Novel peptide marker corresponding to salivary protein gSG6 potentially identifies exposure to Anopheles bites

Abstract

Background: In order to improve malaria control, and under the aegis of WHO recommendations, many efforts are being devoted to developing new tools for identifying geographic areas with high risk of parasite transmission. Evaluation of the human antibody response to arthropod salivary proteins could be an epidemiological indicator of exposure to vector bites, and therefore to risk of pathogen transmission. In the case of malaria, which is transmitted only by anopheline mosquitoes, maximal specificity could be achieved through identification of immunogenic proteins specific to the Anopheles genus. The objective of the present study was to determine whether the IgG response to the Anopheles gambiae gSG6 protein, from its recombinant form to derived synthetic peptides, could be an immunological marker of exposure specific to Anopheles gambiae bites.

Methodology/principal findings: Specific IgG antibodies to recombinant gSG6 protein were observed in children living in a Senegalese area exposed to malaria. With the objective of optimizing Anopheles specificity and reproducibility, we designed five gSG6-based peptide sequences using a bioinformatic approach, taking into consideration i) their potential antigenic properties and ii) the absence of cross-reactivity with protein sequences of other arthropods/organisms. The specific anti-peptide IgG antibody response was evaluated in exposed children. The five gSG6 peptides showed differing antigenic properties, with gSG6-P1 and gSG6-P2 exhibiting the highest antigenicity. However, a significant increase in the specific IgG response during the rainy season and a positive association between the IgG level and the level of exposure to Anopheles gambiae bites was significant only for gSG6-P1.

Conclusions/significance: This step-by-step approach suggests that gSG6-P1 could be an optimal candidate marker for evaluating exposure to Anopheles gambiae bites. This marker could be employed as a geographic indicator, like remote sensing techniques, for mapping the risk of malaria. It could also represent a direct criterion of efficacy in evaluation of vector control strategies.

Figure 1. IgG antibody response specific to gSG6 recombinant protein.

The IgG antibody level was evaluated in children (n = 16) living in an endemic area for malaria. Individual ΔOD results (ΔOD as described in the “Materials and Methods” section) at the peak of the season of Anopheles exposure (September) are reported. Samples are ordered according to the intensity of the individual ΔOD value.

Figure 2. Amino-acid sequence of gSG6 Peptides.

Amino-acid sequence of the SG6 protein of Anopheles gambiae (gi:13537666) is presented and sequences of the selected peptides, gSG6-P1 to gSG6-P5, are underlined. Signal peptide (SP) sequence is indicating by dotted underline.

Figure 3. IgG antibody response according to gSG6 peptides.

For each gSG6 peptide, the IgG antibody level was evaluated in a subsample of children living in the studied area. Results at the peak of the season of Anopheles exposure are reported according to gSG6 peptides. Results are presented by box plot graph where lines of the boxes represent the 75th percentile, median and 25th percentile of individual average ΔOD values; whiskers represent the lower and upper adjacent values.

Figure 4. IgG response to gSG6-P1 and gSG6-P2 according to intensity of exposure to Anopheles gambiae bites.

Individual ΔOD values in September (peak of the season of Anopheles exposure) are shown for the three different exposure groups. Results are presented for the same children (n = 241) for gSG6-P1 (A) and gSG6-P2 (B). Exposure groups were defined by entomological data. Bars indicate median value for each exposure group. Statistical significance between the 3 groups is indicated (non-parametric Mann-Whitney U-test).