A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants

Abstract

Background: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy.

Methods: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants.

Results: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs.

Conclusions: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

Keywords: Booster dose; COVID-19; Cross-protectivity; Hybrid vaccine; Next generation vaccine; Omicron vaccine; SARS-CoV-2; Variants of concern; mRNA vaccine.

Fig. 1

Wild-type RBD mRNA vaccine induces protective and Th1-biased immune responses against SARS-CoV-2. A Immunization, blood draw, AAV/hACE2 transduction, and wild-type SARS-CoV-2 (Wuhan strain) challenge schedule. B Serum IgG binding to recombinant SARS-CoV-2 RBD measured by ELISA. C Serum neutralizing activity against SARS-CoV-2 D614G, Beta, and Delta pseudovirus measured by pseudovirus neutralization assay. Plotted values represent geometric mean of 50% neutralizing titers (NT₅₀). D Serum neutralizing activity against SARS-CoV-2 wild-type, Beta, and Delta virus measured by live virus micro-neutralization assay. Plotted values represent geometric mean of 50% neutralizing titers (NT₅₀). E Body weight change of SARS-CoV-2 challenged mice. F Infectious viral load in lung of the challenged mice measured by Median Tissue Culture Infectious Dose (TCID₅₀) assay 5 days post challenge. G Serum IgG1 or IgG2a binding to recombinant SARS-CoV-2 wild-type RBD measured by ELISA. H, I IFN-γ (H) and IL-4 (I) secretion of RBD-stimulated splenocytes of control and vaccinated mice measured by Multiplex assay. Statistical comparisons between control and vaccinated mice were determined by unpaired T test. Statistical comparisons across groups were determined by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, non-significant. Dotted line indicates the limit of detection. See also Additional file 1: Fig. S1

Fig. 2

Characterization of WT and variant RBD mRNA and RBD mRNA-LNP vaccines. A Mutation sites of wild-type (WT), Delta, Omicron, and Hybrid RBD mRNA constructs. UTR untranslated region, SP signal peptide. B RNA identity and integrity of in vitro transcribed WT, Delta, Omicron, and Hybrid RBD mRNA measured by Fragment analysis. LM lower marker, RLU relative fluorescence units, nt nucleotide. C, D Binding capacity of WT and variant RBD expressed from mRNA (C) or mRNA-LNP (D) transfected cell supernatants to cells expressing human angiotensin-converting enzyme 2 (ACE2) or mouse ACE2 measured by flow cytometry. See also Additional file 1: Fig. S2

Fig. 3

RBD-specific IgG binding and neutralizing antibodies in sera of naïve mice immunized by WT and variant RBD mRNA vaccines. A Immunization and blood draw schedule. B Serum IgG binding to recombinant SARS-CoV-2 RBD of WT, Beta, Delta, and Omicron strain measured by ELISA. Plotted values represent mean endpoint titers. Fold change between groups with statistically significance were shown after asterisks. C Serum neutralizing activity against SARS-CoV-2 D614G, Beta, Delta, and Omicron pseudovirus measured by pseudovirus neutralization assay. Plotted values represent geometric mean of 50% neutralizing titers (NT₅₀). Fold change between groups with statistically significance were shown after asterisks. Statistical comparisons across groups were determined by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Dotted line indicates the limit of detection. See also Additional file 1: Fig. S3

Fig. 4

IFN-γ secretion of CD8⁺ cells in naïve mice immunized by WT and variant RBD mRNA vaccines. Mice were immunized with various RBD mRNA and the Bivalent vaccines as described in Fig. 3. A, B IFN-γ secretion capacity of CD8⁺ cells against SARS-CoV-2 (CoV-2) spike RBD peptide measured by ELISpot assay. Representative spot images were shown in A and summary spot counts shown in B. SFC, Spot forming cells. Statistical comparisons across groups were determined by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns non-significant. See also Additional file 1: Fig. S4

Fig. 5

RBD-specific IgG binding and neutralizing antibodies in sera of long-term WT vaccinated mice boosted by WT and variant RBD mRNA vaccines. A Immunization and blood draw schedule. B Pre- and post-booster serum IgG binding to recombinant SARS-CoV-2 RBD of WT, Beta, Delta, and Omicron strain measured by ELISA. C Pre- and post-booster serum neutralizing activity against SARS-CoV-2 D614G, Beta, Delta, and Omicron pseudovirus measured by pseudovirus neutralization assay. D Post-booster serum neutralizing activity against SARS-CoV-2 D614G, Beta, Delta, and Omicron pseudovirus measured by pseudovirus neutralization assay. Plotted values represent geometric mean of 50% neutralizing titers (NT₅₀). Statistical comparisons between pre- and post-booster were determined by repeated-measures two-way ANOVA with Sidak’s multiple comparisons test. Statistical comparisons across groups were determined by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Dotted line indicates the limit of detection. See also Additional file 1: Fig. S5