(533) Discovery of immune biomarkers for detection of exposure to Anopheles stephensi, an invasive and highly competent Plasmodium falciparum malaria vector

Introduction/Rationale: The recent invasion of Anopheles stephensi (Ans), a highly competent, urbanized vector of Plasmodium, into Africa threatens malaria prevention efforts. Epidemiological studies are necessary to track the mosquitoes. Immunological biomarkers, such as antibody in human plasma targeting Ans-specific salivary antigens, could be the basis of serologic screening assays indicating the invasive mosquito's presence and impact on local transmission. These antigens have been used to search for serological biomarkers of mosquito exposure, but the use of shorter peptides allows non-conserved epitopes to be targeted.

Methods: Plasma from a US-based controlled human malaria infection study, where participants were exposed to repeated Ans bites, and Ans-naïve Malian children were tested on an overlapping peptide microarray with single-amino acid resolution. The microarray included six Ans salivary antigens (ce5, D7, SG1b, SG5, SG6, Trio). Maximum median fluorescence intensity for sequential 10-mers peptides across each protein was used to construct receiver operator characteristic (ROC) curves to determine each peptide’s utility in distinguishing Ans-exposed from Ans-naïve plasma.

Results: Three 10-mers with the highest area under the ROC curve (AUC) were selected. D7290-314, D7182-206, and SG5309-333 and yielded AUCs of 0.77, 0.69, and 0.57, respectively. Using the highest MFI for any peptide among these three as the true signal for each sample yielded an AUC of 0.89. Sequence analysis of each peptide and the surrounding regions confirmed that two (SG5309-333 and D7182-206) are divergent from major African malaria vectors. The third peptide, D7290-314, is much more conserved but differs in two potentially crucial loci.

Conclusion: Sequence homology among Anopheles proteins makes a peptide-level search for serological biomarkers essential. Three peptides from the D7 and SG5 salivary gland antigens are strong candidates for serological assays to monitor Ans range and exposure.