Phd in ADVANCES IN INFECTIOUS DISEASES, MICROBIOLOGY, LEGAL
MEDICINE AND PUBLIC HEALTH SCIENCES

Thesis title: Temporal and spatial profiling of Aedes albopictus immune responses to chikungunya virus (CHIKV) and characterization of a novel salivary biomarker of human exposure

Vector-borne diseases represent a major global health challenge, accounting for over 17% of infectious diseases and causing more than 700,000 deaths each year. Among these, arboviral infections – including dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) – are of particular concern due to their high pathogenicity and the lack of effective preventive and therapeutic measures. Aedes aegypti and Aedes albopictus are currently the main vectors of these pathogens, and the rapid global expansion of Aedes albopictus further increases its threat to public health. Following the acquisition of an infected blood meal, the virus enters the mosquito midgut, starting a journey inside the vector that requires overcoming several anatomical and immune barriers to survive and reach the salivary glands for transmission to a subsequent vertebrate host. Although mosquitoes lack an adaptive immune system, they are capable of mounting a sophisticated innate immune response against pathogens, underscoring the importance of understanding vector–virus interactions to inform the development of effective vector control strategies. In recent years, studies of vector–vertebrate host interactions have also enabled the development of innovative approaches to monitor human exposition to vector activity and, consequently, the associated risk of arboviral transmission, through the direct measurement of antibody responses to specific mosquito salivary antigens injected during blood feeding. This thesis presents two studies conducted during my doctoral research, both focusing on the vector Aedes albopictus. Chapter 1, entitled “Temporal and spatial profiling of Aedes albopictus immune responses to chikungunya virus (CHIKV) infection”, investigates transcriptional changes in Ae. albopictus in response to CHIKV infection. The study focuses on two key time points: (i) one day post-infection in the midgut, corresponding to the early phase when the mosquito mounts its initial immune response following viral entry into the gut, and (ii) five days post-infection in the hemocoel, representing a later stage when viral dissemination triggers a second wave of immune responses mediated by hemocytes and fat body cells. Adult female Ae. albopictus were experimentally infected with CHIKV; midguts at one day post-infection and carcasses (gut-depleted) at five days post-infection were collected and analyzed by RNA-seq, using mosquitoes fed on uninfected blood as controls. Results showed that the midgut plays a critical role during the early phase, activating immune pathways such as RNA interference, autophagy, and ubiquitination, while also modulating key metabolic processes. At later stages, following viral dissemination into the hemocoel, systemic immune responses predominate, including activation of immune receptors, secretion of antimicrobial peptides, and melanization. RT-qPCR validation of selected genes identified by RNA-seq confirmed their expression profiles. This transcriptomic analysis provides a detailed map of tissue-specific and temporal molecular responses of Aedes albopictus to CHIKV infection. Chapter 2, entitled “A novel biomarker of human exposure to Aedes albopictus based on the Ag5-3 salivary protein from the tiger mosquito”, presents research aimed at validating the salivary antigen alAg5 as a marker of human exposure to Aedes albopictus bites using ELISA. In a previous study conducted in our laboratory (Buezo Montero et al., 2020), the salivary protein al34k2 was validated as a biomarker of human exposure to tiger mosquito bites. In 2017, sera were collected from healthy donors in two areas of the Veneto region (Italy), Padua and Belluno, which differ in Aedes albopictus density, colonization history, and co-occurrence of other Aedes species, allowing for comparative analyses. Sampling was conducted in two phases: before the summer, corresponding to low vector activity, and after the summer, when mosquito exposure peaks. Entomological surveillance data were collected in parallel. Using the same sera, this study measured human antibody responses to the salivary protein alAg5-3, alone or in combination with al34k2, to evaluate its potential as a marker of exposure. A pilot study indicated that alAg5-3 is more immunogenic than al34k2 and suitable for detecting variations in human exposure. Analysis of the full sample set showed that anti-alAg5-3 IgG levels increased significantly after the summer, while differences between the two areas were detected only when both antigens were combined, suggesting that their combination better reflects spatial and temporal exposure variations. Future studies will further clarify the species- and genus-specificity of Ag5-3. In conclusion, both studies contribute to a better understanding of the complex vector–pathogen–human host interactions and provide new perspectives for improving current vector control and arboviral surveillance strategies.