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Development of Messenger RNA platform-based Zika virus vaccine candidate encapsulated in Lipid nanoparticle

  • 김기수 (서강대학교 일반대학원)

초록 (요약문)

Zika virus (ZIKV), a member of the Flaviviridae family transmitted by mosquitoes, is known to cause microcephaly in newborns and Guillain–Barré syndrome in adults. Global concern regarding ZIKV intensified following the significant outbreak in the Americas in 2014; however, a vaccine for ZIKV infection remains unavailable. In this study, I developed a ZIKV mRNA vaccine candidate encoding the pre-membrane and envelope (prM-E) glycoproteins of the ZIKV strain from French Polynesia (H/PF/2013). The ZIKV prM-E mRNA was encapsulated within lipid nanoparticles (LNPs), which are a well-established delivery vehicle for mRNA vaccines. When the ZIKV mRNA vaccine candidate was transfected into human embryonic kidney (HEK) 293T cells, the mRNA was successfully translated into protein, resulting in the formation of virus-like particles (VLPs). Furthermore, in 6-week-old female C57BL/6 mice that were intramuscularly administered two doses of 2 µg, 10 µg, or 20 µg of the mRNA at two-week intervals, my ZIKV vaccine candidate elicited the production of envelope-specific binding antibodies and neutralizing antibodies in all dosage groups. These results indicate that my ZIKV mRNA vaccine candidate possesses immunogenicity and has the potential to protect humans against ZIKV infection. In addition, further experiments have observed that beta-tropomyosin2 and 3 (TPM2 & TPM3) UTR increase luciferase protein expression compared to the previously used hemoglobin beta (HBB) UTR, and have found the potential to improve the immunogenicity of ZIKV mRNA vaccine candidate.

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초록 (요약문)

Flaviviriadae family에 속하고 모기에 의해 전염되는 지카 바이러스(ZIKV)는 신생아에서 소두증을 유발하고 성인에서 길랑-바레 증후군을 유발한다고 알려져 있다. 2014년 아메리카 대륙에서 심각한 발병이 발생한 이후 ZIKV에 대한 전세계적인 우 려가 대두되었지만, 그에 대한 백신은 현재까지 상용화되어있지 않다. 본 연구에서 는 ZIKV strain French Polynesian (H/PF/2013)의 전막(pre-membrane) 및 외피(envelope) 단백질(prM-E) 을 코딩하는 ZIKV mRNA 백신 후보물질을 개발하고자 하였다. 제작 된 ZIKV prM-E mRNA는 mRNA 백신용으로 잘 확립된 전달수단인 지질나노입자 (Lipid nanoparticle)로 캡슐화되었다. ZIKV mRNA 백신 후보물질을 인간 배아 신장 (HEK) 293T 세포에 이식했을 때, mRNA가 단백질로 성공적으로 번역되어 바이러스 유사 입자(VLP)를 형성하는 것으로 관찰되었다. 또한 6주령 암컷 C57BL/6 생쥐에 2 µg, 10 µg, 20 µg의 mRNA를 2주 간격으로 총 2회 근육주사로 투여한 결과, 모든 투여 량 그룹에서 ZIKV envelope 단백질에 특이적인 결합 항체와 중화 항체가 검출되었 다. 이러한 결과는 개발한 ZIKV mRNA 백신 후보 물질이 면역원성을 가지며 ZIKV 감염으로부터 인간을 보호할 가능성이 있음을 나타낸다. 또한 추후 실험을 통해 beta-tropomyosin2와 3(TPM2 & TPM3) UTR이 기존에 사용한 hemoglobin beta (HBB) UTR보다 luciferase 단백질 발현을 증가시킨다는 것을 관찰하였고, ZIKV mRNA 백 신 후보 물질의 면역원성을 더욱 향상시킬 수 있는 가능성을 발견하였다.

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목차

II. Introduction 1
III. Materials and methods 7
1. Materials 7
2. Vector construction 7
3. In vitro transcripstion (IVT) 8
4. LNP formulation 8
5. Dynamic light scattering (DLS) 9
6. Measurement of mRNA concentration & encapsulation efficiency 9
7. Cell culture 10
8. In vitro transfection 11
9. Immunoblotting 11
10. Purification of ZIKV VLP and transmission electron microscopy (TEM) 12
11. Mouse immunization 12
12. Enzyme-linked immunosorbent assay (ELISA) 13
13. Focus reduction neutralization test (FRNT) 14
14. Analysis for antigen-specific T cell responses 15
15. Preclinical safety test 16
16. In vivo Renilla luciferase assay 17
IV. Results 18
1. Design and production of HBB-ZIKV prME mRNA-LNP 18
2. The stability of mRNA LNP at various temperatures 21
3. ZIKV E protein was detected in HEK 293T cells transfected with HBB-ZIKV prM-E mRNA 23
4. The ZIKV prM-E mRNA vaccine candidate induced immune responses in C57BL/6 mice 26
5. The ZIKV prM-E mRNA vaccine candidate demonstrated safety in SD rats 31
6. New 5' & 3' UTR composition may further enhance the immunogenicity of the ZIKV prM-E mRNA vaccine candidate 35
V. Discussion 39
VI. References 45

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