Promiscuous glycan site recognition by antibodies to the high-mannose patch of gp120 broadens neutralization of HIV

Abstract

Broadly neutralizing monoclonal antibodies (bnmAbs) that target the high-mannose patch centered around the glycan at position 332 on HIV Env are promising vaccine leads and therapeutic candidates because they effectively protect against mucosal SHIV challenge and strongly suppress SHIV viremia in established infection in macaque models. However, these antibodies demonstrate varying degrees of dependency on the N332 glycan site, and the origins of their neutralization breadth are not always obvious. By measuring neutralization on an extended range of glycan site viral variants, we found that some bnmAbs can use alternate N-linked glycans in the absence of the N332 glycan site and therefore neutralize a substantial number of viruses lacking the site. Furthermore, many of the antibodies can neutralize viruses in which the N332 glycan site is shifted to the 334 position. Finally, we found that a combination of three antibody families that target the high-mannose patch can lead to 99% neutralization coverage of a large panel of viruses containing the N332/N334 glycan site and up to 66% coverage for viruses that lack the N332/N334 glycan site. The results indicate that a diverse response against the high-mannose patch may provide near-equivalent coverage as a combination of bnmAbs targeting multiple epitopes. Additionally, the ability of some bnmAbs to use other N-linked glycan sites can help counter neutralization escape mediated by shifting of glycosylation sites. Overall, this work highlights the importance of promiscuous glycan binding properties in bnmAbs to the high-mannose patch for optimal antiviral activity in either protective or therapeutic modalities.

Figure 1. Neutralization of viruses by bnMAbs to the high-mannose patch with the N332 glycan site shifted to the 334 position

Neutralization potency was measured on a 6-virus indicator panel for which the glycan site at N332 was shifted to the 334 position. Presented values are neutralization IC₅₀ in μg/ml and colored according to the listed scale. Values listed as “ND” were not determined.

Figure 2. BnMAbs to the high-mannose patch neutralize many isolates with the glycan site at the 332 position but also some bnMAbs can neutralize isolates with the site at the 334 position and some with no glycan site at either position

Percent neutralization breadth and median IC₅₀ were determined at an IC₅₀ cutoff of 50 μg/mL for the (A) PGT121 antibody family (B) PGT128 antibody family and (C) PGT135. Viruses were separated into those naturally containing a glycan site at N332 or N334 or without a glycan site at either position.

Figure 3. A substantial fraction of isolates in a large panel are neutralized by high-mannose patch bnMAbs when the N332 or N334 glycan sites are eliminated by alanine mutagenesis

Antibody families were tested on an 80-virus panel, whose members naturally include a glycan site at N332/334 (A) and on the same panel with the glycan site at N332/334 removed by alanine mutagenesis (B). Listed are percent viruses neutralized at the indicated neutralization IC₅₀ cutoffs. Cumulative distribution frequency (CFD) of percent viruses neutralized in the 80-virus panel with and without the N332/334 glycan site present at different IC₅₀ cutoffs for the (C) PGT121 antibody family and the (D) PGT128 antibody family.

Figure 4. PGT121 can utilize glycans in the V1/V2 loops to neutralize isolates when the glycan site at N332 or N334 is removed by alanine mutagenesis

(A) Neutralization potency of PGT121 was measured on 92BR020, 92RW020 and CAP45 glycan mutant viruses for which N-linked glycosylation sites have been removed singly (solid line) or in combination with N332 (dashed line) by alanine mutagenesis. PGV04 was included as a control. (B) Neutralization potency of PGT121-123 was measured on virus isolate 92BR020 N332A mutant virus (red) made in HEK 293T cells (solid line) and a GnT1^−/− deficient HEK 293S cell line (dotted line) and the potency compared to WT virus (black).

Figure 5. PGT128 family antibodies can utilize alternate glycan sites in the high-mannose patch to neutralize isolates when the N332 or N334 glycan sites are removed by alanine mutagenesis

Neutralization potency of the PGT128 antibody family was measured on a panel of (A) 92RW020, (B) 92TH021 and (C) JR-CSF glycan mutant viruses for which N-linked glycan sites in the V3 region were removed alone or in combination with other N-linked glycan sites.

Figure 6. bnMAbs to the high-mannose patch can utilize alternate glycan sites to neutralize viruses that naturally do not have a glycan site present at the N332/334 position

(A) PGT121, (B) PGT123, and (C) PGT124 were tested for neutralization of ZM53 glycan site mutants. (D) PGT125, (E) PGT127, and (F) PGT128 were tested for neutralization of BG505 glycan site mutants. Mutants were generated by site-directed mutagenesis.

Figure 7. Structural modeling of high-mannose patch glycan sites and calculations of glycan site frequencies

Glycan models to rationalize PGT128 promiscuity. (A) Close-up view of PGT128 binding to eODmV3 including the glycans at position N301 and N332, as defined by the crystal structure of eODmV3+PGT128. (B) Model for an N-linked Man₈GlcNAc₂ glycan at position N295 on eODmV3. (C) Model for an N-linked Man₈GlcNAc₂ glycan at position N334 on eODmV3. Coloring is as follows: PGT128, magenta; eODmV3, red; N301 glycan, magenta; N332 glycan, red; N295 glycan, yellow; N334 glycan, white. Cross-clade frequency of glycans at different positions on Env. (D) Frequency of critical N-linked glycan sites in 31,788 viruses in the Los Alamos database. A glycan site was determined as being present accordingly to the Nx(T or S) motif, where x is not a proline. Only clades for which > 100 sequences were available are included in the graph. The line represents the mean of the frequency for each glycan site. (E–F) Frequency of combinations of the N-linked glycan sites shown to be important for bnMAb neutralization in the same set of 31,788 virus sequences.

Figure 8. Combinations of antibodies targeting the high-mannose patch increase neutralization coverage compared to single antibodies

(A) Summary of neutralization breadth and potency on the 120-virus panel for individual antibodies and theoretical antibody combinations. (B) Summary of neutralization breadth and potency on an 80 wild-type virus subset of the 120-virus panel and (C) on the same 80-virus panel with the glycan site at N332/334 removed by alanine mutagenesis. The analysis includes individual antibodies and theoretical antibody combinations. (D) Cumulative frequency distribution at different IC₅₀ cutoffs for individual antibodies and theoretical antibody combinations on the 120-virus panel. (E) Cumulative frequency distribution at different IC₅₀ cutoffs for individual antibodies and theoretical antibody combinations on the 80-virus subset of the 120-virus panel with (solid) and without (dotted) the glycan site at N332/334.