Mapping of mutations on gp140 structure.

<p>(A) Surface view of the structure of a BG505 SOSIP.664 gp140 monomer. The gp120 unit is shown in light blue and the gp41 unit in light pink. Glycans are shown as blue sticks, the CD4bs is orange and the V1/2 loop domain golden. Mutated residues found in clone 3.3.1 only are shown in red and residues found in clones 4.3.B01 and 4.3.D01 are shown in green. (B) The molecule was rotated 90° around the x-axis and residues found in 4.3.B01 and 4.3.D01 are shown in green. (C) Cartoon view of mutated residues found within gp120, putative hydrogen bonds are denoted as dashed lines.</p

Directed Evolution of a Yeast-Displayed HIV-1 SOSIP gp140 Spike Protein toward Improved Expression and Affinity for Conformational Antibodies

<div><p>Design of an envelope-based immunogen capable of inducing a broadly neutralizing antibody response is thought to be key to the development of a protective HIV-1 vaccine. However, the broad diversity of viral variants and a limited ability to produce native envelope have hampered such design efforts. Here we describe adaptation of the yeast display system and use of a combinatorial protein engineering approach to permit directed evolution of HIV envelope variants. Because the intrinsic instability and complexity of this trimeric glycoprotein has greatly impeded the development of immunogens that properly represent the structure of native envelope, this platform addresses an essential need for methodologies with the capacity to rapidly engineer HIV spike proteins towards improved homogeneity, stability, and presentation of neutralizing epitopes. We report for the first time the display of a designed SOSIP gp140 on yeast, and the <i>in vitro</i> evolution of derivatives with greatly improved expression and binding to conformation-dependent antibodies. These efforts represent an initial and critical step toward the ability to rapidly engineer HIV-1 envelope immunogens via directed evolution.</p></div

Expression analysis of different gp140 fragments.

<p>(A) Schematic representations of expressed HIV-1 gp140 fragments, including gp120 inner and outer domain (inner and outer), CD4 binding site (CD4bs), V1/2 loop region (V1/2), the V3 loop (V3) and helical regions 1 and 2 of gp41 (H1, H2). (B) Western blot analysis of (1) yeast-secreted YU2 gp120 core, (2) gp120 residues 90–492, (3) gp120, (4) JR-FL SOSIP gp140, (3*) PNGaseF-treated YU2 gp120 with marked main band. Lane (3*) is non-adjacent but originates from the same blot. (C) Western blot analysis of (5) yeast-secreted JR-FL gp41 and (6) gp41 lacking the fusion peptide. (D) Amino acid sequences of the original and the modified fusion peptide, with modified residues in bold text.</p

Pool binding analysis of selection rounds.

<p>(A) Scatter plots obtained from 20,000 yeast cells displaying no HIV envelope protein (-), d-SOSIP gp140 D368R (d-SOSIP D368R), d-SOSIP or selected pools after cycles C3.3 and C4.3, respectively, probed for binding to 27 nM VRC01. (B) Binding isotherms obtained from pools selected after round 1.3, 2.4, 3.3 and 4.3 (cycle 1, 2, 3, 4), and reference clones d-SOSIP gp140 (d-SOSIP) and d-SOSIP D368R to HIV mAbs VRC01 or b6. Expression-normalized median fluorescence intensity (nMFI) is plotted against antibody concentration. Error bars represent standard deviations of triplicate measurements.</p

Single clone binding analysis.

<p>Expression-normalized median fluorescence intensity (MFI) is plotted against logarithmic Ab concentrations for d-SOSIP (gray) and clones 4.3.B01 (black) and 4.3.D01 (green) selected after DNA shuffling. Binding isotherms to a human IgG1k control Ab are included as reference. Error bars represent standard deviations of triplicate measurements.</p

Binding analysis of yeast-displayed spike protein variants.

<p>(A) Schematic illustration of yeast-displayed gp140 that is C-terminally fused to Aga2. (B) Overlay of histograms obtained from <i>S</i>. <i>cerevisiae</i> displaying JR-FL d-SOSIP gp140 (d-SOSIP), JR-FL d-SOSIP gp140 D368R mutant (d-SOSIP D368R), YU2 gp120 core (gp120 core) or an Hepatitis C Virus reference protein (HCV E2) probed for display level. Counts are plotted against fluorescence on logarithmic scale. (C) Overlay of histograms of yeast-displayed constructs binding to 100 nM HIV bnAb VRC01. (D) Median fluorescence intensities (MFI) normalized for yeast display levels (nMFI) are plotted for a panel of HIV Abs, grouped by epitope (CD4bs = CD4 binding site; V loop = variable loops V1/2/3; MPER = membrane proximal extracellular region; and G = glycan-specific). Error bars represent standard deviations of triplicate measurements.</p

Levels and IgG subclass composition differentiate HIV+ subject groups.

<p><b>A.</b> Titer (Mean Fluorescent Intensity, MFI) of gp120-specific IgG present in each subject group. <b>B</b>. The percent of subjects in each group positive for gp120-specific responses of each IgG subclass. <b>C.</b> Spearman correlation matrix between subclass responses across subjects. Strength and significance, as calculated in Graphpad Prism, are represented as color intensity and size, respectively. <b>D.</b> The levels of gp120-specific responses observed across cohort groups for each IgG subclass. Differences between groups were assessed by Kruskal-Wallis ANOVA and corrected for multiple comparisons using Dunn’s test in Graphpad Prism.</p

Functional coordination within HIV-infected subject groups.

<p><b>A.</b> The extent of functional coordination within groups was assessed by calculating Spearman’s rank correlation coefficients across each pair of independently assessed functional assays. Differences between subject groups were evaluated using a Friedman ANOVA corrected for multiple comparisons using Dunn’s Test in Graphpad Prism. <b>B</b>. Prevalence of functional correlations by strength for each subject group. <b>C</b>. Correlation matrix for each pairwise combination of functions tested, in which strong positive correlations appear blue while inverse correlations appear red, for each subject group. Correlative relationships and significance were calculated and visualized using R, with unadjusted p values indicated to facilitate relative comparisons.</p

Antibody functionality can be predicted by subclass composition.

<p><b>A</b>. Correlative relationships between levels of total gp120-specific IgG or gp120-specific antibodies of each subclass to antibody function were assessed by determination of Spearman’s rank correlation coefficients between activity and antibody MFI within each subject group. <b>B</b>. Correlative relationships between <i>relative</i> levels of gp120-specific antibodies of each subclass (i.e., MFI of subclass/MFI of total IgG) were assessed for each functional activity, over all subjects. Positive associations are noted in blue and inverse associations in red, with the magnitude of correlation depicted by intensity. Correlative relationships and significance were calculated and visualized using R, with unadjusted p values indicated to facilitate relative comparisons. <b>C</b>. The magnitude and direction of the contribution of SF162-specific antibody subclass assessments to cross-validated predictive models of polyfunctional activity.</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