Analysis of a clonal lineage of HIV-1 envelope V2/V3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors

Abstract

V2/V3 conformational epitope antibodies that broadly neutralize HIV-1 (PG9 and PG16) have been recently described. Since an elicitation of previously known broadly neutralizing antibodies has proven elusive, the induction of antibodies with such specificity is an important goal for HIV-1 vaccine development. A critical question is which immunogens and vaccine formulations might be used to trigger and drive the development of memory B cell precursors with V2/V3 conformational epitope specificity. In this paper we identified a clonal lineage of four V2/V3 conformational epitope broadly neutralizing antibodies (CH01 to CH04) from an African HIV-1-infected broad neutralizer and inferred their common reverted unmutated ancestor (RUA) antibodies. While conformational epitope antibodies rarely bind recombinant Env monomers, a screen of 32 recombinant envelopes for binding to the CH01 to CH04 antibodies showed monoclonal antibody (MAb) binding to the E.A244 gp120 Env and to chronic Env AE.CM243; MAbs CH01 and CH02 also bound to transmitted/founder Env B.9021. CH01 to CH04 neutralized 38% to 49% of a panel of 91 HIV-1 tier 2 pseudoviruses, while the RUAs neutralized only 16% of HIV-1 isolates. Although the reverted unmutated ancestors showed restricted neutralizing activity, they retained the ability to bind to the E.A244 gp120 HIV-1 envelope with an affinity predicted to trigger B cell development. Thus, E.A244, B.9021, and AE.CM243 Envs are three potential immunogen candidates for studies aimed at defining strategies to induce V2/V3 conformational epitope-specific antibodies.

Fig. 1.

Neutralization screening of primary IgG⁺ memory B cell cultures. IgG⁺ memory B cells were isolated from the peripheral blood of a broad neutralizer African subject chronically infected with a clade A HIV-1 strain. IgG⁺ memory B cells were cultured at a density of 8 cells/well in 3,600 culture wells for 14 days as described in Materials and Methods. At the end of stimulation, crude supernatants were tested for neutralizing activity against reporter tier 2 HIV-1 clade C isolate CAP45, a difficult-to-neutralize virus that was effectively neutralized by this subject's serum (Tomaras et al., unpublished). Solid dots represent the percentage of neutralization of each of the 3,600 cultures. Monoclonal antibodies CH01 to CH04 were isolated from the cultures represented by circled red dots. Positive controls (HIV Ig) are shown as open circles on the far right.

Fig. 2.

Neutralization profile of monoclonal antibodies CH01 to CH04. The neutralizing activity of MAbs CH01 to CH04 was tested on a panel of 91 pseudotyped lentiviruses, which comprised tier 1 and tier 2 isolates as well as transmitted founder viruses from multiple clades. Results are shown as IC₅₀s (μg/ml) and color coded as shown in the key.

Fig. 3.

Determination of the dissociation constant (K_d) from the gp120 E.A244 envelope glycoprotein by surface plasmon resonance (SPR) analysis. Serial dilutions ranging from 100 to 10 μg/ml of MAbs CH01 to CH04 (A to D, respectively), PG9 (E), PG16 (F), and putative reverted unmutated ancestor antibodies of the clonal lineage of CH01 to CH04 (G and H) were tested by SPR to identify the dissociation constants from the gp120 E.A244 envelope glycoprotein. The global curve-fitting χ² values were between 1.9 and 0.08. The data shown are representative of duplicate experiments.

Fig. 4.

Neutralizing activity of the putative reverted unmutated ancestor antibody candidates of the CH01 to CH04 clonal family antibodies. Two putative reverted ancestor antibody sequences (0219-RUA1 and 0219-RUA2) common to MAbs CH01 to CH04 were inferred and expressed as described in Materials and Methods. The two RUAs differed by a single nonsynonymous nucleotide mutation. The breadth of neutralization of 0219-RUA1 and 0219-RUA2 was tested against a panel of 24 pseudotyped lentiviruses comprising tier 1 and tier 2 isolates. Results are shown as IC₅₀s (μg/ml) and color coded as shown in the key.