gp41 (16,50), the occlusion of epitopes in other domains is presumably caused by the close proximity of gp120 subunits within the oligomeric gp120-gp41 complex.…”

Oral mucosal Langerhans’ cells as target, effector and vector in HIV infection

“…In this study, therefore, active expression of p17 indicates the switch from latent to productive infection of HIV-targeted LCs within the lesional epithelium of oral hairy leukoplakia. Another possible explanation for failure to detect p17 in autologous control specimens is HIV genetic diversity, particularly the sequence variation in the env loop V2 region that may significantly affect HIV infectivity and tropism (41,42). In our previous study, a striking finding was the higher rate of G to A mutation in the V2 loop in oral hairy leukoplakia lesions, compared with nonlesional autologous tissue from the same patients (6).…”

Comparison of nucleic acid and protein immunization for induction of antibodies specific for HIV-1 gp120

“…The MoAbs used in this study are listed below, with their epitopes shown in brackets after the clone nomenclature: 11/65a (C1-aa102-121); 11/41a (C5-aa471-491); 11/4c (V2-aa152-181) [4,5]; 11/68b (V1/V2 discontinuous) [4,5]; 10/36c (V3-aa311-321); 10/54ow (V3-aa311-321); 11/85b (V3-aa311-321); ICR41 (V3-discontinuous); ICR38.1a (C4-aa427-436) [6]; ICR39.13 g (CD4 binding site discontinuous) [7] and 8/19b (gp120 discontinuous) [8].…”

“…3). Finally, sera were tested for reactivity with a truncated gp120 molecule containing the N-terminal and V1/V2 regions of the envelope protein [5]. It is interesting to note that the V1/V2 region has been reported to be a target for neutralizing antibodies and is immunogenic in infected individuals [4].…”