Glycosite-deleted mRNA of SARS-CoV-2 spike protein as a broad-spectrum vaccine

Chung-Yi Wu,Chi-Huey Wong,Cheng-Wei Cheng,Che Ma,Chih-Chuan Kung,Kuo-Shiang Liao,Jia-Tsrong Jan

doi:10.1073/pnas.2119995119

Abstract

Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein invitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8+ T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8+ T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.

Highlights

I n 1796, Edward Jenner created the first vaccine in the world to protect against smallpox and successfully rescued millions of people, and, since vaccination has been recognized as the best way to protect against pathogens [1]
As part of our efforts to develop broadly protective vaccines and antibodies against SARS-CoV-2 and the emerging variants, we have shown that immunization of wild-type (WT) S protein with all 22 N-glycosites trimmed down to N-acetylglucosamine (GlcNAc) as the mono-GlcNAc decorated S protein (Smg) elicited broadly protective immune responses, including antibody and CD4+ as well as CD8+ T cell responses against the alpha, beta, gamma, and delta variants
To translate this finding into the messenger RNA (mRNA) vaccine design for development of a broadspectrum mRNA vaccine, here we show the study of S protein mRNA vaccine with deletion of specific glycosites in receptor-binding domain (RBD) and subunit 2 domain (S2) with N to Q and S/T to A replacement and investigation of their protein expression and immune response as well as breadth of protection

Summary

Result

Glycosylation of S Protein Affected Antibody Production. S protein is frequently mutated and highly glycosylated (with 2 N- and 2 O-glycosites in RBD, 11 N-glycosites in S1, and 9 N-glycosites in S2) to evade host immune response (SI Appendix, Fig. S1) [15]. Sera from mice immunized by the mRNA with all S2 N-glycosites deleted (S-(deg-S2)) or with all S2 N-glycosites except N-1194 deleted (S-(S2-1194)), showed lower IgG titer against the fully glycosylated WT S protein (Fig. 1C), S2 (Fig. 1D), RBD (Fig. 1E), or deglycosylated S protein (Fig. 1F), but had higher IgG titer against the deglycosylated S2 antigen (Fig. 1G) in enzyme-linked immunosorbent assay (ELISA) as compared to the unmodified mRNA. The mice immunized with the mRNA with all RBD glycosites deleted (S-(deg-RBD)) elicited slightly lower IgG titer against the fully glycosylated RBD, but higher IgG titer to recognize the deglycosylated RBD antigen (Fig. 1H), suggesting that glycosylation on S protein affected the production of antibody and its binding specificity.

H Deglycosylated RBD anƟ-β-acƟn

Discussion

Findings

Materials and Methods