A Novel RNA Vaccine Platform Using Circular RNA Against Chikungunya

WP3: Preclinical small animal efficacy studies: 01 Nov 2024 – 01 April 2025 Small animal preclinical studies will be performed using the A129 mouse model, which is a commonly utilized small rodent model for CHIKV infection. We will test three circRNA-LNP vaccine candidates with various doses in the mice to allow us to obtain small animal immunogenicity and efficacy data, and to prioritize our vaccine candidates for testing the two best in cynomolgus macaque efficacy studies. For the mouse studies, animals will be observed daily following the prime and boost vaccinations of three separate experimental vaccines and one well-characterized positive control vaccine (CHIKV/IRES), to determine if the vaccines are well tolerated. Animal sera will be collected after the prime and boost vaccinations to determine the levels of neutralizing antibody. At 28 days after the final vaccination, the animals will receive a lethal dose of CHIKV, administered intradermally in the hind left footpad. The animals will be observed throughout the remainder of the study, including footpad swelling measurements, two separate days of live bleeds on days 1-4 after challenge (staggered due to limits on blood collection) to measure viremia, and overall clinical scoring and survival. On day 14 post-infection, the experiment will be terminated, and necropsies will be performed. Protection will be evaluated based on endpoints of prevention of weight loss, cytokine alterations, foodpad swelling, mortality, viremia, clinical disease appearance and histopathologic lesions. Lab safety studies will be performed to assess for any signals of toxicity (this is not a formal tox study). A129 and 129 Immunogenicity studies of three novel vaccines, 10 mice per cohort, 3 doses per vaccine, plus positive control = 200 mice. WP5: Large animal efficacy studies: 01 April 2025 – 31 Jul 2025 These studies will be done using cynomolgus macaques. This nonhuman primate model is well characterized and documented for CHIKV infection, with very reproducible endpoints of fever and viremia. Initially, the animals will be implanted with temperature loggers to record body temperature in nearly real-time. Following the minor surgical procedure, the animals will be given one week to recover, and the vaccinations will proceed if there is no sign of infection. For vaccination, two cohorts immunized with the newly developed experimental vaccines and one mock-vaccinated group will be utilized (N-4 per group). The animals will be given the vaccines in the same prime/boost strategy as utilized in the small rodent model. The animals will be bled and measured for changes in cytokines and observed daily for 4 days to ensure the vaccines are well tolerated. Then, on days 7, 14 and 28 after the final vaccination, IgM, IgG and neutralizing antibodies will be assayed after both prime and boost vaccinations. As with mice, our primary immunogenicity endpoint will be neutralizing antibody titers of 20 or greater on day 28, which correlates with protection in humans. Then 28 days after the final vaccinations, all the animals will receive 104 plaque-forming units of CHIKV administered subcutaneously. The animals will be observed twice each day for clinical scoring. The animals will also be bled daily 1-5 days after challenge to measure viremia, CBC’s and cytokine responses, with the desired endpoints being a lack of significant changes. Animal temperatures will be recorded manually each day via rectal measurement and nearly continuously by a temperature logger with data collected at the end of the study. Historic data from Dr. Weaver’s previously developed CHIKV vaccines will serve as positive controls, or we will add 4 animals. The main endpoints for efficacy will be a significant reduction in viremia and fever. At the termination of the study, animals will undergo necropsy recorded manually each day via rectal measurement and nearly continuously by a temperature logger with data collected at the end of the study. At the termination of the study, animals will undergo necropsy and have various organs and tissues removed for analysis of histopathological lesions. Cynomolgus macaque immunogenicity/efficacy studies of 2 lead vaccines and sham-vaccinated controls, one dose, 4 animals per cohort = 12 animals Immunological large animal efficacy study reports, including measuring IgM and IgG antibody-antigen binding and plaque-reduction neutralization titers 7 and 28 days after one or 2 vaccine doses. Assumptions The University of Texas Medical Branch (UTMB) at Galveston makes certain assumptions regarding the Research Strategy, given that the research-grade experiments will be conducted in a high biocontainment facility (i.e., BSL-4 laboratory) and that UTMB is primarily an academic research institution. The assumptions are: • No GLP studies will be performed. The budget provided by UTMB is not for a stringent study, as work will be done in BSL-4. Any such requirement will necessitate a detailed list of specific requirements and rebudgeting. • Due to the global issue with acquiring NHPs, their purchase price has fluctuated drastically since 2020. The UTMB team will communicate with Houston Methodist and CEPI to ensure sufficient funds are available for NHP purchase prior to the start of WP5.