Barriers to dog rabies vaccination during an urban rabies outbreak: Qualitative findings from Arequipa, Peru - Fig 1

<p>Urban (A) and peri-urban communities (B) where FG were conducted.</p

Participant characteristics.

<p>Participant characteristics.</p

Summary of factors that influence canine rabies vaccination uptake.

<p>Summary of factors that influence canine rabies vaccination uptake.</p

Community dwellers, not focus group participants, with their dogs at a rabies vaccination point in Mariano Melgar, February 2016.

<p>Note harness made of plastic bag (A) and lack of leash and holding dog by ears (B).</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

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