Neutralizing Potency of Prototype and Omicron RBD mRNA Vaccines Against Omicron Variant

Abstract

The newly emerged Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains more than 30 mutations on the spike protein, 15 of which are located within the receptor binding domain (RBD). Consequently, Omicron is able to extensively escape existing neutralizing antibodies and may therefore compromise the efficacy of current vaccines based on the original strain, highlighting the importance and urgency of developing effective vaccines against Omicron. Here we report the rapid generation and evaluation of an mRNA vaccine candidate specific to Omicron, and explore the feasibility of heterologous immunization with WT and Omicron RBD vaccines. This mRNA vaccine encodes the RBD of Omicron (designated as RBD-O) and is formulated with lipid nanoparticle. Two doses of the RBD-O mRNA vaccine efficiently induce neutralizing antibodies in mice; however, the antisera are effective only on the Omicron variant but not on the wildtype and Delta strains, indicating a narrow neutralization spectrum. It is noted that the neutralization profile of the RBD-O mRNA vaccine is opposite to that observed for the mRNA vaccine expressing the wildtype RBD (RBD-WT). Importantly, booster with RBD-O mRNA vaccine after two doses of RBD-WT mRNA vaccine can significantly increase neutralization titers against Omicron. Additionally, an obvious increase in IFN-γ, IL-2, and TNF-α-expressing RBD-specific CD4⁺ T cell responses was observed after immunization with the RBD-WT and/or RBD-O mRNA vaccine. Together, our work demonstrates the feasibility and potency of an RBD-based mRNA vaccine specific to Omicron, providing important information for further development of heterologous immunization program or bivalent/multivalent SARS-CoV-2 vaccines with broad-spectrum efficacy.

Keywords: SARS-CoV-2; mRNA vaccine; neutralizing antibody; omicron variant; receptor-binding domain.

Figure 1

Design and characterization of LNP-encapsulated RBD mRNA vaccines against SARS-CoV-2. (A) Schematic diagram of RBD-WT (top panel) and RBD-Omicron (bottom panel) mRNA framework. Note that 15 mutations are located within Omicron RBD. UTR, untranslated regions; SP_IL-10, human interleukin-10 signal peptide. (B) In vitro transcribed mRNA was transfected into HEK293T cells and the expression of RBD proteins within the cells were analyzed by immunofluorescence staining analysis with mouse anti-RBD polyclonal antibody and anti-mouse Alexa Fluor^® 488 secondary antibody. Ctrl, mRNA encoding luciferase. RBD-O, mRNA encoding RBD-Omicron. Overlay, merge of the blue (DAPI) and green (RBD) channels. Scale bars = 20 μm. (C) The culture supernatants of mRNA-transfected HEK293T cells were analyzed for RBD expression by western blotting with HRP-conjugated anti-His tag antibody and anti-RBD polyclonal antibody as detection antibodies. M, marker.

Figure 2

Immunization with mRNA vaccines elicited neutralizing antibodies in mice. (A) Mice immunization schedule. Groups of BALB/c mice were injected i.m. with 10 μg of RBD-WT mRNA (n = 8), RBD-O mRNA (n = 8), or luciferase-mRNA (Ctrl; n = 5) vaccines at weeks 0 and 2. Serum samples were collected from individual mice at weeks 2 and 4. (B) The week-2 and week-4 antisera were diluted 1:100 and analyzed for RBD-WT-binding activity by ELISA. (C) Neutralizing titers of the week-4 antisera samples from each group against WT-, Delta-, and Omicron-PV (pseudovirus). Serum samples that exhibited less than 50% neutralization at the lowest serum dilution (1:200) were assigned a NT₅₀ value of 100 for statistical analysis. Each symbol represents one mouse. (D) Neutralizing titers of the week-4 antisera from RBD-WT mRNA group (left panel) and RBD-O mRNA group (right panel) against SARS-CoV-2 PV. The geometric mean of NT50 values was shown. For panels (B, C), data are presented as mean ± SEM. p values were analyzed with unpaired t-test and indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

The influence of homologous and heterologous booster immunization programs on serum neutralization titers and breadth. (A) Mice booster immunization schedule. The original RBD-WT mRNA vaccine group was divided into two (n = 4 mice per group): group I (the RBD-WT+WT+WT mRNA group) mice were boosted with the RBD-WT mRNA vaccine, and group II (the RBD-WT+WT+O mRNA group) mice with the RBD-O mRNA vaccine. The original RBD-O mRNA vaccine group (re-named the RBD-O+O+O mRNA group) received a third dose of the RBD-O mRNA vaccine. Serum samples were collected at week 8. (B) Neutralizing titers of the week-4 and week-8 antisera samples from each group against WT-, Delta-, and Omicron-PV (pseudovirus). If serum samples show less than 50% neutralization at the 1:200 dilution, a NT₅₀ value of 100 was assigned for statistical analysis. ns, not significant; *p < 0.05; **p < 0.01. (C) Neutralizing titers of the week-8 antisera from the three vaccine groups against SARS-CoV-2 PVs. The geometric mean of NT50 values was shown.

Figure 4

RBD-WT- and RBD-O-specific CD4 T-cell intracellular cytokine staining (ICS) assays. The splenocytes isolated from control or vaccinated individual mice were stimulated with Omicron-RBD or WT-RBD proteins overnight, and the expression of the intracellular cytokines IFN-γ, IL-2, and TNF-α in CD4 T cells was analyzed by flow cytometry. (A) Representative gating strategy for the identification of antigen-specific CD4 T cells within the mouse spleen. (B) Frequencies of RBD-WT- and RBD-O-specific CD4 T cells in spleen were measured by intracellular cytokine staining. Each symbol represents one mouse. Data are presented as mean ± SEM. p values were analyzed with unpaired t-test and indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.