Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target

Abstract

Broadly neutralizing antibodies (bNAbs), which develop over time in some HIV-1-infected individuals, define critical epitopes for HIV vaccine design. Using a systematic approach, we have examined neutralization breadth in the sera of about 1800 HIV-1-infected individuals, primarily infected with non-clade B viruses, and have selected donors for monoclonal antibody (mAb) generation. We then used a high-throughput neutralization screen of antibody-containing culture supernatants from about 30,000 activated memory B cells from a clade A-infected African donor to isolate two potent mAbs that target a broadly neutralizing epitope. This epitope is preferentially expressed on trimeric Envelope protein and spans conserved regions of variable loops of the gp120 subunit. The results provide a framework for the design of new vaccine candidates for the elicitation of bNAb responses.

Fig. 1. Antigen binding properties of PG9 and PG16

(A) Binding of PG9 and PG16 to monomeric gp120 and artificially trimerized gp140 constructs as determined by ELISA. Antigens were coated directly onto ELISA wells in the experiments shown, but similar results were also obtained when antigens were captured onto wells with antibodies against non-competitive epitopes. (B) Binding of PG9 and PG16 to Env expressed on the surface of 293T cells as determined by flow cytometry. b12 is used as a control for ELISA assays. The bNAb b12, which binds with similar affinity to both cleaved and uncleaved forms of Env, and the non-neutralizing antibody b6, which only binds to uncleaved Env, are included in the cell surface binding assays to show the expected percentages of cleaved and uncleaved Env expressed on the cell surface (19). Experiments were performed in duplicate and data are representative of at least three independent experiments.

Fig. 2. Mapping the PG9 and PG16 epitopes

(A) Competition of PG9 and PG16 with each other and with the CD4 binding site (CD4bs) antibody b12 for cell surface Env binding. The competitor antibody is indicated at the top of each graph. (B) Effect of soluble CD4 (sCD4) on the binding of PG9 and PG16 to cell surface Env. 2G12 is included to control for CD4-induced shedding of gp120. (C) Competition of PG9 with antibodies against the V2 loop (10/76b), V3 loop (F425/b4e8), and the CD4i site (X5) for gp120 binding. Antigens were coated directly onto ELISA wells in the experiments shown, but similar results were also obtained when antigens were captured by antibodies against non-competitive epitopes. (D) Binding of PG9 and PG16 to variable loop deleted HIV-_1JR-CSF variants expressed on the surface of 293T cells. 2G12 is included to control for cell surface Env expression. All experiments were performed in duplicate and data represents an average of at least two independent experiments.