Table 1.
Background information and neutralization sensitivity of broadly resistant HIV-1 strains.
Fig 1.
Signature substitutions distinguishing between the broadly resistant and sensitive HIV-1 strains in the contact regions of CD4bs bnAbs.
(A)Alignment of the broadly resistant strains studied here (red, n = 9) and reported elsewhere (green, n = 10) against the standard sensitive strains HXB2, SF162 and JRFL.The locations of the contact regions such as the inner domain, Loop D and β23/loop V5/β24 along the HIV-1 gp120 and the actual contact residues are indicated above the aligned sequences. Each colored dot represents a single contact residue, with red for bnAbs of the VRC01 class (VRC01, 3BNC117 and N6) and blue for non-VRC01 classes (CH103, 8ANC131, VRC13 and VRC16). Residue differences from the HIV-1 HXB2 sequence are indicated by colors representing their different biochemical properties: green for polar, blue for basic, red for acidic, black for hydrophobic and purple for N or Q residues. Dots represent identical residues and dashes represent gaps introduced to preserve the alignment. (B) Identification of signature substitutions by quantitative comparisons between the broadly resistant strains and the sensitive ones using the program WebLogo. A total of 113 sensitive HIV-1 strains that can be neutralized by VRC01, 3BNC117, N6 and VRC13 were identified from reported research. The signature substitutions disproportionally represented among the resistant and sensitive strains are highlighted with their indicated locations. The colored scheme for the signature substitutions is the same as in (A). All residue numbers are based on the HIV-1 HXB2 sequence.
Fig 2.
Neutralizing activity of N6 against wild-type and mutant HIV-1.
Neutralization curves of N6 against nine wild-type and 58 mutant HIV-1 pseudoviruses. Data from one of three independent experiments is presented.
Table 2.
Restoration of the sensitive phenotype in broadly resistant HIV-1 strains through site-directed mutagenesis.
Fig 3.
Correlations between the neutralizing activity of bnAbs(IC50 in (A) and IC80 in (B)) and binding to the Env trimers expressed on the cell surface. Correlations for each and all antibodies together (far right) were analyzed by determining the Spearman correlation coefficient (R), with statistical significance (P) indicated in red. Different resistant clones and their derivatives are indicated by different colors.
Fig 4.
Introduction of signature substitutions transformed the sensitive HIV-1 JRFL into the resistant phenotype.
(A) Neutralization curves of VRC01 class, non-VRC01 class antibodies and ibalizumab against JRFL mutants containing a combination of signature substitutions from CNE6, CNE23, CNE63/64, CNE66 and BJOX2000. Data from one of three independent experiments is presented. (B) IC50 and IC80 values for each of the tested antibodies against wild-type and mutant HIV-1 JRFL. Receptor usage based on Cf2Th cell lines is also indicated. All data is presented as the means of at least three independent experiments. The color scheme used is identical to that in Tables 1 and 2.
Fig 5.
Structural basis of the signature resistance mutations.
(A) Major epitope regions of CD4-binding site antibodies on the HIV-1 Env in its pre-fusion closed conformation. The inner domain contact, Loop D, CD4-binding loop, Loop V5 and the outer domain exiting loop are colored cyan, orange, yellow, magenta and marine blue respectively. (B) Overlay of the boundaries of epitopes for VH1-2-derived VRC01-class antibodies (green line) and VRC13 (blue line) onto the different structural regions. (C) Resistance conferred by mutations that disrupt salt bridges and hydrogen bonds between gp120 and CD4bs antibodies. HIV-1 gp120 residues were colored and labeled according to their structural regions and the antibody residues were labeled by source and location, such as Asp100 of antibody N6. (D) Resistance conferred by mutations providing steric hinderance on loop D and the outer domain exiting loop. Potential clashes between resistance mutations and the antibodies were marked as red discs. The heavy chains of VRC01 class antibodies were colored in shades of green and CDR H3 of VRC13 was colored red. Light chains were colored slate. (E) Resistance conferred by mutations providing steric hinderance on β23. (F) Resistance conferred by mutations providing steric hinderance through bulkier residues or a glycan on loop V5.
Fig 6.
Polymorphisms and shifts at the signature residues before and after treatment with 3BNC117 and VRC01.
The logograms show the frequency/height of each signature residue between (A) the sensitive strains retrieved from the HIV database and the broadly resistant HIV-1 strains highlighted in the current study, (B, C) pre- and post-treatment with 3BNC117, and (D,E, and F) pre- and post-treatment with VRC01. The trial registration numbers and the actual number of HIV-1 sequences retrieved and analyzed are indicated.Those that demonstrated substantial changes (> 10-fold or newly emerged) are indicated in red boxes. All numbers are based on the HIV-1 HXB2 sequence. The signature residues are colored according to their distinct biochemical properties as in Fig 1.