A modular three in one mucosal vaccine against three antigenic clusters of ACE2 using sarbecoviruses

Figures

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  Fig. 1. Construction of 3Rs-NC containing RBDs from three distant sarbecovirus clusters. A Phylogenetic tree of sarbecovirus RBD sequences by maximum likelihood analyses. The scale bar indicates the genetic distance scale of 5%. B Principal coordinate analysis (PCoA) of antigenic distances among viruses of clade 1a and clade 1b sarbecovirus. The axes represent the two highest variance components. Each dot corresponds to a viral strain as labeled. C Schematic diagram of the recombinant protein 3Rs-NC containing triple RBDs. RBD genes of SARS-CoV-2 Omicron BA.1 strain, SARS-like virus WIV1, and SARS-like virus RsRSHC014 were connected by the gene of scaffold NC to generate 3Rs-NC gene. D 3D structure of the protein 3Rs-NC predicted by AlphaFold 3. E Diagram of the RBD binding with four classes of SARS-CoV-2 RBD-specific neutralizing monoclonal antibodies (mAbs). F Binding ability of SARS-CoV-2 Omicron BA.1 strain RBD (Ro), WIV1 RBD (Rw), RsRSHC014 RBD (Rs), and 3Rs-NC with the representative neutralizing mAbs analyzed by ELISA. Data shown represent one of two independent experiments, each with a duplicate.

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  Fig. 2. Immunogenicity of 3Rs-NC in the presence of aluminum adjuvant in BALB/c mice. A The AlphaFold 3 predicted 3D structure of 3Rs-NC, and 3D structure of 3Ro-NC that contains one RBD of the SARS-CoV-2 Delta strain and two RBDs of Omicron BA.1 strain in the scaffold NC. B Diagram scheme of immunization and sampling (n = 6 female mice per group). C–E RBD-specific IgG responses in serum after the 2nd and 3rd immunizations. F Neutralizing antibody responses in serum after the 3rd immunization against Omicron BA.1, WIV1, and rRsSHC014S viruses. G Geometric mean titers (GMT) of neutralizing antibodies against three authentic viruses and two pseudotyped viruses SARS-CoV-1 and SARS-CoV-2 variants BA.4/5. Data are represented as mean ± SEM and are representative of at least two independent experiments. In (C–F), groups were compared using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ns nonsignificant.

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  Fig. 3. Antibody and T cell responses induced by intranasal immunization of 3Rs-NC adjuvanted with KFD or CpG in BALB/c mice. A Diagram scheme of immunization and sampling (n = 6 female mice per group). B RBD-specific IgG responses in serum after the 3rd immunization. RBD-specific mucosal IgA responses after the 3rd immunization in saliva (C), vaginal lavage fluid (D), and nasal turbinate lavage fluid (E). RBD-specific T cell responses after the 3rd immunization in lung (F) and spleen (G) tested by ELISpot. Background signals without stimulating protein/peptide were deducted. Data are represented as mean ± SEM and are representative of at least two independent experiments. Groups were compared using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ns nonsignificant.

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  Fig. 4. Protection of 3Rs-NC immunization against SARS-CoV-2 Omicron BA.1 and WIV1 infection in female HFH4-hACE2 mice. A Diagram scheme of immunization and virus challenge. Each group (n = 12) of mice was divided into two subgroups after immunization and challenged by Omicron BA.1 or WIV1, respectively. B RBD-specific serum IgG after the 1st, 2nd, and 3rd immunizations. C RBD-specific salivary IgA after the 2nd and 3rd immunizations. D, F Neutralization antibody responses against authentic SARS-CoV-2 Omicron BA.1 in serum after the 3rd immunization and ahead of the challenge. E, G qPCR tested RNA copies of N gene in lung and turbinate at 3 days post-infection. Groups were compared using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ns nonsignificant, LOD limit of detection.

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  Fig. 5. Protection of 3Rs-NC immunization against virus rRsSHC014S in male and female HFH4-hACE2 mice. A Diagram scheme of immunization and virus challenge (n = 8–9, including 4 female mice). RBD-specific serum IgG (B) and salivary IgA (C) after the 3rd immunization. D Neutralization antibody responses against rRsSHC014S in serum after the 3rd immunization and ahead of the challenge. Survival curves of total mice (E) and female mice (F) within 10 days post-infection. G Weight changes of total mice within 10 days post-infection. Groups were compared using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ns nonsignificant.

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  Fig. 6. Long-lasting antibody responses and protection of 3Rs-NC immunization against SARS-CoV-2 Omicron BA.1. A Diagram scheme of immunization and virus challenge (n = 5–6 female BALB/c mice per group). B, C RBD-specific serum IgG and salivary IgA at 0.5 months (0.5 M) and 12 months (12 M) after the 3rd immunization. D, E qPCR tested RNA copies of SARS-CoV-2 E gene in the lung and turbinate at 4 days post-infection. F, G Correlation of virus copy numbers in lung and turbinate tissues with the titers of RBD-specific serum IgG and salivary IgA antibodies. Groups were compared using one-way ANOVA (A–D) or assayed by simple linear regression (F, G). *p < 0.05; **p < 0.01; ***p < 0.001.