Mapeamento de epítopos e produção de proteínas quiméricas como estratégia para o desenvolvimento de uma vacina multiepítopo contra Schistosoma mansoni

Abstract

Schistosomiasis is an endemic disease in 78 countries and presents the highest morbidity among helminth infections. Therefore, an effective vaccine against this disease would be a valuable addition to current control strategies and, ultimately, to its elimination. Most vaccines investigated to date have been based on single antigens, and none have reached the final stages of development. We believe that using multiple immunogenic epitopes associated with protective immune responses represents a more promising approach for developing an effective vaccine. It is possible to identify protection-associated epitopes in murine models immunized with attenuated cercariae and in self-curing rhesus macaques. Additionally, in endemic areas, some individuals develop resistance to reinfection after multiple treatments with praziquantel (PZQ). This thesis is divided into four chapters. In Chapter 1, a systematic review was conducted using articles indexed in PubMed, totaling 396 studies evaluated with respect to various aspects of vaccine development against schistosomiasis. It was observed that the emergence of new technologies has significantly impacted preliminary studies in animal models. The majority of published studies used S. mansoni, with over 80% employing mice as the animal model. Furthermore, most of the proteins evaluated originated from the parasite’s tegument. In Chapter 2, epitope mapping was performed in rhesus macaques experimentally infected with S. mansoni. The animals were classified as high or low responders according to the rate of parasite clearance. Subsequently, sera from these animals were used in peptide microarray assays targeting proteins at the parasite–host interface (tegument, esophageal gland, and gut lining). The targeted epitopes were similar between the groups; however, the intensity of the immune response was greater in high responders. Overall, animals exhibited a stronger response against gastrointestinal tract-associated antigens. Based on these findings, 18 candidate epitopes were selected for further analysis. In Chapter 3, a convenience study was conducted in the endemic region of Conde-BA, involving individuals classified as resistant to reinfection (RR) and susceptible to reinfection (SR) after PZQ treatment. Serological analysis revealed that RR individuals had significantly higher levels of IgG1 against tegumental membrane extract (SmTEG). Epitope mapping using microarrays identified three epitopes preferentially recognized by the RR group, located in the tegumental proteins Sm25 and ADP- ribosyl cyclase. When compared to animal models (mice immunized with attenuated cercariae and infected rhesus macaques), 4 shared epitopes were identified across all approaches, which warrant further investigation. In Chapter 4, three chimeric proteins were designed (P1 – Esophageal gland, P2 – Tegument, P3 – Gastrodermis) each containing epitopes previously mapped in the murine model immunized with attenuated cercariae. The proteins were expressed recombinantly and purified via nickel affinity chromatography. P1 yielded the highest production, while P2 exhibited the highest purity. Initially, these proteins were used to immunize BALB/c mice, aiming to generate polyclonal antibodies through hybridoma production. P1 induced the greatest number of positive clones, while P2 and P3 yielded fewer. Sera from immunized animals were able to recognize native parasite proteins, as demonstrated by immunohistochemistry assays. The next step will involve functional evaluation of these polyclonal antibodies against schistosomula in vitro. Finally, an immunization and challenge assay were conducted in C57BL/6 mice, which received three doses of the chimeric proteins (administered individually or in combination), formulated with Alum as an adjuvant. After 21 days, the animals were challenged with S. mansoni. A robust IgG1 response against the proteins was observed. The group immunized with P1 showed a significantly higher ratio of IgG1 response compared to IgG2c, especially when compared to the P3 and pool groups. There was a trend of reduced parasitic load across all groups. Group P1 showed a significant reduction in egg count and female fecundity, while Group P2 exhibited a significant reduction in the adult worm burden. Therefore, we intend to further investigate the protective potential of these proteins.