Differential Evolutionary Selection and Natural Evolvability Observed in ALT Proteins of Human Filarial Parasites.

The abundant larval transcript (ALT-2) protein is present in all members of the Filarioidea, and has been reported as a potential candidate antigen for a subunit vaccine against lymphatic filariasis. To assess the potential for vaccine escape or heterologous protection, we examined the evolutionary selection acting on ALT-2. The ratios of nonsynonymous (K(a)) to synonymous (K(s)) mutation frequencies (ω) were calculated for the alt-2 genes of the lymphatic filariasis agents Brugia malayi and Wuchereria bancrofti and the agents of river blindness and African eyeworm disease Onchocerca volvulus and Loa loa. Two distinct Bayesian models of sequence evolution showed that ALT-2 of W. bancrofti and L. loa were under significant (P<0.05; P < 0.001) diversifying selection, while ALT-2 of B. malayi and O. volvulus were under neutral to stabilizing selection. Diversifying selection as measured by ω values was notably strongest on the region of ALT-2 encoding the signal peptide of L. loa and was elevated in the variable acidic domain of L. loa and W. bancrofti. Phylogenetic analysis indicated that the ALT-2 consensus sequences formed three clades: the first consisting of B. malayi, the second consisting of W. bancrofti, and the third containing both O. volvulus and L. loa. ALT-2 selection was therefore not predictable by phylogeny or pathology, as the two species parasitizing the eye were selected differently, as were the two species parasitizing the lymphatic system. The most immunogenic regions of L. loa and W. bancrofti ALT-2 sequence as modeled by antigenicity prediction analysis did not correspond with elevated levels of diversifying selection, and were not selected differently than predicted antigenic epitopes in B. malayi and O. volvulus. Measurements of ALT-2 evolvability made by χ2 analysis between alleles that were stable (O. volvulus and B. malayi) and those that were under diversifying selection (W. bancrofti and L. loa) indicated significant (P<0.01) deviations from a normal distribution for both W. bancrofti and L. loa. The relationship between evolvability and selection in L. loa followed a second order polynomial distribution (R2 = 0.89), indicating that the two factors relate to one another in accordance with an additional unknown factor. Taken together, these findings indicate discrete evolutionary drivers acting on ALT-2 of the four organisms examined, and the described variation has implications for design of novel vaccines and diagnostic reagents. Additionally, this represents the first mathematical description of evolvability in a naturally occurring setting

Selection and Evolvability of ALT-2.

<p>Values for selection (ω) and evolvability (E) were calculated for each amino acid residue in the ALT-2 sequence. The single E value was plotted against the ω value for pairwise comparisons between all species. Residues that were both significantly evolvable and under diversifying selection appear in the blue-shaded boxes. The two conserved ALT-2 sequences (<i>O</i>. <i>volvulus</i>, [filled circles] and <i>B malayi</i> [filled diamonds], <b>l A</b>) and the two diversified ALT-2 sequences (<i>L</i>. <i>loa</i> [open squares] and <i>W</i>. <i>bancrofti</i> [open triangles], <b>B</b>) served as positive and negative controls, respectively. Selection and evolvability of ALT-2 from <i>L</i>. <i>loa</i> followed a second-order polynomial distribution when compared to either <i>B</i>. <i>malayi</i> (<b>C</b>) or <i>O</i>. <i>volvulus</i> (<b>E</b>). Only a small number of residues in <i>W</i>. <i>bancrofti</i> were both diversified and evolvable when compared to <i>B</i>. <i>malayi</i> (<b>D</b>), but none were detected when compared to <i>O</i>. <i>volvulus</i> (<b>F</b>).</p

Phylogenetic Analysis.

<p>The evolutionary history of ALT-2 consensus sequences from <i>B</i>. <i>malayi</i>, <i>W</i>. <i>bancrofti</i>, <i>O</i>. <i>volvulus</i>, and <i>L</i>. <i>loa</i> was inferred using (<b>A</b>) the Neighbor-Joining method and (<b>B</b>) the Maximum Parsimony method (N = 500 bootstrap replicates each). Regardless of method, the branching order was the same and was consistent with ribosomal RNA-derived branching order.</p

Pairwise Evolvability (E) Analysis Design.

<p>Pairwise Evolvability (E) Analysis Design.</p

Evolutionary Selection Analysis.

<p>Calculated ω values are presented for each amino acid residue (X axis, marked by position number) for ALT-2 from the indicated species using the M8 model. The cutoff ω value of 1 is depicted as a black line; values extended above the line represent residues under diversifying selection, and values below the line represent residues under purifying selection. Statistically significant diversifying selection was detected globally in ALT-2 of <i>L</i>. <i>loa</i> (<i>P</i><0.001) and <i>W</i>. <i>bancrofti</i> (<i>P</i><0.05). Residues encompassing the signal peptide are beneath the solid lines, and those encompassing the VAD are beneath the dashed lines.</p

ALT-2 Evolvability in Filarial Parasites.

<p>Calculated evolvability (E) values (Y axis) are presented for each amino acid residue (X axis) for ALT-2 from the indicated pairs of species using the calculated ω values from each organism at each site. Cutoffs for significant deviations between conserved (normal distribution) and diversified sequences are shown as red lines (<i>P</i><0.05 or <i>P</i><0.001); values extended above the line represent residues that are significantly evolvable. Goodness-of-fit analyses between the two conserved ALT-2 sequences (<i>O</i>. <i>volvulus</i> and <i>B malayi</i>, <b>A</b>) and the two diversified ALT-2 sequences (<i>L</i>. <i>loa</i> and <i>W</i>. <i>bancrofti</i>, <b>B</b>) served as positive and negative controls, respectively. Multiple residues of <i>L</i>. <i>loa</i> were significantly evolvable when examined for deviation from <i>B</i>. <i>malayi</i> (<b>C</b>) or <i>O</i>. <i>volvulus</i> (<b>E</b>). Multiple residues of <i>W</i>. <i>bancrofti</i> were significantly evolvable when examined for deviation from <i>B</i>. <i>malayi</i> (<b>D</b>), but not <i>O</i>. <i>volvulus</i> (<b>F</b>).</p

Antigenicity and Selection Projection.

<p>Calculated ω values using the M8 model (Y axis, bar graph) are projected onto graphical measurements of antigenicity predictions (Z axis, line graph) across all sites in ALT-2 (X axis, marked by position number). Predicted antigenic epitopes are denoted with arrows. The cutoff ω value of 1 is depicted as a black line; values extended above the line represent residues under diversifying selection, and values below the line represent residues under purifying selection. Areas under diversifying selection in ALT-2 of <i>L</i>. <i>loa</i> and <i>W</i>. <i>bancrofti</i> have minimal overlap with predicted antigenicity.</p