R&D1801 and RoAb13 recognise overlapping core epitopes in the CCR5 N-terminal domain.

<p>A, R&D1801 or RoAb13 were incubated overnight with competing peptides 1–24 (1 μg/ml) in wells coated with hCCR5_1–31, and binding (shown as OD 405 nm) of antibody was then measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128381#pone.0128381.g001" target="_blank">Fig 1a</a> using a rabbit anti-mouse second layer. C—PBS used in place of competitive peptide. B, The peptide sequence of the 24 peptides tested in a. The blue bars shows all peptides which inhibited the binding by more than 50%. C, Diagrammatic representation of the core binding epitope recognized by the two antibodies, defined by peptides which inhibited binding to the whole N-terminal peptide by more than 50%.</p

Two monoclonal antibodies recognize a linear epitope in the N-terminal domain of CCR5.

<p>A, Dilutions of the four monoclonal antibodies shown were tested for binding to hCCR5_1–31coding for the N-terminal extracellular domain of CCR5 by ELISA as described in M&M. The left panel shows binding (as measured by OD at 405 nm) to plates coated with the N-terminal peptide coupled to avidin, while the right panel shows binding to avidin alone. B, Dilutions of R&D1801 and RoAb13 were tested for binding to CCR5-transfected CHO cells by indirect immunofluorescence and flow cytometry. The results are normalized and expressed as % maximum binding for each antibody. Binding to untransfected controls or to CHO cells transfected with mouse CCR5 was less than 5% of maximum binding in each case.</p

Immunisation with a chimeric peptide coding a linear CCR5 antibody epitope together with a helper epitope from tetanus toxoid can stimulate antibodies which recognize native CCR5.

<p>A, The sequence of the chimeric peptide used for immunization, showing the CCR5 B cell epitope, the linker sequence and the T cell helper epitope from tetanus toxoid. B, Sera from 5 immunized mice were collected after priming and boosting (see M&M) with the peptide shown in a), and tested in ELISA for binding to hCCR5_1–31. Binding is shown as OD at 405 nm. U: serum from an unimmunized mouse (preimmune sera showed equivalent binding). RoAb13: supernatant from the RoAb13 hybridoma diluted as shown. C, Sera from the same five mice were tested for binding to CCR5 transfectants (red line) or controls at a dilution of 1:50.</p

RoAb13 inhibits monocyte chemotaxis.

<p>PBMC were incubated in the upper chamber of transwells, while the lower chamber contained medium alone, supernatant of unactivated macrophages, or supernatant of macrophages activated with LPS (see M&M for details). RoAb13 Fab fragment or a control Fab fragement were added to some wells as shown. Migrating cells were collected from the lower chamber after three hours and the cells phenotyped as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128381#pone.0128381.s002" target="_blank">S2 fig</a>. Migration is shown as the proportion of cells in the PBMC premigration sample which migrate to the lower chamber during the assay. Each experimental condition was set up in triplet and * shows significant difference from control (T test, p<0.05).</p

The sequence and crystals of RoAb13.

<p>A, The protein sequence of RoAb13 (lower sequence) compared to the closest germline sequence for heavy (right) and light (left) chains (as determined by the V-Quest). Conserved residues are shown in red. B, Crystals of RoAb13.</p

Species specificity of RoAb13 and R&D1801.

<p>A, The sequence of the N-terminal extracellular domain of CCR5 from human, rhesus Macaque or mouse CCR5. * identical amino acids—insertion in mouse sequence. B, Flow cytometry showing binding of R&D1801 or RoAb13 to CHO cells transfected with mouse (filled) or human (red line) CCR5. C, Flow cytometry showing binding of R&D1801 or RoAb13 or control to PBMC from rhesus macaques (indirect immunofluorescence, using Goat anti-mouse PE conjugate as second layer). The first panel shows binding of a commercially available PE-conjugated anti-rhesus CCR5 antibody (positive control).</p

Affinity of RoAb13 binding to native cell-surface CCR5 and a peptide coding for the N-terminal extracellular domain of CCR5.

<p>A, RoAb13 was incubated with CCR5-expressing transfected cells or controls at different concentrations and binding measured by indirect immunofluorescence and flow cytometry. The binding was converted to absolute number of bound antibody molecules by using Ig calibration beads as described in M&M and shown in Supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128381#pone.0128381.s001" target="_blank">S1 Fig</a>. The graph shows the concentration of bound antibody molecules plotted against the ratio of bound/free antibody (a classical Scatchard plot). The equation showing the relationship between bound and bound/free derived from the law of mass action is shown below the figure, together with the affinity and number of receptors calculated respectively from the slope and intercept of the equation. B, RoAb13 was incubated with different concentrations of hCCR5_1–31N-terminal domain peptide overnight. The remaining amount of free antibody was estimated by binding to hCCR5_1–31 in ELISA and comparison to a standard curve. Bound antibody was calculated as total—free. The resulting bound and bound/free ratio was plotted as in a) and the affinity calculated from the slope.</p