Docking simulations indicate that the antigen-binding site of 7C8 binds primordially to the FHSQ epitope on gp125.

<p>Representation of the two docking solutions of the crystal structure of 7C8 in complex with the putative gp125 trimer. The two solutions, displayed in cyan and grey respectively, interact mainly with the solvent-exposed FHSQ epitope. The interactions proposed by the two solutions also suggest a mechanism for neutralization since the size of the bound molecules would not allow for subsequent engagement of the gp125 trimer to the co-receptor on target cells.</p

The deep and narrow antigen-binding site of 7C8, well fitted for elongated epitopes, is highly hydrophobic.

<p>The surface of the antigen-binding site of 7C8, displayed from the perspective of a bound antigen, is colored according to its electrostatic surface potential. To the left, the six CDR loops that form the antigen-binding site are colored according to the sequence-panel. To the right, the relative arrangement and the conformation of the CDRs result in the formation of a deep and narrow highly hydrophobic cleft surrounded by the prominent CDRL1, CDRH1 and CDRH3 loops. Each side of the deep antigen-binding cleft is positively (in blue) and negatively (in red) charged, respectively, while the middle section is highly hydrophobic. A selection of light and heavy chain residues that form the antigen-binding site are indicated in white and black, respectively.</p

The epitope FHSQ is localized centrally on the V3-region of gp125.

<p><b>A</b>) The molecular model of gp125 is very similar to its HIV-1 counterpart gp120. The conserved inner face and the more variable outer face of gp125, which includes the V3 region, are highlighted in grey and blue, respectively. The 15-mer gp125 V3-peptide used to raise 7C8 is displayed in red. The localization and solvent exposure of the epitope GPGR on gp120 and the epitope FHSQ on gp125 are highlighted in orange. <b>B</b>) A putative trimeric model of gp125 displayed with the V3 region oriented towards the target cell indicates the accessibility and solvent exposure of the FHSQ epitope.</p

Overall three-dimensional structure of the HIV-2 neutralizing Fab fragment 7C8.

<p>A) Overall view of the crystal structure of the 7C8 Fab fragment. The light and heavy chains are displayed in light and dark grey, respectively. The six CDR loops are in dark blue (CDRL1), light blue (CDRL2), blue (CDRL3), brown (CDRH1), red (CDRH2) and pink (CDRH3). A black dashed line indicates the disordered loop within the first constant region of the C_H1 domain. B) Stick representation of the stretch of residues 93 to 100C corresponding to the unusually long third complementary determining region of the heavy chain (CDRH3) with the electron density carved around the model at 1.2 σ. <b>C</b>) Ribbon representation of the two monomers found in the assymmetric unit indicating the large variation in elbow angle.</p

Data collection and refinement statistics.

a<p>Number in parentheses indicate the outer-resolution shell.</p>b<p><i>R</i>_merge  =  ∑<i>_hkl</i> ∑<i>_i</i> |<i>I_i</i> (<i>hkl</i>) - 〈<i>I</i> (<i>hkl</i>) 〉|/∑<i>_hkl</i> ∑<i>_i I_i</i> (<i>hkl</i>), where <i>I_i</i>(<i>hkl</i>) is the <i>i</i>th observation of reflection <i>hkl</i> and 〈<i>I</i> (<i>hkl</i>) 〉 is the weighted average intensity for all observations <i>i</i> of reflection hkl.</p>c<p><i>R</i>_cryst  =  Σ_hkl  =  ∑_hkl|<i>F</i>_obs − <i>F</i>_calc|/Σ_hkl |<i>F</i>_obs|.</p>d<p><i>R</i>_free is the same as <i>R</i>_cryst except for 5% of the data excluded from the refinement.</p>e<p>Sum of the TLS and Residual B-factor contributions.</p

Biologic responses to Can f 1-2-4-6 among dog allergic patients.

<p>Allergen-specific basophil degranulation monitored by addition of serial dilutions of rCan f 1 or equimolar amounts of either rCan f 2, rCan f 4, rCan f 6, an equimolar mix or Can f 1-2-4-6 (x-axis) to whole blood from three dog allergic patients. Positivity was defined by analysis of CD63 and CD203c double positive cells (y-axis) using flow cytometry.</p

Biochemical analysis of the linked molecule.

<p>(A) SDS-PAGE of Can f 1-2-4-6 or the single recombinant lipocalins visualized by coomassie staining under reducing conditions. MW markers (lane 1,7, MW (kDa)), rCan f 1 (lane 2), rCan f 2 (lane 3), rCan f 4 (lane 4), rCan f 6 (lane 5) and Can f 1-2-4-6 (lane 6). (B) Analytical size exclusion chromatography of rCan f 1-2-4-6 and the single lipocalin allergens. Molecular weight markers, bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and Ribonuclease A (13.7 kDa). (C) Far-UV CD analysis of Can f 1-2-4-6 or an equimolar mix of the single recombinant allergens Can f 1, Can f 2, Can f 4 and Can f 6. The spectra are expressed as mean residue ellipticities (θ) at a given wavelength.</p

Antibody responses after immunization with allergens.

<p>(A) quantification of IgE-responses in mice (n = 6) immunized with each rCan f 1, rCan f 2, rCan f 4, rCan f 6, an equimolar mix or Can f 1-2-4-6 (x-axis in graphs) to plates coated with anti-mouse IgE, mouse antiserum and biotinylated allergen (graph heading) by sandwich ELISA. (B) Comparison of IgG1 and IgG2a-antibodies to each lipocalin induced by solid phase Can f 1-2-4-6 or a mix. Immunoglobulin responses are presented as OD405 nm, y-axis. Boxes with median values and horizontal bars denote 50% of values and 1 standard deviation respectively. * p<0.05, ** p<0.01, *** p<0.001, analyzed with Kruskal-Wallis with Dunn's multiple comparison test (A, B, C) and Mann Whitney test (D).</p

Human serum IgE blocking capability of IgG antibodies induced by the linked construct.

<p>Pooled sera from mice (n = 6) immunized with each of rCan f 1, rCan f 2, rCan f 4, rCan f 6, Can f 1-2-4-6 or an equimolar mix was preincubated in wells coated with either rCan f 1, rCan f 2, rCan f 4 or rCan f 6. Sera from multi-sensitized subjects (n = 6) was added to the wells and IgE-responses recorded. The percentage of IgE inhibition is displayed on the y-axis. Median values are indicated in the boxes. The boxes and horizontal bars denote 50% of values and 1 standard deviation respectively.</p

IgE-binding properties of the linked construct.

<p>(A) IgE-reactivity of 100 consecutive dog-sensitized subjects (e5, >0.35kU_A/L, ImmunoCAP, Thermo Fisher scientific) towards dog extract, Can f 1-2-4-6, the single allergens rCan f 1, rCan f 2, rCan f 4 and rCan f 6, or an equimolar mix of the same allergens. Bars denote mean values. Data are presented as adjusted kU_A/L. (B) Pair-wise comparison of accumulated IgE-reactivities to Can f 1, Can f 2, Can f 4 and Can f 6 among positive subjects with reactivity to Can f 1-2-4-6. *** p<0.001 (Wilcoxon matched pairs test) (C) Competitive inhibition ELISA with sera from subjects sensitized to rCan f 1, rCan f 2, rCan f 4 and rCan f 6. After pre-incubation of sera with 10-fold dilutions of equimolar amounts of each recombinant allergen, mix or Can f 1-2-4-6, IgE-responses were measured. Inhibition was calculated as response in percentage (%) of response to the coated antigen after allergen addition (y-axis). Subject 1 and 2 respectively correspond to subject 8 and 21, Table S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111041#pone.0111041.s001" target="_blank">File S1</a>.</p