Anopheles gambiae salivary proteins as epidemiological markers of human exposure to malaria vectors: humoral response to the gSG6 and cE5 proteins in a malaria hyperendemic area of Burkina Faso

Hematophagous arthropods saliva injected into hosts during blood feeding evokes a humoral response which may be used as marker of human exposure to disease vectors and, therefore, may be a useful tool to evaluate disease risk and to monitor efficacy of vector control programs. We have previously analyzed the IgG response to the anopheline-specific salivary protein gSG6 in a population from a malaria hyperhendemic area of Burkina Faso and reported that the Anopheles gambiae gSG6 may be exploited as marker of human exposure to the three main Afrotropical malaria vectors: An. gambiae, An. arabiensis and An. funestus (Rizzo C et al, 2011 PLoS ONE, 6: e17980; Rizzo C et al, 2011, Malaria J, 10: 206). More recently we characterized the An. gambiae salivary protein cE5, a highly specific thrombin inhibitor of the anophelin family (Ronca R et al, 2012 Insect Biochem Mol Biol - in press). AIM. (i) evaluate immunogenicity to humans of the cE5 protein; (ii) compare IgG, IgG1 and IgG4 response to the An. gambiae salivary proteins gSG6 and cE5; (iii) measure the seasonal variation of the anti-cE5 IgG response. MATERIALS AND METHODS. Surveys were carried out in the village of Barkoumbilen, a malaria hyperendemic area of Burkina Faso ~35 km NE of Ouagadougou where the main malaria vectors are An. gambiae and An. funestus. Antibody levels (IgG, IgG1 and IgG4) were measured by ELISA in the sera of exposed individuals (Rimaibé and Mossi ethnic groups, n=208 or n=121) and of European unexposed controls (n=59 or n=68) at the beginning (Aug’94) and at the end (Oct’94) of the high transmission/rainy season, as well as during the following low transmission/dry season (Mar’95). RESULTS. Despite the individual heterogeneity cE5 was more immunogenic than gSG6. Consistently with a previous report (Rizzo C et al, 2011 PLoS ONE, 6: e17980) the anti-gSG6 IgG response was high in children and progressively decreased with age, most likely because of tolerance due to prolonged exposure. On the contrary the anti-cE5 humoral response showed a trend to increase during adulthood. Intriguingly, the humoral response to these two proteins also differed for the dominant IgG subclass: the response to gSG6 was characterized by high IgG4 levels (IgG4>IgG1), whereas in the case of cE5 IgG1 was largely the prevalent antibody (IgG1>>IgG4). Finally, there was no significant variation of the anti-cE5 IgG response between the rainy (high transmission) and the dry (low transmission) seasons. CONCLUSIONS. The relatively long-lasting nature of the anti-cE5 humoral response points out that cE5 is not a good candidate as marker of human exposure to malaria vectors. However, taking into account that IgG1 and IgG4 antibodies are often considered as markers of Th1- and Th2-type responses, respectively, we conclude that our study allowed to identify two salivary antigens with strikingly different properties. On one side gSG6 seems to triggers a short-lived response of the Th2-type, with high IgG4 levels and induction of tolerance. On the other side cE5 apparently elicits a Th1-type response which lasts longer and is characterized by high IgG1 levels and no development of tolerance. Previous reports in murine malaria models indicated that pre-immunization with Anopheles saliva may provide protection from Plasmodium infection by up-regulation of Th1-type response (Donovan MJ et al, 2007 Infect Immun, 75: 2523; Fonseca L et al, 2007 Malaria J, 6: 77). In this context the An. gambiae gSG6 and cE5 salivary proteins may represent useful tools to study both the effects of mosquito saliva on early host response to Plasmodium infection and the mechanisms underlying the development of tolerance to insect saliva.