bNAb individuals have higher HIV-specific IgG subclass diversity.

<p>(<b>A</b>) A multiplex assay was used to measure levels of HIV-specific IgG subclasses present in 6 month samples from bNAb and no-bNAb individuals to 12 different HIV antigens. Median abundance of antigen-specific IgG2, IgG3 and IgG4 (orange, yellow and purple respectively) are represented as a ratio to IgG1 calculated using median fluorescence intensities. Data are representative of 2 independent experiments. Spearman´s correlations between subclass diversity score and (<b>B</b>) neutralization breadth and (<b>C</b>) Fc polyfunctionality are shown. The score was calculated as the ratio of gp120 ConC IgG2 and IgG4 relative to IgG1 levels. bNAb individuals are shown in red and no-bNAb in blue with dotted trend lines.</p

Fc effector function early in HIV infection is higher in individuals that develop bNAbs.

<p>(<b>A</b>) Purified IgG from 13 bNAb, 10 no-bNAb and 5 HIV-negative individuals (in red, blue and grey respectively) at 6 months post-infection was tested for antibody dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular trogocytosis (ADCT) and cellular cytoxicity (ADCC) using three HIV-specific antigens gp120 ConC, gp140 C.ZA.1197MB and gp120 CAP45.G3. Significant differences between groups determined by the Mann-Whitney U test are indicated by *p<0.05; **p<0.001. (<b>B</b>) Medians and IQR of different Fc effector functions for bNAb and no-bNAb individuals against all tested antigens over 36 months of infection are indicated as cumulative Fc effector function. Data are representative of 3 independent experiments. (<b>C</b>) Each Fc function was standardized by calculating a Z-score and polyfunctionality determined by addition of the Z-scores for all functions for each individual. Bars above the x-axis indicate Fc polyfunctional individuals, while those below indicate poor Fc polyfunctionality. bNAb and no-bNAb individuals are indicated in red and blue respectively. (<b>D</b>) Spearman´s correlation coefficient for the relationship between the Fc polyfunctionality Z-score and % neutralization breadth calculated by a 44 multi-clade virus panel is shown. The dashed diagonal line indicates the trend of the relationship.</p

IgG isolated from individuals that develop bNAbs shows increased gp120-specific binding to Fc receptors and complement proteins.

<p>(<b>A</b>) Binding gp120 ConC-specific IgG isolated from bNAb (red) and no-bNAb (blue) individuals to Fc receptors and C1q measured by an antigen-specific Fc receptor multiplex array. Significant differences (calculated by Mann-Whitney U test) in binding are shown as *p<0.05; **p<0.001; ***p<0.0001. Data are representative of 2 independent experiments. (<b>B</b>) The ratio of activating FcγRIIa (either H131 or R131) to inhibitory FcγRIIb receptor binding at 6 months post infection for bNAb and no-bNAb individuals. Medians are shown and significance was calculated by the Mann-Whitney U test. (<b>C</b>) Correlations between ADCT or ADCD and binding to Fc receptors and C1q shown as MFI. Significant Spearman´s correlation coefficients are indicated. Lines indicate the trend of the correlations.</p

Multivariate classifications reveal that individuals who develop bNAbs can be reliably identified by their Fc features at 6 months of infection.

<p>(<b>A</b>) Principal components analysis of 13 bNAb (red) and 10 no-bNAb (blue) using 17 variables. Individual CAPRISA identifiers are shown, with component 1 and 2 explaining 52.3% of the variance in the data set. (<b>B</b>) Confusion matrix showing the classification of bNAb and no-bNAb individuals achieved by random forest classification. Shown are the numbers of individuals for each predicted or observed group with correct classifications indicated in color and misclassifications indicated in white. The 2 bNAb (CAP257 and CAP292) and 2 no-bNAb (CAP88 and CAP228) individuals that were incorrectly classified can be seen in 5A. (<b>C</b>) Importance of the features employed in the random forest classification is indicated by the mean decrease in Gini importance weighting. (<b>D</b>) The model was verified by permutation testing following random shuffling of the classification data 100,000 times. The dashed line indicates the accuracy of the proposed model (82.6%), with shuffles resulting in accuracy greater than this shown as a proportion of the total shuffles (0.38%).</p