자가 증폭 mRNA 플랫폼을 이용한 구제역 백신의 개발 및 효능평가 = Development and Evaluation of the Efficacy of a Foot-and-Mouth Disease Vaccine Using A Self-Amplifying mRNA Platform

다국어 초록 (Multilingual Abstract)

Foot-and-mouth disease (FMD) is a highly contagious viral infection of cloven-hoofed livestock that causes substantial economic losses worldwide. Although chemically inactivated vaccines are effective, they are limited by biosafety concerns related to live virus handling, the requirement for biosafety level 3 (BSL-3) facilities, manufacturing complexity, and lack of DIVA compatibility. To address these limitations, we evaluated a Venezuelan equine encephalitis virus (VEEV)–derived self-amplifying mRNA (saRNA) vaccine platform encoding FMDV antigens. In vitro analyses demonstrated that all three saRNA constructs supported efficient intracellular translation, expressed FMDV antigens, and sustained RNA replication, resulting in detectable RNA and protein expression for up to 120 hours. However, in mice, only the LPX-formulated capsid precursor (CP) saRNA induced FMDV-specific and neutralizing antibody responses comparable to those elicited by a commercial vaccine. Importantly, the efficacy of LPX-formulated CP saRNA was further demonstrated in swine, a primary target species for FMD vaccination. Following booster immunization, LPX-formulated CP saRNA induced humoral immune responses comparable to those of the commercial vaccine and, unlike the commercial vaccine, additionally elicited cellular immune responses. Following FMDV challenge, LPX-formulated CP saRNA provided robust clinical protection in swine, comparable to that of the commercial vaccine, and effectively suppressed viremia and viral shedding. Collectively, these findings demonstrate the potential of LPX-formulated CP saRNA as a promising next-generation vaccine candidate for the control of foot-and-mouth disease.

목차 (Table of Contents)

• CHAPTER 1. FMDV VACCINE OVERVIEW 1
• 1. General information 2
• 2. FMDV vaccine 4
• 2-1. Inactivated vaccine 4
• 2-2. Live attenuated vaccine 5
• 2-3. DNA vaccine 7
• 2-4. Live viral vector vaccine 9
• 2-5. Peptide vaccine 10
• 2-6. Virus-like particle vaccine 12
• 3. Specific aims 14
• Reference 15
• CHAPTER 2. Characterization and immunogenicity assessment of a self-amplifying mRNA encoding Foot-and-mouth disease virus structural proteins 31
• 1. INTRODUCTION 32
• 2. MATERIALS AND METHODS 36
• 2-1. Cells and virus 36
• 2-2. Construction of saRNA encoding FMDV antigens 37
• 2-3. Immunoprecipitation analysis of the interaction between VP2 and a VP2-specific scFv 38
• 2-4. Expression of saRNAs encoding FMDV antigens 39
• 2-5. Western blot analysis 40
• 2-6. Immunofluorescence assay 40
• 2-7. Transmission electron microscopy 41
• 2-8. RNA isolation and quantitative RT-PCR 42
• 2-9. Cellular Replication and Sustained Protein Expression from saRNA in Mammalian Cells 43
• 2-10. Lipid nanoparticle formulation 43
• 2-11. Nanoparticles formulated saRNAs characterization 45
• 2-12. Cytotoxicity test in nanoparticle-formulated saRNA 45
• 2-13. Immunization of mice with nanoparticle-formulated saRNAs encoding FMDV antigens 46
• 2-14. Nanoparticle-formulated saRNAs vaccination and virus challenge in swine 47
• 2-15. Detection of FMDV-specific antibodies in sera 48
• 2-16. Assessment of neutralizing antibody titers using sera 48
• 2-17. Measurement of total IgG and IgM levels in sera by ELISA 49
• 2-18. Cytokine immunoassay from vaccinated swine sera 50
• 2-20. Statistical Analysis 50
• 3. RESULT 51
• 3-1. Construction of saRNAs encoding FMDV antigens 51
• 3-2. Verification of VP2-scFv interaction using immunoprecipitation 52
• 3-3. Functional characterization of saRNAs encoding FMDV antigens 52
• 3-4. Transcriptional and protein expression analysis of saRNAs encoding FMDV antigens in mammalian cell lines 55
• 3-5. Comparative physicochemical profiling of LNP and LPX saRNA delivery systems 56
• 3-6. Comparative analysis of delivery efficiency and functional performance of FMDV saRNAs across delivery platforms 56
• 3-7. Humoral immune responses following inoculation with saRNAs encoding FMDV antigens in mice 58
• 3-8. Humoral and cellular immune responses induced by saRNA vaccination in swine 60
• 3-9. Protective efficacy of saRNA vaccination against FMDV challenge in swine. 62
• 4. DISCUSSION 66
• 6. REFERENCE 101
• 국문초록 111