Dottorato in MALATTIE INFETTIVE, MICROBIOLOGIA E SANITA' PUBBLICA

Titolo della tesi: Aedes albopictus 34k2 salivary protein as epidemiological tool for the assessment of human exposure to the tiger mosquito

Arthropod-borne viruses (arboviruses) such as dengue, chikungunya and Zika have nowadays a large impact on the human population worldwide. Their public health relevance significantly increased in recent decades, both due to their re-emergence in tropical areas and to their appearance in more temperate regions. These arboviruses are transmitted between humans by the bite of infected Aedes mosquitoes, mainly Aedes aegypti and Aedes albopictus. Their rapid worldwide spreading, especially of the tiger mosquito Ae. albopictus, is expanding the risk of arboviral transmission also to temperate areas and emphasize the need for improved monitoring and control. Evaluating human- vector contact is essential to assess the risk of transmission of Aedes mosquito-borne diseases and to guide planning and implementation of vector control. This is currently achieved by classical entomological measurements (ovitraps, larval/pupal indices, adult traps, human landing catches), although they provide only an indirect evaluation and have their drawbacks and limitations. However, transcriptome studies on blood feeding insects salivary glands highlighted the existence of mosquito genus-specific salivary proteins, which may be ideal candidates for the development of novel tools to evaluate human exposure to vector bites. In fact, while feeding on their hosts, blood sucking arthropods inject a cocktail of salivary proteins with anti-hemostatic and anti-inflammatory activities. These salivary proteins whose also evokes in humans specific antibody responses, whose measurement can provide a direct evaluation of host exposure to disease vectors. The culicine-specific 34k2 salivary protein from Ae. albopictus (al34k2) and its ortholog from Ae. aegypti (ae34k2) were here evaluated as novel specific biomarkers of human exposure to Aedes mosquitoes. Evidence of al34k2 and ae34k2 immunogenicity was first obtained in a murine model, and preliminary indications of immunogenicity to humans acquired through the use of a single human serum hyperimmune to Ae. albopictus saliva. The al34k2 antigen was then validated measuring by ELISA the anti-al34k2 IgG responses in sera collected from healthy individuals naturally exposed to the tiger mosquito in two areas of Northeast Italy (Padova and Belluno), during two different time periods (at the end of the low- and shortly after the high-density mosquito seasons). IgG responses to al34k2 appeared suitable to evaluate spatial and temporal variation of human exposure to Ae. albopictus. Analysis of IgG1 and IgG4 subclasses suggested that the al34k2 protein evokes in naturally exposed individuals an IgG1-dominated antibody response that may be indicative of a Th1-type polarization. A tendency of the anti-al34k2 IgG response to decrease with age was also found. This trend was evident in individuals from Padova but only hardly detectable in those from Belluno, possibly because of the different history of colonization of the study areas. Finally, to get further insights into the species-specificity of IgG responses to 34k2 salivary proteins and to validate them in epidemiological settings with ongoing arboviral transmission, the IgG responses to the al34k2 and the ae34k2 salivary antigens were measured in cohorts of individuals from the Réunion Island (only exposed to Ae. albopictus) and from Bolivia (only exposed to Ae. aegypti). Individuals from Réunion Island showed significantly higher IgG responses to al34k2 than to ae34k2 validating this antigen as a good and specific marker in an endemic area where Ae. albopictus is present. On the contrary, ae34k2 IgG responses showed in both areas a low specificity and a relatively high background, perhaps due to cross-reactivity with some unknown antigen. The results reported in this thesis emphasize the potential use of the IgG antibody responses to the al34k2 salivary protein as a specific biomarker of human exposure to Ae. albopictus. Serological assays as the one described here have the advantage of providing a direct measure of human vector contact and may be useful to assess the efficacy of vector control interventions. Such a complementary tool can also be employed for epidemiological studies and possibly for estimation of transmission risk.