Fig 1.
Ab-specific variations in neutralization potency against HIV-1 strains assigned to different neutralization tiers.
All IC50 data available from the Los Alamos National Laboratory database (http://hiv.lanl.gov/catnap; as of October 2024) was downloaded for the antibodies listed (grouped by epitope as indicated) against all HIV-1 strains that had been attributed to a neutralization tier (tier 1A: most sensitive; tier 3: most resistant, see S1 Table for the neutralization data available per Ab and tier). A. Distribution of IC50 values for each antibody and for HIV-1 strains of different tiers (dots: median, boxplot: values within the interquartile range; whiskers indicating minimum and maximum values were omitted for clarity). B. Summary of differences between median potencies of Abs against Tier 1A and Tier 2 strains. Significant differences (p < 0.05, Mann-Whitney test) are indicated by a red star.
Fig 2.
Characterizing the generalized neutralization sensitivity of JR-CSF envelope point mutants.
The neutralization sensitivity of 126 JR-CSF envelope (Env) mutants was determined in a TZM-bl based pseudovirus inhibition assay. A. Each mutant virus was titrated with eleven plasma samples from HIV-1 chronically infected individuals (nine subtype B infected, two infected with non-B subtypes). All eleven plasmas neutralized JR-CSF wild-type virus at an inhibitory dilution (ID50) > 100, the minimal dilution probed. The distribution of ID50 ratios (mutant/wt) is shown for each mutant (center line: median; box limits extend from the 25th to 75th percentiles; whiskers indicate minimum and maximum values). A dotted line at ID50 ratio (mutant/wt) = 1 indicates equal neutralization sensitivity of the mutant Env compared to the wildtype reference. Thresholds used for categorizing mutants into highly (ID50 ratio (mutant/wt) >10; colored red) and moderately (10 > ID50 ratio (mutant/wt) >4; colored blue) sensitive phenotypes are indicated by dotted lines. B. IC50 values determined for each mutant against CD4 binding site (CD4-IgG2 and mAbs b6 and b12) and V3-crown (mAbs 1–79 and 447-52D) directed inhibitors were compared with median plasma ID50s as determined in A. Colorcode as in A. Titrations of each plasma and antibody on all viruses was done once, except for the JR-CSF wt reference for which plasma ID50s and nAb IC50s were derived by fitting a curve to pooled datapoints from n ≥4 titrations.
Fig 3.
HIV-1 Env generalized neutralization sensitivity impacts differentially on the potency of broadly neutralizing antibodies (bnAbs).
The whole JR-CSF Env mutant panel was tested for neutralization sensitivity against bnAbs targeting four major epitopes (CD4bs: VRC01, VRC07-523, N6 and N49P7; V3-glycan: PGT121, PGT128, PGT130, PGT135; 2G12; V2-glycan: PGT145; MPER: 2F5, 4E10, 10E8). A. Graphs show the log IC50 ratio (mutant/wt) for each Env mutant. IC50 ratios corresponding to minimal and maximal concentrations of inhibitors probed are indicated with lines. Weak-nAb data from Fig 2B is shown for comparison. The order of Env mutants and shaded regions of interest are indicated on top. Titrations of most antibodies on all viruses was done once (n = 1), except for the JR-CSF wt reference (n ≥ 4). mAbs PGT128, PGT145, 10E8 and 2G12 were titrated twice (n = 2) on all mutants. B. Spearman correlation matrix and hierarchical clustering of neutralization fingerprints based on IC50 data in A and Fig 2. For correlation with PWH plasma the median reciprocal ID50 ratio (mut/wt) over 11 JR-CSF wt neutralizing plasma (Fig 2A) was used. All significant correlations (p-value ≤ 0.05) are indicated with a circle with bigger circles indicating stronger support (lower p-values).
Fig 4.
Characterizing the exposure of the co-receptor binding site by neutralization sensitive Env mutants.
A. Schematic illustrating the experimental sCD4-induced opening of cell surface expressed Env leading to exposure of the co-receptor binding site. For detection by flow-cytometry mAb 17b was used as a co-receptor mimic. B. Top panel: Opening of cell surface expressed Envs by increasing concentrations of sCD4 was monitored by staining with mAb 17b. Env mutants were divided into two groups showing enhanced 17b binding in absence of sCD4 (right) or not (left) compared to JR-CSF wt Env. The N332A mutant served as additional control. All titrations were performed once. Color code of mutant sensitivity as in Fig 2. Middle panel: magnification of top panel data indicating data points for 17b staining of individual Env mutants. Bottom panel: The bar graph depicts area under the curve values derived from the normalized MFI curves of the top panel graphs. C. Spearman correlation between 17b neutralization sensitivity of JR-CSF wildtype and mutant viruses and 17b binding according to B. Mutations directly affecting 17b binding [65] were not included (see also S2C Fig). D. Alanine-substitutions leading to moderate (blue) or high (red) generalized neutralization sensitivity of the JR-CSF Env were mapped onto the trimeric closed prefusion structure of the closely related JR-FL Env ectodomain (PDB: 5FYK; V1V2: yellow, V3: orange, β20-β21: brown, gp120: light grey, gp41: dark grey). Glycans on neutralization sensitive positions N156, N262 and N301 are depicted. The N197 glycan is not shown as JR-FL naturally lacks this PNGS.
Fig 5.
Neutralization fingerprinting of nAbs on a neutralization sensitive Env mutant panel (SENSE-19).
Pseudovirus neutralization data on TZM-bl cells. A. IC50 ratios (mutant/wt) of an extended panel of nAbs were displayed as a heatmap. Abs are ordered by epitope. All titrations were performed in one technical replicate. B. Spearman correlation matrix and hierarchical clustering of SENSE-19 neutralization fingerprints from Abs, bnDs and PWH plasma (median reciprocal ID50 ratio (mut/wt) over 11 JR-CSF wt neutralizing plasma (Fig 2A)). All significant correlations (p-value ≤0.05) are indicated with a circle with bigger circles indicating stronger support (lower p-values). C. Comparison of three Ab quality parameters: neutralization breadth, potency and SENSE-19 score. Breadth and potency were determined on a cross-subtype 40 virus panel.
Fig 6.
The generalized neutralization sensitivity phenotype is equivalent in different assay formats.
JR-CSF wt virus and Env mutants were tested for neutralization sensitivity in three assay formats differing in the virus/cell combination: JR-CSF pseudovirus/TZM-bl, JR-CSFrc/TZM-bl, JR-CSFrc/PBMC. A. Heatmap comparing IC50 values from the three assay types. Titrations in the pseudovirus assay were performed once, except for the JR-CSF wt reference (n ≥ 4). Titrations in the JR-CSFrc/TZM-bl assay were set up in triplicates. For the JR-CSFrc/PBMC assay results are shown from one of two independent experiments set-up in triplicate for each mAb respectively. B. Spearman correlation of shifts in neutralization sensitivity (log IC50 ratio (mutant/wt)) between the JR-CSFrc/PBMC and JR-CSF pseudovirus/TZM-bl assays (corresponding to data shown in S8A Fig).
Fig 7.
Validation of a virus mini-library neutralization assay for mAb fingerprinting with sequencing-based readout.
The virus mini-library comprised 15 JR-CSFrc mutant viruses including 11 of high generalized neutralization sensitivity from the SENSE-19 panel and 4 PNGS mutants (controls). Wt JR-CSFrc was also included as reference. A. Schematic explaining the assay set-up and readout by Illumina sequencing. B. Comparison of results from the virus mini-library neutralization assay with a separately conducted PBMC based assay format testing JR-CSFrc mutants individually against four different antibodies. Results indicating resistance and sensitivity to a nAb are colored red and blue respectively. Left panels for each Ab: Enrichment and depletion of mutant viruses in the mini-library in presence (25 µg/ml) versus absence of Ab are depicted as Z-scores (error bars: STD), with Z-score (Env variant y)replicate z = (arF(Env variant y)replicate z with Ab – mean arF(Env variant y) without Ab)/standard deviation arF(Env variant y) without Ab, wherein arF(Env variant y) is the relative read frequency of Env variant y after adjustment according to the proportion of total JR-CSF reads versus total of reads including the internal control recovered (for details see materials and methods). A positive Z-score indicates enrichment and therefore resistance to the Ab, a negative Z-score indicates depletion and therefore sensitivity to the Ab (n = 3). The dotted lines indicate the threshold at which the enrichment/depletion in presence versus absence of antibody exceeds two times the standard deviation of read frequencies in absence of antibody (Z-scores = 2 and −2). Right panels for each Ab: Resistance of individually tested mutants in the PBMC-based assay was attributed if the IC50 was above 25 µg/ml (dotted line).