Title Page
Contents
ABSTRACT 9
Chapter 1. Introduction 10
1.1. Literature Review 10
1.2. Introduction 21
Chapter 2. Materials and Methods 25
2.1. Source of SARS-CoV-2 Genome Data 25
2.2. Prediction of Antigenic Proteins 25
2.3. Epitope Mapping 25
2.4. Epitope Analysis 26
2.5. Analysis of SARS-CoV-2 Mutations 27
2.6. Structure Prediction 28
2.7. Cloning, Expression, and Purification of Polypeptide Antigens 29
2.8. Anti-spike SARS/Cov-2 Antibody assay 29
Chapter 3. Results 31
3.1. Reverse-Vaccinology Workflow for Predicting SARS-CoV-2 Antigens 31
3.2. Selection and Analysis of SARS-CoV-2 Proteins as Immune Cell-Responsive Antigens 32
3.3. Analysis of Epitopes Derived from Antigenic SARS-CoV-2 Proteins 35
3.4. Analysis of Mutational Variants in the Selected Epitopes 38
3.5. Structural Insights into Epitopes with Mutations 41
3.6. Cloning, Purification, Evaluation of Potential Protective Antigens Using Serum Samples from Vaccinated and Naturally Infected Individuals 44
Chapter 4. Discussion 47
Chapter 5. Conclusion 53
References 54
Appendices 68
[Appendix 1] B-cell target antigenicity scores 68
[Appendix 2] T-cell target antigenicity scores 68
[Appendix 3] ToxinPred results (B-cell epitopes are in blue, T-cell epitopes are in black, and epitopes with mutations are in red) 68
[Appendix 4] Mutations collected from CoV-GLUE, that are within the selected epitopes (replaced mutations indicated as a red-colored background; intensity indicating density). 70
Abstract (in Korean) 103
Table 1.1. Vaccine examples with their type, development strategy, target pathogen and respective year. 12
Table 3.1. B-cell antigens of SARS-CoV-2 and their corresponding antigenicity scores*, epitope density, number of mutations, and mutation density. 33
Table 3.2. T-cell antigens of SARS-CoV-2 and their corresponding antigenicity scores*, epitope density, allele coverage, number of mutations, and mutation density. 33
Table 3.3. B-cell epitopes of SARS-CoV-2 and their corresponding antigenicity scores 36
Table 3.4. T-cell epitopes of SARS-CoV-2 and their corresponding antigenicity scores, IFN-γ induction, and HLA coverage (CD8+) and/or MCP (CD4+) 37
Table 3.5. Epitopes selected for reanalysis with their respective site mutation densities and the highest frequent replacement. New sequences of epitopes are listed with their antigenicity... 41
Table 3.6. Amino acid sequence of four spike antigens 44
Figure 3.1. Our reverse vaccinology workflow, employed for the in-silico screening of immunogenic SARS-CoV-2 viral epitopes, is illustrated as follows: Data Retrieval: The process... 31
Figure 3.2. Proportion of SARS-CoV-2 mutations, presented as a percentage per protein (Mutations compiled from CoV-GLUE). 39
Figure 3.3. Structural estimating of epitopes featuring amino acid substitutions (Table 3.5) was accomplished using the PEP-FOLD3, which provided their local structural prediction profiles,... 43
Figure 3.4. Recombinant antigens, purified through a two-step process involving mini-Talon and HPLC, were subjected to 10% SDS-PAGE and silver staining. The respective HPLC fraction... 45
Figure 3.5. Binding of antigens to serum antibodies: In the upper panel, the ELISA plate visually demonstrates the interaction between the antigens and antibodies from sera obtained from... 46