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Immunologic Responses to an Extracellular Vesicle-Based Vaccine Expressing the Full Suite of SARS-CoV-2 Structural Proteins

초록/요약

The emergence of severe acute respiratory syndrome coronavirus 2 （SARS-CoV-2） and the resulting COVID-19 pandemic highlighted the need for vaccine platforms capable of inducing broad humoral and cellular immune responses in a murine model, as assessed by pseudotyped virus neutralization and antigen-specific T cell stimulation assays. Extracellular vesicles （EVs） have emerged as promising vaccine delivery vehicles because of their biocompatibility, intrinsic stability, and ability to efficiently present antigens to the immune system. In this study, a novel EV-based SARS-CoV-2 vaccine was developed by engineering EVs to simultaneously express the four structural proteins of SARS-CoV-2: spike （S）, nucleocapsid （N）, membrane （M）, and envelope （E）. The spike protein incorporated receptor-binding domain （RBD） sequences derived from the BA.2 and BA.5 variants while maintaining a prefusion-stabilized spike backbone. Stable producer 293T cells expressing viral structural proteins were generated, and engineered EVs were isolated from conditioned culture media. Biochemical and structural analyses confirmed the successful incorporation of spike trimers, nucleocapsid, membrane, and envelope proteins into the EVs. The immunogenicity of the EV-based vaccine was evaluated in mice following intramuscular immunization with 10, 25, or 50 μg of EVs. Vaccination induced antigen-specific IgG responses against both spike and nucleocapsid proteins, which were significantly enhanced following booster immunization. Furthermore, sera from immunized mice demonstrated neutralizing activity against SARS-CoV-2 spike pseudotyped vesicular stomatitis virus （VSV）. Cellular immune analyses revealed the induction of antigen-specific CD4⁺ and CD8⁺ T cell responses, including memory and polyfunctional T cell populations. These responses were most pronounced in the 25 and 50 μg immunization groups. In conclusion, this study demonstrates the feasibility of an EV-based multi-antigen SARS-CoV-2 vaccine platform capable of inducing broad humoral and cellular immune responses in mice. Simultaneous presentation of the four structural proteins of SARS-CoV-2 through EVs represents a promising strategy for broadening vaccine- induced immunity and supports the further development of EV-based vaccines against SARS-CoV-2 and other emerging infectious diseases. Keywords: SARS-CoV-2; EVs; Multi-Antigen Vaccine; Prefusion-Stabilized Spike; Cellular Immunity; Humoral Immunity; Neutralizing Antibodies; COVID-19 Vaccine.

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목차

I- INTRODUCTION 1
1. Overview of SARS-CoV-2 1
2. Vaccines for SARS-CoV-2 2
3. Limitations of the developed vaccines 4
4. Need for Multi-Antigen Vaccine Strategies 5
5. Introduction to EVs 6
6. EVs from Dying Cells 8
7. Incorporation of M and E proteins 9
8. EV-Based Vaccines against SARS-CoV-2 10
9. Study Rationale and Knowledge Gap 11
10. Hypothesis 13
11. Aim and Objectives 14
II- MATERIALS AND METHODS 15
1. Cells, antibodies, and other reagents 19
2. Expression constructs of SARS-CoV-2 structural proteins and lentiviral transduction 20
3. 3D modeling of SARS-CoV-2 spike fusion protein 21
4. Preparation of cell lysates and western blots 21
5. Preparation of EVs 22
6. Nanoparticle tracking analysis (NTA) 23
7. Transmission electron microscopic analysis of EVs 23
8. Confocal microscopy 24
9. Flow-cytometric analysis of EVs 24
10. Sandwich enzyme-linked immunosorbent assay 25
11. OptiPrep density gradient centrifugation 26
12. Immunization of mice and collection of samples 26
13. Indirect Enzyme-linked immunosorbent assay 27
14. T cell immune responses in splenocytes 28
15. Pseudotyped virus preparation and neutralization assay 29
16. Statistical analyses 30
III- RESULTS 31
1. Design and Validation of Prefusion-Stabilized SARS-CoV-2 Spike Constructs 31
1.1. Multiple mutations to generate the stable prefusion form of spike 31
1.2. 3D modeling to ensure the formation of stable trimeric structure 32
1. 3. Spike glycoprotein expression in the form of both trimer and monomer 32
2. Characterization of Extracellular Vesicles Expressing SARS-CoV-2 Structural Proteins 39
2.1. Establishing lentiviral expression of structural proteins 39
2.2. Expression of spike glycoprotein in cytosol. 40
2.3. Quantitative expression of spike proteins in EVs and cell lysates 40
2.4. Morphology and size distribution of the EVs expressing spike glycoprotein. 42
2.5. Quantification of amount of spike protein loaded onto EVs 43
3. Humoral Immune Responses Induced by EV-Based SARS-CoV-2 Vaccination 53
3.1. Schedule of the immunization and sampling of mice 53
3.2. Antibody responses against nucleocapsid were more robust than spike proteins 54
3.3. Booster-immunized mice demonstrated higher neutralizing activity against the spike proteins 55
4. T cell responses to spike and nucleocapsid protein peptide pools 60
5. Induction of polyfunctional effector/memory T cells via immunization with EVs vaccine expressing SARS-CoV-2 structural proteins 76
6. T cell responses to membrane and envelope glycoproteins in mice immunized with EVs expressing structural proteins of SARS-CoV-2 78
IV- DISCUSSION 90
V- LIMITATIONS 95
VI- CONCLUSION 98
REFERENCES 100

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