Fig. 2: Bioinformatic mining of single-mutation NGS data from SSM library screens revealed multiple mutations that provided enhanced HIV-1 neutralization potency and breadth. | Nature Communications

Fig. 2: Bioinformatic mining of single-mutation NGS data from SSM library screens revealed multiple mutations that provided enhanced HIV-1 neutralization potency and breadth.

From: Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site

Fig. 2: Bioinformatic mining of single-mutation NGS data from SSM library screens revealed multiple mutations that provided enhanced HIV-1 neutralization potency and breadth.The alternative text for this image may have been generated using AI.

A Enrichment ratios (ER) are plotted for single mutant antibody sequences derived from Round 3 high-affinity sorted libraries. Mutations were defined by determining the percent identity match to the template gene and denoting the substituted amino acid residue relative to the template sequence, with Base 1 corresponding to the start of the antibody variable region. NGS analysis of single mutant library screens highlighted multiple amino acid substitutions across VRC34.01 antibody variable regions that could enhance diverse HIV-1 FP recognition. B A 20-virus panel was optimized for predictive correlation with neutralization breadth on a larger 208-virus panel as a pre-screening tool to down-select promising candidates prior to 208-virus panel neutralization analysis. Template antibody VRC34.01 recognized 60% of strains in the 20-virus panel and ~50% in the 208-virus panel. PGT151, the broadest previously reported antibody that interacts with FP, recognized 70% of strains in the 20-virus panel and ~60% of the broader 208 strains. One-sided p-value for significance of data points deviating from a straight line is shown. No adjustments were made for multiple comparisons. C Four single mutations discovered by SSM screening (VH_T59Y, VH_T59F, VH_E2K, VH_E2P) were expressed as soluble IgG and revealed neutralization to 75% of the 20-virus panel. Rational combinations of these top single-mutant variants further improved neutralization breadth, with a maximum 85% breadth on the 20-virus panel achieved for VH_E2K_T59F (also referred to as VRC34.01-Combo1, Kabat numbering VH_E2K_T59F). Antibodies were considered neutralizing if their IC50 potency was <50 μg/mL. D 208-virus panel data revealed enhanced breadth for Combo1 (VH_E2K_T59F) to 68% of all strains, with strong neutralization of FP_v1 strains (98%), moderate neutralization of FP_Thai strains (61%), and gain-of-function for neutralization of FP_v3 and FP_v4 strains that were not neutralized at all by VRC34.01 (43% and 11%, respectively). Combo1 showed improvements compared to the template VRC34.01 antibody against all HIV-1 FP subclasses.

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