Title Page
Contents
Overall Abstract 15
Ⅰ. Overall Introduction 17
Ⅱ. Chapter 1. Peptides Derived from S and N Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 Induce T Cell Responses: A Proof of Concept for T Cell Vaccines 33
1. Abstract 34
2. Introduction 35
3. Materials and Methods 38
4. Result 49
5. Discussion 69
Ⅲ. Chapter 2. Analysis of the immunostimulatory effects of cytokine-expressing internal ribosome entry site-based RNA adjuvants and their applications 72
1. Abstract 73
2. Introduction 74
3. Materials and Methods 76
4. Result 86
5. Discussion 107
Ⅳ. Chapter 3. Development of mRNA vaccine using ZIKV Envelope domain III 112
1. Abstract 113
2. Introduction 114
3. Materials and Methods 117
4. Result 127
5. Discussion 149
Ⅴ. Chapter 4. ssRNA expressing cytokines induces anticancer effects in an ovarian cancer peritoneal dissemination model 153
1. Abstract 154
2. Introduction 156
3. Materials and Methods 160
4. Result 165
5. Discussion 180
Ⅵ. Overall Discussion 184
Ⅶ. References 189
국문 초록 211
Table 1. Size and zeta potential of mRNA-encapsulated LNPs 78
Table 2. Serum biochemical data for mice immunized with the ssRNA-3×EnvIII 145
Table 3. Hematological data for mice immunized with the ssRNA-3×EnvIII 146
Figure 1. Cap-dependent and internal ribosome entry site-dependent initiation, two alternative mechanisms of translation 19
Figure 2. Generation classification of vaccines 22
Figure 3. Classifications and mechanisms of vaccine adjuvants 24
Figure 4. Molecular targets of adjuvants. 27
Figure 5. mRNA therapeutics in cancer immunotherapy 32
Figure 6. Overview of the 20 peptides derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S and N proteins.... 50
Figure 7. Analysis of T cell activation after immunization with the SARS-CoV-2 20-peptide mixture and ssRNA adjuvant.... 52
Figure 8. Analysis of T cell activation after immunization with the SARS-CoV-2 20-peptide mixture and ssRNA adjuvant.... 55
Figure 9. Analysis of IFN-γ-producing T cells after stimulation with each single peptide. 56
Figure 10. Analysis of T cell activation after immunization with the four SARS-CoV-2 peptides and ssRNA adjuvant.... 59
Figure 11. SARS-CoV-2 peptide-specific CTL responses in vivo. (A, D) Overview of the experimental groups. (B, E) Schedule of mice immunization.... 61
Figure 12. Immunization with SARS-CoV-2 peptides partially reduced the viral titer in hamsters and conferred protection to hACE2 mice against SARS-CoV-2 infection.... 64
Figure 13. Binding prediction of the SARS-CoV-2-derived peptides to human HLA and in vitro CTL response in human PBMCs.... 65
Figure 14. SARS-CoV-2 peptide vaccine does not induce toxicity in C57BL/6 mice. (A) Overview of the experimental groups. (B) Schedule of mice vaccination. (C)... 68
Figure 15. Construction and characterization of EMCV-IRES-based GM-CSF-or IL-18-expressing ssRNA.... 87
Figure 16. Effects of ssRNA-GM-CSF and ssRNA-IL-18 on co-stimulatory molecule expression on DCs and macrophages.... 89
Figure 17. Gating strategy for costimulatory expression on DC and Macrophage 90
Figure 18. T-cell responses increased after treatment with cytokine-expressing ssRNA adjuvants.... 93
Figure 19. Gating strategies used for the immune cell analysis. (A) Gating strategy for the effector memory and effector T cells. (B) Gating strategy... 94
Figure 20. Evaluation of humoral responses after immunization with cytokine-expressing ssRNA and influenza vaccines.... 97
Figure 21. Evaluation of cellular responses after immunization with cytokine-expressing ssRNA and influenza vaccines.... 100
Figure 22. Immunization with cytokine-expressing ssRNAs together with influenza vaccines induced potent protective immune responses.... 103
Figure 23. cytokine production of antigen-stimulated splenocytes from mice immunized with IIV alone or IIV and ssRNA adjuvants after virus infection.... 105
Figure 24. Effects of cytokine-expressing ssRNA adjuvants on the cross-protective immunity in mice... 106
Figure 25. Increased expression of MDA-5 and RIG-I by ssRNA adjuvants 1 × 10⁶ Raw 264.7 cells were transfected with 2.5µg of ssRNA adjuvants and cells... 108
Figure 26. Construction and characterization of ZIKA 3×ENVIII-expressing ssRNA (A) The structure of Zika virus genome and design of ZIKV 3×ENVⅢ as antigen. (B)... 128
Figure 27. Evaluation of humoral immunity induced by ssRNA-3×EnvIII (A) Group design and (B) immunization schedule of mice. (C) The level of ZIKV E-... 129
Figure 28. Evaluation of cellular immunity induced by ssRNA-3×EnvIII (A) Effector memory CD8⁺ and CD4⁺ T cells (TEM) and (B) effector CD8⁺ and CD4⁺...[이미지참조] 131
Figure 29. ssRNA-3×EnvIII provide protective immunity against ZIKV challenge (A) Group design and (B) immunization and challenge schedule of mice. (C) The level... 134
Figure 30. ssRNA-3×EnvIII provide T cell responses in protective immunity against ZIKV challenge... 136
Figure 31. Analysis of long-term immunity by ssRNA-3×EnvIII against ZIKV (A) Group design and (B) immunization and challenge schedule of mice. (C) The... 139
Figure 32. T cell responses of long-term immunity by ssRNA-3×EnvIII against ZIKV (A) Effector memory and (B) effector CD8⁺ and CD4⁺ T cells in the spleen and (C) the... 140
Figure 33. Toxicity and safety test of ssRNA-3×EnvIII (A) Group design and (B) immunized and sacrificed schedule of mice. (C) The levels... 142
Figure 34. The body and organ weight in the toxicity test. n=5. Data represent the mean ± SD. *p≤0.05, **p≤0.01, ***p≤0.005 and ****p≤0.001. 147
Figure 35. Evaluation of the toxicity in vivo. Histological data (H&E staining) obtained in the major organs (brain, heart, kidney,... 148
Figure 36. Construction of ssRNA expressing IL-12sc, IL-15sc, IL-18, and Caspase-1 and in vivo expression of each cytokine... 166
Figure 37. Distribution of ssRNA expression in immune cells in the abdominal cavity C57BL/6 mice were administrated intraperitoneally 50ug of LNP-formulated ssRNA-... 168
Figure 38. Anti-tumor effects induced by ssRNA-IL-12 and ssRNA-IL-12/15/18. C57BL/6 mice were injected with 1x10⁷ ID8-Fluc cells intraperitoneally and after 2... 171
Figure 39. Anti-metastasis induced by ssRNA-IL-12 and ssRNA-IL-12/15/18. (A-C) The luminescence in each organ were measured at the time of sacrifice by in vivo... 172
Figure 40. ssRNA-IL-12 and ssRNA-IL-12/15/18 induced cell population changes in the spleen, ascites and eWAT.... 174
Figure 41. Immunological profiling of T cell responses in spleen, ascites, and eWAT following IL-12-, IL-15-, and IL-18-expressing ssRNA Treatment.... 177
Figure 42. Analysis of NK cell and NKT cell percentages in spleen and ascites following IL-12-, IL-15-, and IL-18-expressing ssRNA Treatment.... 179