A single residue within the V5 region of HIV-1 envelope facilitates viral escape from the broadly neutralizing monoclonal antibody VRC01

Abstract

VRC01, a broadly neutralizing monoclonal antibody, is capable of neutralizing a diverse array of HIV-1 isolates by mimicking CD4 binding with the envelope glycoprotein gp120. Nonetheless, resistant strains have been identified. Here, we examined two genetically related and two unrelated envelope clones, derived from CRF08_BC-infected patients, with distinct VRC01 neutralization profiles. A total of 22 chimeric envelope clones was generated by interchanging the loop D and/or V5 regions between the original envelopes or by single alanine substitutions within each region. Analysis of pseudoviruses built from these mutant envelopes showed that interchanging the V5 region between the genetically related or unrelated clones completely swapped their VRC01 sensitivity profiles. Mutagenesis analysis revealed that the asparagine residue at position 460 (Asn-460), a potential N-linked glycosylation site in the V5 region, is a key factor for observed resistance in these strains, which is further supported by our structural modeling. Moreover, changes in resistance were found to positively correlate with deviations in VRC01 binding affinity. Overall, our study indicates that Asn-460 in the V5 region is a critical determinant of sensitivity to VRC01 specifically in these viral strains. The long side chain of Asn-460, and potential glycosylation, may create steric hindrance that lowers binding affinity, thereby increasing resistance to VRC01 neutralization.

FIGURE 1.

Genotypic and phenotypic comparison between CNE47 and CNE48. The gp160 amino acid sequences of CNE47 and CNE48 are shown aligned against the HXB2 reference strain. Regions known to interact with VRC01, including the CD4-binding loop, loop D, and V5 regions, are highlighted. Hyper-variable regions V1, V2, V3, V4, V5, and the trans-membrane domain are boxed (A). Pseudoviruses expressing full-length Env from either CNE47 or CNE48 were assayed for neutralization sensitivity and binding affinity with VRC01, sCD4, b12, and HIV-1 B′C plasma pool (B). CNE47 pseudoviruses (solid circle) were distinctly more resistant (IC₅₀ = 9.27 μg/ml) to VRC01 relative to CNE48 pseudoviruses (open circle) (IC₅₀ = 0.39 μg/ml). ELISA results showed CNE47 gp160 had a much weaker binding affinity for VRC01 (EC₅₀ of 12.21 μg/ml) compared with that of CNE48 (EC₅₀ of 2.40 μg/ml). No measurable differences were found between the wild-type Env pseudoviruses in terms of their sCD4, b12, or B′C plasma pool neutralization and binding profiles.

FIGURE 2.

Comparisons of mutant viruses containing either swapped regions (A) or single alanine substitutions (B) in terms of neutralization sensitivity to or binding affinity with VRC01. Mutant CNE47 viruses containing the CNE48 V5 region (clones 3–5) became more sensitive to VRC01, although swapping only the loop D region (clone 2) did not measurably affect sensitivity. Conversely, mutant CNE48 viruses containing the CNE47 V5 region (clones 13–15) showed greater resistance to VRC01. Altering only the loop D region had a minimal effect on sensitivity (A, upper panels). Differences in ELISA binding profiles complemented the observed trend in neutralization sensitivities, although changes were not as pronounced (A, lower panels). Among CNE47 mutant viruses containing single alanine mutations, only CNE47-N460A (orange) showed significantly increased neutralization sensitivity to VRC01 relative to wild-type (B, upper panels). The CNE48 Env had only one PNGS, Asn-463, for analysis in the V5 region (CNE48-N463A), and mutation here did not alter neutralization sensitivity (B, upper panel). Again, trends across ELISA binding profiles reflected those for neutralization sensitivity, with changes less pronounced (B, lower panels).

FIGURE 3.

Correlative analysis of VRC01 neutralization and binding. Binding activities of monomeric gp120 derived from CNE47 and CNE48 pseudoviruses were compared between VRC01-sensitive (IC₅₀ <5 μg/ml) and VRC01-resistant (IC₅₀ >5 μg/ml) groups (A). Soluble CD4 (sCD4) neutralization sensitivities were compared across the same groups (B). The horizontal lines indicate mean EC₅₀ or IC₅₀ values. The Mann-Whitney test was used to derive p values, with a statistical significance of p < 0.05.

FIGURE 4.

Comparisons of CNE47- and CNE48-derived mutant viruses in terms of their neutralization sensitivities to b12, sCD4, and ibalizumab and their binding affinities with sCD4 and the HIV-1 B′C plasma pool. None of these mutations altered b12 neutralization sensitivity or binding affinity with either sCD4 or the HIV-1 B′C plasma pool compared with parent wild-type (columns I, IV, and V). Pseudoviruses CNE48-N463A and CNE47-N460A/N463A, in which the PNGS at position 460 and/or 463 was eliminated, showed increased resistance to ibalizumab (C and D, and column II). All of the mutant CNE47 pseudoviruses became more resistant to sCD4 neutralization (A, column III) while those CNE48 mutants remained largely unchanged (B, column III), although sCD4 binding profiles were unchanged (A, B, column IV).

FIGURE 5.

Docking of CNE47 (A) and CNE48 (B) onto VRC01, focusing on the interaction between the V5 region of gp120 and VRC01. An ensemble of 10 V5-modeled CNE47 and CNE48 were docked onto VRC01, as based on the reported crystal structure of VRC01 with gp120 monomer (26). The heavy chain and light chain of VRC01 are shown on the surface with green and cyan colors, respectively. The Arg-61 residue on the heavy chain of VRC01 that plays significant roles in binding is colored in yellow. The expanded panels display close-up views of the interaction between VRC01 Arg-61 and the surrounding residues of the gp120 V5 region.