Graphical representation of the low VB variability.

<p><b>A</b>: Trees were generated for each of the variable blocks in DBL6ε, branch annotations are the average percent pairwise identity (APPI) for VB1 sequences in this subtree. <b>B</b>: For various APPI (on the x-axis), branches (i.e. groups) are counted and marked on y-axis. Graphic representation of variability of the DBL6ε domain (black circle), the envelope protein of HIV (purple triangle), VB1 (blue square), VB2 (black diamond), VB3 (green triangle), VB4 (red diamond), VB5 (gray square), VB6 (light blue triangle) and VB7 (orange circle). In <b>C</b>, a histogram was constructed to determine the variability of the various blocks composed of more than one consensus sequence. HIV represents the histogram obtained with the Env glycoprotein of HIV. For comparison, global APPI of all the sequences are indicated at the top of each histogram.</p

Reactivity of purified IgG against variable blocks.

<p>The level of purified IgG responses expressed as optical density (OD) units at 450nm against the various peptides is shown for women at delivery living in endemic area. The top, bottom, and middle lines of the box correspond to the 75th percentile, 25th percentile, and 50th percentile (median), respectively. The whiskers extend from the 10th percentile and top 90th percentile.</p

Summary of sequences from strains and isolates.

<p>*Sequence too short; the carboxy-terminal region of the DBL6ε is missing.</p><p>29 strains and isolates DBL6ε accession number, their origin and length.</p

Alignment of the entire sequence of the DBL6ε domain from various isolates.

<p>With respect to the 3D7 PFL0030c residue numbering and combined the alignment, ID5 is located from M2287 to K2328, DBL6ε from G2329 to P2651, transmembrane domain from N2677 to P2710 and ATS from K2711 to the C-terminus. The highly conserved amino acids are coloured in red, the lowly conserved amino acids are in blue, and the highly variable amino acids are in black. The seven polymorphic blocks are highlighted in gray. At the top of the sequences, cysteine residues are numbered from 1 to 12 in bold. (*) indicates the sequence of the expressed and purified CYK48-DBL6ε domain. On the left, all strains and isolates are presented; all are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011276#pone-0011276-t001" target="_blank">Table 1</a>.</p

Graphic representation of the variability tolerated for a sequence that remains dominant epitope at various identity percentages.

<p>Experimental responses of purified IgG against the VB are shown in percent on the x-axis and are summarised in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011276#pone-0011276-t003" target="_blank">Table 3</a> (Reactivity of IgG). The occurrence of each VB sequences in the population was counted. For example, in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011276#pone-0011276-g004" target="_blank">Figure 4B</a> at APPI>85% (here 86.1%) we counted 14 strains (or 14/29*100%). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011276#pone-0011276-g004" target="_blank">Figure 4</a>, the number of sequences identical to CYK48 (indicated with*) were counted. Percentage of isolates with the same sequence as CYK48 for each VB is indicated on y-axis at 80% (black circle), 85% (blue square), 90% (green triangle), or 95% (red diamond) APPI. For example, see values for APPI = 85% in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011276#pone-0011276-t003" target="_blank">Table 3</a> (% of isolates). Experimental distribution around the linear curve is linked to the APPI tolerated to have an identical IgG response to two sequences. The best Pearson's r (0.74) for a linear model is calculated for 85% APPI. This graph indicates a cut-off of 85%. In our ELISA, two sequences sharing this APPI will have the same antibody response.</p

Synthesised peptide sequences from CYK48-DBL6ε VB1 to 7.

<p>*Identical at 85% of average percentage pairwise identity.</p>§<p>In % on 80 purified IgG.</p>$<p>B: Biotin; Ct: Carboxyterminal.</p><p>Sequence of variable blocks from CYK48-DBL6ε. For example, the number of isolates identical to CYK48 VB1 determined from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011276#pone-0011276-g004" target="_blank">Figure 4B</a>. At the branch where APPI>85% (here 86.1%), 14 strains are counted (for a total of 29 strains: 48% of them have the same sequence at APPI>85%). The other VB values were determined as the same way.</p

Alignment of the variable blocks.

<p>Alignments of variable blocks VB1, VB2, VB3, VB4, VB5, VB6 and VB7 shows that consensus sequence groups within each VB are limited in number. The sequence of CYK48-DBL6ε is indicated by (*). Green: 100% similar; khaki: 80 to 100% similar; Orange: 60 to 80% similar; Gray: less than 60% similar.</p

Purity of various proteins.

<p>CYK48-DBL6ε domain and antibodies observed after purification on 10% acrylamide gel (Invitrogen) stained with Coomassie blue. Reduced CYK48-DBL6ε domain (lane 1) after two purification steps and non-reduced or reduced antibody from protein G purification (respectively lanes 2 and 3). Molecular weight on left lane (Biorad).</p

Structure model of CYK48-DBL6ε domain.

<p>This structure was performed with HHMM software using the DBL6ε (2WAU) domain of Kundrae <i>et al</i> as template. This model was used to determine the limits of variable blocks and to design relevant peptides. Cysteine residues are shown in yellow and variable blocks on the surface of the domain are in red. Conserved sequence is coloured in gray and is located in the core of the domain.</p