Sequencing primers overview.

<p>Sequencing primers overview.</p

Scatter plots of FC of subtype C amplicons recombined in subtype B and subtype C backbones.

<p>(A–D) X-axis: Subtype B and Y-axis: subtype C for 1346 pairs; Black line x = y; (A) all drug classes (R² = 0.88); (B) NRTIs (R² = 0.88); (C) NNRTIs (R² = 0.90); (D) PIs (R² = 0.87); (E) Analysis of the pair-wise comparison of differences in FCs per clone and per drug, Ratio FC_{Subtype B}/FC_{Subtype C} (Average, Red squares) and P-value (Black diamonds).</p

Experimental flow.

<p>Flow of the testing of the subtype C GPRT amplicons in the pGEM-HIV-1-C-Δgprt-BstEII-V (pHIV-1-C-Δgprt) and the pGEM-HXB2-Δgprt-BstEII (pHIV-1-B-Δgprt) backbones. “TRF”: transfection (Amaxa); “FC”: Fold Change; Red boxes: phenotypes; Green boxes: genotypes.</p

Boxplot illustrating the effect of RAM 184V on the NRTI FC in a subtype B and C backbone.

<p>Blue = HIV-1 subtype B backbone; Green = HIV-1 subtype C backbone; “+” = mutation 184V is present in RT; number under block = number of observed FC. P values have been calculated for each subtype for FC with mutation vs. FC without mutation.</p

Kinetics of HIV-1 subtype C virus production.

<p>A. viral load and pictures of fluorescence (day 4(1); day 6 (2); day 11 (3)); B: p24 measurements for the three clones of sample 4.</p

Subcloning strategy of the vector containing the HIV-1 subtype C-Δgprt backbone.

<p>Fragment I (A) and Fragment II (B) were digested using BstEII and EcoRI and religated resulting in an HIV-1 subtype C clone lacking a part of GAG, protease and reverse Transcriptase and most of ENV (Fragment I-II (C)). Fragment I-II was linearized using PacI and AccIII to insert the Env region from Fragment III (D) resulting in a final clone, pGEM-HIV-1-C-Δgprt-BstEII-V, that can be linearized using BstEII/EcoRV, ready for In-Fusion cloning with the 1.7 kb GPRT amplicon. • pGEM-HIV-1-C-Δgprt-BstEII-V+GPRT (wild type sequence).</p

Overview of the clones per sample, day of harvest (subtype C and B), VL and p24 measurements as well as the corresponding resistance-associated mutations per clone (as referenced by IAS-USA, ANRS) per sample.

<p>“DTH” Days to harvest.</p

Citrullination only infrequently impacts peptide binding to HLA class II MHC

<div><p>It has been hypothesized that HLA class II alleles associated with rheumatoid arthritis (RA) preferentially present self-antigens altered by post-translational modification, such as citrullination. To understand the role of citrullination we tested four RA-associated citrullinated epitopes and their corresponding wild-type version for binding to 28 common HLA class II. Binding patterns were variable, and no consistent impact of citrullination was identified. Indeed, in one case citrullination significantly increased binding compared to the WT peptide, in another citrullination was associated with a reduction in promiscuity by 40%. For a more comprehensive analysis, we tested over 200 citrullinated peptides derived from vimentin and collagen II for their capacity to bind the RA-associated shared epitope alleles DRB1*01:01 and DRB1*04:01. The overall effect of citrullination on binding was found to be relatively minor, and only rarely associated with 3-fold increases or decreases in affinity. Previous studies have suggested that citrullination of MHC anchor residues, in particular P4, is associated with generation of novel RA-associated epitopes. However, analysis of the predicted MHC-binding cores of all peptides tested found that in modified peptides with increased binding affinity the citrullinated residue was predicted to occupy an anchor position in only a minority of cases. Finally, we also show that identification of citrullinated peptide binders could be facilitated by using the NetMHCIIpan 3.1 algorithm, representing citrullination as a wildcard. Our studies identify a total of 117 citrullinated peptides that bound RA-associated alleles with an affinity of 1000 nM or better.</p></div

Prediction of the DRB1*01:01 and DRB1*04:01 binding capacity of citrullinated peptides.

<p>The DRB1*01:01 (left panel) and DRB1*04:01 (right panel) binding capacity of citrullinated peptides were predicted using NetMHCIIpan version 3.1 predictions by substituting the citrullinated residues with the wildcard “X”. Trend lines are show in red.</p

Predicted HLA DR binding cores of four known citrullinated RA-associated epitopes.

<p>Predicted HLA DR binding cores of four known citrullinated RA-associated epitopes.</p