Kinetics of gp41-specific antibody titers and contribution of Fc mediated gp41 specific antibody responses following SHIV-SF162P3 challenge.

<p>The level of gp41 specific antibody subclass over time was measured by Luminex in the plasma of the 4 infected monkeys that received the EC antibodies as well as in one monkey that received the HIV-NEG antibodies and is reported as mean fluorescence intensity (A). Antibodies were purified from the rhesus monkeys that received the EC antibodies or the HIV-NEG antibodies and tested for their ability to recruit monocytes to mediate phagocytosis (B) or to activate NK cell measured as a release of CD107a, MIP1β and IFNγ (C) 2 weeks prior the antibody transfer and at day 1, 3, 7, 14, 21 and 28 post antibody transfer.</p

Polyclonal antibodies from naturally infected donors have a wide range of non-neutralizing anti-viral activities.

<p>Antibodies purified from the plasma of 15 different elite controllers (labeled EC-1 to EC15) were tested for their ability to bind to gp120 by ELISA, shown as the inverse of EC50 (A), to mediate ADCVI, measured as a percent of inhibition of viral replication (B) and to neutralize HIV virus (JRCSF), measured as an inverse of EC50 (C). The correlation plots show the relationship between gp120-specific antibody titers from elite controllers and ADCVI activity (D), or virus neutralization (E) as well as the association between the neutralization and ADCVI activity of antibodies (F). The elite controller from which the purified antibodies had the greatest innate immune recruiting properties in the absence of neutralization, EC10, was selected for passive transfer into macaques and is highlighted in gray. A minimum of 2 separate experiments was assessed to select the EC antibodies that had the best activity.</p

Kinetics of plasma viremia following SHIV-SF162P3 challenge.

<p>50 mg/kg of antibodies purified from the selected elite controller (A) or from HIV-negative individuals (B) were passively transferred intravenously one day prior to the challenge with SHIV-SF162P3 in 5 rhesus macaques each. The plasma viremia was measured 14 days before and 1, 3, 7, 14, 21, 28, 56 and 84 days after antibody transfer. No viremia was detected in the animal that received 25 mg/kg of the b12 monoclonal antibodies used as a positive control. The area under the curve (AUC) of viral loads was measured in the 4 infected monkeys that received the EC antibodies and the 3 infected monkeys that received HIV-NEG antibodies (C). An unpaired two-tailed t-test with 95% confidence intervals was used to compare the AUC between both groups.</p

Antibodies purified from the elite controller selected for the <i>in vivo</i> study display a wide range of innate immune recruiting properties.

<p>Large quantities of antibodies were purified from the plasma of EC10 and assessed for their neutralizing capacity in a TZM-bl assay (A) and in a primary CD4 T cell assay (B). IgG HIV-binding titer against YU-2, SF162 gp120s, SHIVgp140 SF162p3 and gp41 was assessed (C) along with ADCVI activity (D) against a tier 2 (JRCSF), a tier 1A (SF162) and the challenge virus (SHIV-SF162P3). Purified b12 and a pool of antibodies purified from HIV-negative individuals were used as positive and negative controls, respectively. HIVIG was used as a positive control for testing gp41-binding titer. Antibodies purified from the plasma of EC10 were also evaluated for their ability to induce ADCP against a tier 2(JRCSF), a tier 1A (SF162) virus as well as against gp41 (E) and for their ability to induce complement activation as measured by C3b deposition on YU-2 or SF162 gp120 pulsed CEM cell line using HIVIG and a pool of antibodies purified from healthy individuals as positive and negative controls, respectively (F). A minimum of 2 separate experiments was performed to confirm the innate immune recruiting properties of the antibodies from the selected EC.</p

Kinetics of gp120-specific antibody titers and innate immune recruiting properties of antibodies following SHIV-SF162P3 challenge.

<p>ELISA was used to test the level of gp120-specific antibodies in the plasma of infected rhesus macaques (A) that received purified antibodies from either the pool of HIV-negative individuals (<i>i</i>) or the elite controller (<i>ii</i>). Optical Density (OD) values are reported. The level of gp120 specific antibody subclass over time was measured by Luminex in the plasma of the 4 infected monkeys that received the EC antibodies as well as in one monkey that received the HIV-NEG antibodies and is reported as mean fluorescence intensity (B–E). Antibodies were purified from the rhesus monkeys that received the EC antibodies (<i>i</i>) or the HIV-NEG antibodies (<i>ii</i>) and tested for their ability to recruit NK cells to mediate ADCC (F) or to recruit monocytes to mediate phagocytosis (G) 2 weeks prior the antibody transfer and at day 1, 3, 7, 14, 21 and 28 post antibody transfer. NK cells isolated from 2 separate donors were used to assess ADCC and 2 separate experiments were performed to measure the phagocytic activity.</p

Global glycan occupancy site utilization across 94 HIV gp120s.

<p>(a) The heat map represents the N-linked glycosylation site occupancy profiles of 94 distinct recombinant gp120 proteins. Site utilization was determined by mass spectrometry, and the frequency of utilized sites at each potential glycosylation site (columns) is presented using a yellow-to-black gradient. The gray boxes depict the absence of a sequon (N-X-S/T, X≠P) at that specific site within that sequence. The right panel shows the average glycosylation site occupancy per protein. N-glycan sites were aligned based on the HXB2 sequence. Canonical N-glycan sites were designated based on the aligned sequence. Non-canonical N-glycan sites, which are not present in the HXB2 sequence, are shown in decimal numbers, based on the previously aligned N-glycan site. (b)-(e) The bar graphs show (b) the frequency of sequons present at each potential N-glycan site across all strains; (c) the mean (± standard deviation) glycan occupancy; (d) the variance of the glycosylation site occupancy (dotted line represents the top 15th percentile). (e) The N-glycan sites with the top 15% highest variance were mapped onto the BG505.SOSIP crystal structure (PDB #: 4NCO) highlighted as red. The approximate binding epitopes of various bNAbs on the Env structure are labeled in hatched circles.</p

Defining the glycosylation site determinants that shape bNAb binding profiles.

<p>(a)-(d) Four different Bayesian MCMC-SVR models were evaluated for their respective abilities to predict PGT121 binding to the 94 proteins. The models include a Bayesian MCMC-SVR model based on: (a) sequon presence (Figure A in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006093#pcbi.1006093.s001" target="_blank">S1 File</a>); (b) protein sequence; (c) glycosylation site occupancy; or (d) glycosylation site occupancy and sequence combined. Cross validation (100-iterative 10-fold) was used to evaluate model performance. Goodness-of-fit was assessed and is reported as the mean squared error (MSE) between predicted and ELISA-measured binding. (e) Heat map shows the binding signatures of individual Abs (rows), where the selected glycan sites (determinants) that mediate effects on Ab binding are highlighted. NAbs that share similar glycan determinants are grouped by hierarchical clustering. (f) The significant glycan site determinants for PGT121, PGT128, and VRC01 are plotted onto a 3-dimensional gp120 monomer structure using the same directional color coding as the heat-map. Additionally, the critical protein residues predicted by our model are shaded in yellow on the same 3D structure. Finally, broad Ab-binding sites were highlighted for each bNAb in hatched circles. (g) Agonistic and antagonistic glycan site determinants and critical protein residues for PGT122 are projected on the BG505 SOSIP.664-PGT122 co-crystal structure (PDB #: 4NCO) with the same color coding. The V3 loop is highlighted in light green shading.</p

Geographic location affects bulk IgG glycosylation.

<p>Bulk IgG glycosylation was assessed in subjects from three regions: Unite States (blue, n = 43), Kenya and Rwanda (maroon, n = 69), and South Africa (yellow, n = 47). (A) Bulk antibody Fc glycosylation in vaccine recipients from each of the three regions was measured via capillary electrophoresis, and the mean proportion of total galactosylated, sialylated, fucosylated, and bisected structures was compared using Kruskal-Wallis one-way ANOVA (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001). (B) Multivariate comparison of antibody Fc glycosylation among the three geographic sites was performed using PCA. The score plot (left panel) depicts the principal component analysis of samples collected in the three regions (each dot represents a vaccinee, and colors are as described above), and the loadings plot of the PCA (right panel) shows the contribution of particular glycan structures to driving the observed separation, where longer arrows signify a greater contribution to separating glycan profiles. This PCA describes 55% of the total variance among these samples.</p

Proof-of-concept glycoengineering of gp120 antigens to selectively enhance antigenicity.

<p>(a) Cartoon depicts the overall <i>de novo</i> antigen optimization design approach. (b) The heat maps, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006093#pcbi.1006093.g003" target="_blank">Fig 3E</a>, depict the glycosylation site determinant profiles preferred by PGT121 and PGT128 including directional glycan coloring across all N-glycan sites (columns). (c) The top heat map represents the original wild-type MG535.W0M.ENV.D11 gp120 sequon site profile (yellow = sequon site absent and black = sequon site present); middle and bottom heat maps indicated the introduced point mutations (brown = sequon site knock-in, light blue = sequon site knock-out) for the gp120s engineered to have increased binding to PGT121 (+PGT121), PGT128 (+PGT128), and both PGT121 and PGT128 (+PGT121+PGT128); also, the gp120s engineered to selectively bind PGT121 but not PGT128 (+PGT121-PGT128), or PGT128 but not PGT121 (-PGT121+PGT128 and -PGT121+PGT128 2nd). (d) The bar graph depicts comparison of the predicted binding (beige = PGT121 and brown = PGT128) and ELISA-determined binding (light blue = PGT121, dark blue = PGT128, and grey = VRC01 binding) to the wildtype and engineered gp120s. ELISA binding activity was determined as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006093#pcbi.1006093.g002" target="_blank">Fig 2A</a>. In order to compare the model predictions to the experimental results, both the model and actual ELISA values were normalized to wild-type binding values, which were set to 1. Error bars indicate the standard deviation from six replicates. (e) The bar graph shows the degree of steric hindrance found on each antigen by summing all steric glycan site pairs (Figure J in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006093#pcbi.1006093.s001" target="_blank">S1 File</a>), if any site in the pair was considered essential for predicting Ab binding. Pink highlighted region denotes the average and range of the degree of steric hindrance across all the 94 recombinant gp120 proteins.</p

Vaccine-elicited antibody glycosylation profiles are distinct from bulk antibody glycosylation.

<p>Viral vector–induced gp120-specific and influenza specific antibodies were isolated from vaccinees, and the attached glycans were analyzed by capillary electrophoresis. (A) Multivariate PCA was used to compare bulk antibody glycoprofiles (blue, n = 32) and vaccine-elicited antigen-specific antibody glycoprofiles (maroon, n = 20), and both the scores plot (left) and loadings plot (right) are shown. This analysis describes 69% of the variation. (B) The mean proportions of bulk and vaccine-elicited antibody glycan were compared using students two-tailed paired t tests (n = 13 for bulk, n = 20 for gp120 (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001) (C) The mean proportions of vaccine-elicited antibody glycan structures were compared across vaccine groups using Kruskal-Wallis ANOVA (n = 9 for United States, n = 6 for Kenya/Rwanda, n = 4 for South Africa). No statistically significant differences were found. (D) The mean proportions of influenza-specific antibody glycans at baseline (magenta), post-first (green), and post-boost (purple) vaccine timepoints were compared using non-parametric two-way ANOVA (n = 18 for United States, n = 11 for Kenya/Rwanda, n = 5 for South Africa). No significant differences were found between the three timepoints for either antigen or glycan type.</p