Two mosquito salivary antigens demonstrate promise as biomarkers of recent exposure to P. falciparum-infected mosquito bites

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 <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.

Methods
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).