DOI: 10.5061/dryad.cz8w9gjbj
title: Two mosquito salivary antigens demonstrate promise as biomarkers of recent exposure to P. falciparum-infected mosquito bites

description: Measuring malaria transmission intensity using the traditional
 entomological inoculation rate is difficult. Antibody responses to
 mosquito salivary proteins such as SG6 have previously been used as
 biomarkers of exposure to Anopheles mosquito bites. Here, we investigate
 four mosquito salivary proteins as potential biomarkers of human exposure
 to mosquitoes infected with P. falciparum: mosGILT, SAMPSP1, AgSAP, and
 AgTRIO. We tested population-level human immune responses in
 longitudinal and cross-sectional plasma samples from subjects with known
 P. falciparum infection from low and moderate transmission areas in
 Senegal using a multiplexed magnetic bead-based assay. AgSAP and
 AgTRIO were the best indicators of recent exposure to infected mosquitoes,
 with antibody responses to AgSAP in a moderate endemic area, and to AgTRIO
 in both low and moderate endemic areas, significantly higher than healthy
 non-endemic control cohort (p-values = 0.0245, 0.0064, and &lt;0.0001
 respectively). No antibody responses significantly differed between the
 low and moderate transmission area, or between equivalent groups during
 and outside the malaria transmission seasons. For AgSAP and AgTRIO,
 reactivity peaked 2-4 weeks after clinical P. falciparum infection and
 declined 3 months after infection. Reactivity to both AgSAP and
 AgTRIO peaked after infection and did not differ seasonally nor between
 areas of low and moderate transmission, suggesting reactivity is due to
 exposure to infectious mosquitos or recent biting rather than general
 mosquito exposure. Kinetics suggest reactivity is relatively short-lived.
 AgSAP and AgTRIO are promising candidates to incorporate into multiplexed
 assays for serosurveillance of population-level changes in P.
 falciparum-infected mosquito exposure.
description: Multiplex assay We tested IgG responses to
 AgSAP, AgTRIO, mosGILT, and SAMSP1 in a magnetic bead-based assay on the
 Bio-rad Multiplex using the CDC multiplex assay. Plasma samples and
 controls were diluted 1:400 in assay buffer (PBS, 0.05% Tween20, 0.5% BSA,
 0.5% PVA, 0.5% PVP, 0.02% NaN3, 5% casein). For DBS samples, 6 mm punches
 were eluted in 200 μl of assay buffer 4,34. Since this results in the
 equivalent of a 1:40 plasma dilution, the eluted DBS were further diluted
 1:10 in assay buffer for a final equivalent concentration of
 1:400. Five hundred beads per bead region for each of
 the four antigens (each antigen has beads in a specific bead region) in 50
 μl assay buffer were added to each well, the plate was washed twice in PBS
 with 0.05% Tween20, and 50 μl of the diluted sample (plasma or DBS) was
 added. 50 μl of 0.4% biotin-labeled anti-human IgG (Southern Biotech),
 0.16% biotin-labeled anti-human IgG4 (Invitrogen), and 1% Streptavidin-PE
 (Invitrogen) in assay buffer was added to each well. Plates were shaken
 for 60 minutes at 700 rpm, washed 4 times, and 50 μl of assay buffer was
 added per well to ensure unbound proteins were eliminated. Plates were
 further shaken for 30 minutes at 700 rpm, washed twice, and 100 μl of PBS
 was added to each well. Plates were shaken at 1000 rpm for 30 seconds and
 then read on the Bio-Plex 200 in combination with Bio-Plex Manager
 software version 6.1 (Bio-Rad Laboratories). Samples
 were tested in duplicate and the average mean florescent intensity (MFI)
 was calculated by: MFIave = (MFI1 + MFI2 - MFIblank1 - MFIblank1)/2, where
 MFI1 and MFI2 represent the sample duplicates and MFIblank1 and MFIblank1
 represent the blank duplicates. Positive controls (pooled plasma from Mali
 and pooled plasma from Kédougou, Senegal) and negative controls (healthy,
 unexposed US controls) were included on each plate. 
 ELISA On a subset of samples, a standard
 enzyme-linked immunosorbent assay (ELISA) testing for AgSAP and AgTRIO IgG
 was conducted as described previously for comparison with the multiplex
 cytometric bead assay. Plates were read at 450nm and 570nm, and the
 results of each well were calculated as the result at 450nm minus the
 result at 570 to remove the background. For each plate, the average
 optical density (OD) of blanks was calculated and subtracted from the OD
 of the sample. Samples were tested in duplicate and the mean OD was
 calculated. Plates were tested with positive controls (pooled plasma from
 Mali and pooled plasma from Kédougou, Senegal) and negative controls
 (healthy US controls).
description: # Two mosquito salivary antigens demonstrate promise as biomarkers of
 recent exposure to *P. falciparum*-infected mosquito bites
 [https://doi.org/10.5061/dryad.cz8w9gjbj](https://doi.org/10.5061/dryad.cz8w9gjbj) The data contains multiplex serology data for 4 mosquito salivary antigens (AgSAP, AgTRIO, mosGILT, and SAMSP1) and the Sg6 peptide. Samples were tested in duplicate and the average mean florescent intensity (MFI) was calculated by: MFIave = (MFI1 + MFI2 - MFIblank1 - MFIblank1)/2, where MFI1 and MFI2 represent the sample duplicates and MFIblank1 and MFIblank1 represent the blank duplicates. Positive controls (pooled plasma from Mali and pooled plasma from Kédougou, Senegal) and negative controls (healthy, unexposed US controls) were included on each plate. ELISA data is also included for AgSAP and AgTRIO. For each plate, the average optical density (OD) of blanks was calculated and subtracted from the OD of the sample. Samples were tested in duplicate and the mean OD was calculated. Plates were tested with positive controls (pooled plasma from Mali and pooled plasma from Kédougou, Senegal) and negative controls (healthy US controls) * Date of data collection: (2017-06-01 to 2023-08-31)  * Geographic location of data collection: Kédougou, Senegal; Thiès, Senegal; Connecticut, USA ## Description of the data and file structure Lapidus_Mosquito_salivary_antigen_multiplex_Dryad.xlsx and Lapidus_Mosquito_salivary_antigen_multiplex_SG6_Dryad.xlsx describe all Multiplex and ELISA data in the study. This data file contains the data associated with this study, separated by tabs with a Data Dictionary preceding the data tab of interest.  Missing data is denoted as "NA" ## Version Changes On October 14, 2025 an additional file was added to the dataset including data for the SG6 peptide multiplexed with the 4 mosquito salivary antigens (AgSAP, AgTRIO, mosGILT, and SAMSP1). ## Sharing/Access information NA