V1/V2 Neutralizing Epitope is Conserved in Divergent Non-M Groups of HIV-1

Abstract

Background: Highly potent broadly neutralizing monoclonal antibodies (bNAbs) have been obtained from individuals infected by HIV-1 group M variants. We analyzed the cross-group neutralization potency of these bNAbs toward non-M primary isolates (PI).

Material and methods: The sensitivity to neutralization was analyzed in a neutralization assay using TZM-bl cells. Twenty-three bNAbs were used, including reagents targeting the CD4-binding site, the N160 glycan-V1/V2 site, the N332 glycan-V3 site, the membrane proximal external region of gp41, and complex epitopes spanning both env subunits. Two bispecific antibodies that combine the inhibitory activity of an anti-CD4 with that of PG9 or PG16 bNAbs were included in the study (PG9-iMab and PG16-iMab).

Results: Cross-group neutralization was observed only with the bNAbs targeting the N160 glycan-V1/V2 site. Four group O PIs, 1 group N PI, and the group P PI were neutralized by PG9 and/or PG16 or PGT145 at low concentrations (0.04-9.39 μg/mL). None of the non-M PIs was neutralized by the bNAbs targeting other regions at the highest concentration tested, except 10E8 that neutralized weakly 2 group N PIs and 35O22 that neutralized 1 group O PI. The bispecific bNAbs neutralized very efficiently all the non-M PIs with IC50 below 1 μg/mL, except 2 group O strains.

Conclusion: The N160 glycan-V1/V2 site is the most conserved neutralizing site within the 4 groups of HIV-1. This makes it an interesting target for the development of HIV vaccine immunogens. The corresponding bNAbs may be useful for immunotherapeutic strategies in patients infected by non-M variants.

FIGURE 1

Phylogenetic analysis of env sequences. The 16 env sequences of the PIs included in the study were aligned with 72 env sequences from non-M variants that were available in the Los Alamos database. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 752,171,464 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura-Nei method and are in the units of the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1). All ambiguous positions were removed for each sequence pair. There were a total of 3165 positions in the final data set. Evolutionary analyses were conducted in MEGA6.

FIGURE 2

Conservation of amino acids involved in antibody binding epitopes. An alignment of the env protein sequences of the non-M viruses used in the study is depicted, with dashes representing gaps introduced to improve the alignment. HXB2 sequence is shown as reference. Amino acids are colored based on their physicochemical properties. The logo plots denote the conservation of individual amino acids, with the height of each letter indicating the proportion of sequences that contain the residue at that site. Contact residues of VRC01 (blue), JM4SdAb (yellow), and MPER bAbs (brown) are highlighted below the alignment. The Y symbols indicate the positions of the glycans associated with antibody neutralizing activity (see colors in the inserted legend to identify the corresponding bNAbs).

FIGURE 3

Neutralization breadth and potency of PG9-iMab and PG16-iMab, and parental Mabs against the panel of non-M viruses. A, Comparison of potency. For each virus, IC₅₀ are represented with a closed circle for PG9-iMab, an open circle for PG9, a closed square for PG16-iMab, an open square for PG16, and a cross for iMab. B, Percent viral coverage achieved by PG9-iMab, PG16-iMab, PG9, PG16, and iMab. Cumulative frequency distribution of IC₅₀ values of the antibodies tested against the 16 viruses. Symbols as above.