Assessment of antibody responses to Anopheles SG6-P1 and Aedes N-term 34kDa salivary peptides: a human-challenge trial of controlled exposures to vector bites

Summary Background Human antibodies against mosquito salivary proteins are proposed as proxy biomarkers of exposure to vector bites. This trial sought to characterise the boosting and decay dynamics of antibodies against Anopheles SG6-P1 and Aedes N-term 34kDa salivary peptides in a human challenge model of controlled exposure to the main Southeast Asian malaria and global dengue vectors. Methods In this single-centre, open-label, randomised, exploratory factorial trial, healthy volunteers aged 18-60 years with no history of recent travel to rural areas were recruited in Mae Sot, Thailand (ClincalTrials.gov: NCT04478370 ). Participants were randomly assigned to receive either 35 or 305 bites of mosquitos of laboratory-adapted colonies of An. dirus , An. maculatus , An. minimus , Ae. aegypti , and Ae. albopictus using a block randomisation schedule. Samples were collected weekly before, during and after the challenges for 16 weeks. The primary endpoint was total IgG antibodies against Anopheles SG6-P1 peptides measured using high-throughput ELISA and analysed with Generalized Estimating Equations. Outcome assessors were masked to the intervention groups. Findings Between January 21, 2021, and May 10, 2022, 248 volunteers were screened, of whom 210 were randomly assigned to receive either 35 or 305 bites of Ae. aegypti (n=20 and n=19, respectively), Ae. albopictus (n=20, n=21), An. dirus (n=21, n=21), An. maculatus (n=23, n=24), or An. minimus (n=22, n=19), comprising the intention-to-treat population. In participants exposed to 305 An. minimus bites, total anti-gSG6-P1 IgG levels increased 1.14-fold (95%CI: 1.03-1.26) and 1.18-fold (95%CI: 1.05-1.33) during the exposure and post-exposure periods respectively (relative to baseline), with minimal or no boosting observed in other groups. The estimated half-life of anti-gSG6-P1 antibodies was 421 (95%CI: 155-688) days. Seven participants were withdrawn due to an adverse event. Interpretation Anti-gSG6-P1 antibodies were boosted in response to exposure to 305 bites of An. minimus but the magnitude of boosting was small and antibodies decayed slowly. Future research is warranted to identify and validate serological markers of vector biting exposures. Funding Wellcome Trust, NHMRC. Research in Context Evidence before this study Human antibodies against mosquito salivary proteins have been investigated as serological biomarkers of exposure to bites of mosquitos that transmit malaria ( Anopheles ) and dengue ( Aedes ), however their associations with and dynamics following biting exposures remain unknown. On June 3 2020, we searched published articles in PubMed and MEDLINE using the search terms ((Anophel* OR Aede*) AND saliva* AND (antibod* OR sero* OR antigen OR marker* OR biomarker*)). We systematically reviewed studies investigating the An. gambiae salivary gland protein 6 (gSG6), its derivative gSG6-P1 peptide, or the Ae. aegypti N-term 34kDa peptide as an outcome measure of biting exposures. We used multilevel modelling to assess the association between population-level anti-gSG6 IgG antibody seroprevalence and Anopheles human-biting rates reported in 12 studies. The results showed that seroprevalence and vector biting rates are positively associated and that this association is stronger in African settings where An. gambiae is the only dominant vector species than in areas where An. gambiae is absent. Five studies investigated anti- Ae. aegypti N-term 34kDa IgG antibody responses but the associations with Aedes biting rate were not assessed. This review also identified a knowledge gap on the association between antibodies against either gSG6 or Ae. aegypti N-term 34kDa and human-biting rates measured at the individual level or under conditions of controlled exposure to accurately quantify boosting and decay dynamics. Added value of the study This is the first assessment of boosting and decay dynamics of antibody responses directed against mosquito salivary antigens in a human challenge model of controlled exposure to vector bites. Small boosts of long-lasting antibodies directed against gSG6-P1 and Ae. aegypti N-term 34kDa peptides, as well as orthologous peptides designed using published sialomes of An. minimus , An. maculatus , An. dirus and Ae. albopictus , were detected in response to the study challenges. This innovative trial design allows determination of the dose-response relationship between mosquito biting exposures and antibody responses, the rate of antibody decay, and the cross-reactivity of anti-salivary antibody responses across species of biting exposure, thereby providing crucial information for the validation of antibodies against mosquito saliva as a quantitative outcome measure of recent human-vector contact. Implications of the available evidence This study demonstrates minimal boosting and slow decay of antibodies to Anopheles SG6-P1 and Aedes N-term 34kDa salivary antigens following controlled biting exposures in a cohort of participants with relatively high levels of baseline seroreactivity against those peptides. These findings suggest somewhat limited utility of anti-salivary antibodies to measure changes in individual-level biting exposures from these mosquito species over short periods of time, however, their utility to measure population level exposure over longer periods of time is yet to be determined. This will impact how these serological biomarkers can be applied to evaluate the effectiveness of vector control interventions or for serosurveillance, whereby larger samples sizes or longer follow up may be required to accurately capture boosting and decay dynamics.