Characterization of a monoclonal antibody to a novel glycan-dependent epitope in the V1/V2 domain of the HIV-1 envelope protein, gp120

Abstract

Recent studies have described several broadly neutralizing monoclonal antibodies (bN-mAbs) that recognize glycan-dependent epitopes (GDEs) in the HIV-1 envelope protein, gp120. These were recovered from HIV-1 infected subjects, and several (e.g., PG9, PG16, CH01, CH03) target glycans in the first and second variable (V1/V2) domain of gp120. The V1/V2 domain is thought to play an important role in conformational masking, and antibodies to the V1/V2 domain were recently identified as the only immune response that correlated with protection in the RV144 HIV-1 vaccine trial. While the importance of antibodies to polymeric glycans is well established for vaccines targeting bacterial diseases, the importance of antibodies to glycans in vaccines targeting HIV has only recently been recognized. Antibodies to GDEs may be particularly significant in HIV vaccines based on gp120, where 50% of the molecular mass of the envelope protein is contributed by N-linked carbohydrate. However, few studies have reported antibodies to GDEs in humans or animals immunized with candidate HIV-1 vaccines. In this report, we describe the isolation of a mouse mAb, 4B6, after immunization with the extracellular domain of the HIV-1 envelope protein, gp140. Epitope mapping using glycopeptide fragments and in vitro mutagenesis showed that binding of this antibody depends on N-linked glycosylation at asparagine N130 (HXB2 numbering) in the gp120 V1/V2 domain. Our results demonstrate that, in addition to natural HIV-1 infection, immunization with recombinant proteins can elicit antibodies to the GDEs in the V1/V2 domain of gp120. Although little is known regarding conditions that favor antibody responses to GDEs, our studies demonstrate that these antibodies can arise from a short-term immunization regimen. Our results suggest that antibodies to GDEs are more common than previously suspected, and that further analysis of antibody responses to the HIV-1 envelope protein will lead to the discovery of additional antibodies to GDEs.

Analysis of the avian coronavirus spike protein reveals heterogeneity in the glycans present

“…As kifunensine inhibits a cellular enzyme, it is therefore likely that it starts to take effect on the newly synthesized proteins during virus replication. Complex glycans have been implicated in the recognition of HIV-1 glycoproteins by broadly neutralizing antibodies [28] and dependency on glycans and glycosylation in antibody binding has been noted for other viruses including HIV-1 and influenza [31, 80–82], supporting the data described here for recognition of IBV Beau-R S. These data suggest the presence of glycan-dependent epitopes on the surface of the IBV S protein.…”

Immunogenicity and malaria transmission reducing potency of Pfs48/45 and Pfs25 encoded by DNA vaccines administered by intramuscular electroporation

“…In MUT2, the S/T residues at the third position of all the seven consensus NXS/T motif were altered to A. A similar design strategy was recently used, for example, to study N-glycan dependent epitopes of HIV gp120 [56]. …”

Development of a Stable MGAT1− CHO Cell Line to Produce Clade C gp120 With Improved Binding to Broadly Neutralizing Antibodies

“…SDS-PAGE of purified rgp120 proteins was performed as previously described ( 25 ). Purified protein samples were run on a Bis-Tris 4–12% gradient gel (Invitrogen, Carlsbad, CA) and stained with SimplyBlue SafeStain (Invitrogen, Carlsbad, CA).…”