Abstract
The development of a vaccine specific to severe acute respiratory syndrome coronavirus 2 Omicron has been hampered due to its low immunogenicity. Here, using reverse mutagenesis, we found that a phenylalanine-to-serine mutation at position 375 (F375S) in the spike protein of Omicron to revert it to the sequence found in Delta and other ancestral strains significantly enhanced the immunogenicity of Omicron vaccines. Sequence FAPFFAF at position 371–377 in Omicron spike had a potent inhibitory effect on macrophage uptake of receptor-binding domain (RBD) nanoparticles or spike-pseudovirus particles containing this sequence. Omicron RBD enhanced binding to Siglec-9 on macrophages to impair phagocytosis and antigen presentation and promote immune evasion, which could be abrogated by the F375S mutation. A bivalent F375S Omicron RBD and Delta-RBD nanoparticle vaccine elicited potent and broad nAbs in mice, rabbits and rhesus macaques. Our research suggested that manipulation of the Siglec-9 pathway could be a promising approach to enhance vaccine response.
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Data availability
All data supporting the findings of this study are available within the paper. The RNA-seq data for mouse BMDMs are available in the Gene Expression Omnibus database under accession code GSE251973. Structures and EM maps of structures BA.2(S375) spike (S6P)/hACE2 complex (PDB 8WGV, EMDB-37516), local refinement of RBD–ACE2 (PDB 8WGW, EMDB-37517) have been deposited to the PDB and EMDB. Source data are provided with this paper.
Code availability
This study did not generate custom code for the analyses. Standard workflows, and open-source R packages and software were used, which are available at https://github.com/kmayerb/NIA34780B.
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Acknowledgements
This work was supported by National Natural Science Foundation of China (grant nos. 92369205, 92169201, 32100743, 82171825, 82141205 and 82202029), National Key R&D Program of Department of Science and Technology of China (grant no. 2022YFC0870700), Guangdong Basic and Applied Basic Research Foundation (grant nos. 2022B1111020004, 2022A1515010132 and 2023B1515020005) Health Commission of Guangdong Province Program (grant no. 0920220202), Science and Technology Planning Project of Guangzhou (grant no. 202201011469). Emergency Key Program of Guangzhou National Laboratory (grant no. EKPG21-24).
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Contributions
Conceptualization was carried out by X.H. and H.Z. The investigation was conducted by X.H., X.Z., B.W., J.D., Yongli Zhang, A.Z., Y.Y., Y. Lin, A.C., J.F., X. Wang, S.W., Y. Liu, J.L.,Y.W., R.L., C.L., Q.Y., Y. Liang, Q.F., Z.X., W.L., L.L., Z.Z., H.T., Y.P., C.K., X.M., W.C., T.P., B.L., K.D., J.C., J.Z., X. Wei, R.C. and Yiwen Zhang Validation was carried out by X.H., X.Z., B.W., J.D., R.C., Yiwen Zhang and H.Z. Formal analysis was conducted by X.H., X.Z., B.W., J.D., X. Wei., R.C., Yiwen Zhang and H.Z. The structure was developed by X.H., X.Z., Z.Z. and X. Wei. Resources were obtained by X.H., X.Z., B.W., J.D., Y.P., C.K., X. Wang., K.D., J.C. and H.Z. The original draft was written by X.H. and H.Z. Cosupervision was carried out by J.Z., X. Wei., R.C., Yiwen Zhang and H.Z. Supervision was carried out by H.Z. The project administration was the responsibility of X.H. and H.Z.
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Extended data
Extended Data Fig. 1 F375S enhanced immune responses of Omicron RBD nanoparticle.
a. Coomassie Blue staining showing the Delta and BA.2 RBD nanoparticles after the conjugation of RBD and ferritin. b. ELISA showing the respective RBD-specific IgG endpoint titers in the BALB/c mice vaccinated with one dose of 10 ug of Delta or BA.2 RBD nanoparticles at day 14. n = 6 from 3 independent experiments. Student′s t-test was used. c. Neutralizing titer measured for the BALB/c mice vaccinated with one dose of 10 ug of Delta or BA.2 RBD nanoparticles at week 4. n = 6 from 3 independent experiments. Student′s t test was used. d-e. Coomassie Blue staining showing the purification of mutant RBDs from HEK293F and the conjugation of RBD and ferritin. f. ELISA showing the BA.2 RBD-specific IgG endpoint titers in the BALB/c mice vaccinated with one dose of 10 ug of BA.2 RBD nanoparticles harboring the indicated mutation at day 14. n = 3 from 2 independent experiments. Mean ± SD. Adjusted p values were calculated using one-way ANOVA with Tukey′s multiple comparisons test. g. ELISA showing the respective RBD-specific IgG in the BALB/c mice vaccinated with one dose of 10 ug of indicated RBD nanoparticles at day 14. n = 5 in BA.1 and BA.2.12.1 panels, n = 6 in other panels, all from 2 independent experiments. Student′s t test was used. h. ELISA showing the RBD-specific IgG endpoint titers in the BALB/c mice vaccinated with one dose of 10 ug of Delta or Delta(F375) RBD nanoparticles at day 14. n = 6 from 3 independent experiments. Adjusted p values were calculated using Student′s t test. i. Coomassie Blue staining showing the BA.2 and BA.2S(S375) spike from HEK293F cells through Ni-NTA affinity purification. j. ELISA showing the BA.2 spike-specific IgG in the BALB/c mice vaccinated with one dose of 5 ug of BA.2 or BA.2(S375) spike at day 14. n = 5 from 2 independent experiments. P values were calculated using Student′s t test. All data were presented as mean ± SD.
Extended Data Fig. 2 F375S did not change binding of BA.2 spike/RBD to hACE2.
a, b. Flow cytometry analysis showing percentages of CD4 activation (CD69+CD154+) (a) or CD8 activation (CD69+CD137+) (b) in the splenocytes stimulated by a pool of Omicron BA.2 spike peptides for the BA.2(S375) or BA.2 RBD nanoparticles-vaccinated BALB/c mice at day 24 post-dose 2. n = 5 from 2 independent experiments. Data were presented as mean values ± SEM. P values were calculated using Student′s t test. ns not significant. c-d. Flow cytometry analysis showing percentages of intracellular cytokine expression in CD8 T cells of the splenocytes stimulated by BA.2 spike peptides for BQ.1, BQ.1(S375), XBB.1 or XBB.1(S375) RBD nanoparticles-vaccinated BALB/c mice at day 24 post-dose 2. n = 5 from 2 independent experiments. Data were presented as mean values ± SEM. P values were calculated using Student′s t test. ns not significant. e. Representative BIAcore plots showing the binding of hACE2 with the spike trimers (S6P), RBD monomer, RBD nanoparticle from Delta, BA.2, or BA.2(S375) variants. The KD values were calculated using the BIAevaluation software and were presented as the average of three independent experiments. f. Cryo-EM analysis of spike-ACE2. A representative micrograph of the Omicron BA.2(S375) spike (S6P)/hACE2 complex was shown at the beginning. The 2D class averages of the BA.2(S375) spike (S6P)/hACE2 complex were selected, followed by the 3D reconstruction of the BA.2(S375) spike (S6P)/hACE2 complex with a mask applied to RBD-ACE2. The local resolution of RBD-ACE2 is estimated using Resmap.
Extended Data Fig. 3 The Omicron RBD (S375) promoted macrophage phagocytosis.
a. Schematic of RFP-labeled RBD, which can be conjugated onto the nanoparticle surface through a GV/SD covalent isopeptide bond reaction. b. The uptake assay by flow showing RFP percentages of C57BL/6 DCs in vivo or human peripheral monocyte-derived DCs in vitro, which were treated with RFP-BA.2 or RFP-BA.2(S375) RBD nanoparticles. n = 5 in mice and n = 4 in human samples from 2 independent experiments. Data were presented as mean ± SD. P values were calculated using Student′s t test. ns no significance. c. The uptake assay by flow showing RFP percentages of mouse Macs in lymph nodes from BALB/c treated with RFP-BQ.1, RFP-BQ.1(S375), RFP-XBB.1, or RFP-XBB.1(S375) RBD nanoparticles. n = 6 from 2 independent experiments. Data were presented as mean ± SEM. Adjusted p values were calculated using Student′s t test. d. Neutralization assay showing the antisera efficacy from the Clodronate liposomes-pretreated BALB/c mice for one day, which were then analyzed four weeks post-vaccination with one dose of 10 ug of Delta RBD nanoparticles. n = 4 from 2 independent experiments. Data represented as mean ± SEM. Adjusted p values were calculated using Student′s t test.
Extended Data Fig. 4 The Omicron RBD domain inhibited macrophage phagocytosis.
a. Schematic of Omicron or Delta 7 amino acid labeled GFP-Oseq or GFP-Dseq nanoparticles. The Omicron 7 amino acid sequence (371FAPF375FAF)-tagged Ferritin, which can self-assemble to form the nanoparticle. b. The uptake assay by flow showing GFP percentages in BMDMs, which were treated with GFP, GFP-Oseq, or GFP-Dseq nanoparticles for four hours. n = 4 from 3 independent experiments. Data were presented as mean ± SEM. Adjusted p values were calculated using one-way ANOVA with Tukey′s multiple comparisons test. c. ELISA showing GFP-specific IgG in the BALB/c mice at day 14, which were subcutaneously vaccinated with one dose of 10 ug of GFP, GFP-Oseq, or GFP-Dseq nanoparticles. n = 3 from 3 independent experiments. Data were presented as mean ± SEM. Adjusted p values were calculated using one-way ANOVA with Tukey′s multiple comparisons test.
Extended Data Fig. 5 Omicron RBD increased interaction with Siglec-9.
a. WB showing interaction between Siglec-9 and BA.2, BA.2(S375), BA.5, BA.5(S375), BQ.1, BQ.2(S375), XBB.1 or XBB.1(S375) RBD-His proteins via the Ni-NTA pulldown. b. WB showing the interaction between XBB.1 RBD truncations and Siglec-9 via Ni-NTA pulldown. The truncated XBB.1 RBDs were construct by deleting the N-terminal, middle, or C-terminal parts, respectively, as shown in the left panel. c. Neutralization assay showing the antisera efficacy in the BALB/c mice vaccinated with one dose of α2-3,6,8 Neuraminidase-desialylated BA.2 or Delta RBD nanoparticles at day 28. n = 5 from 2 independent experiments. Data represented as mean ± SEM. Adjusted p values were calculated using Student′s t test. d. The relative luminescence unit (RLU) assay showing infectivity of BA.2, BA.2(S375) or Delta spike pseudoviruses in HEK293T/hACE2 or HEK293T/hACE2/Siglec9 cells 48 hours post incubation. n = 4 wells from 3 independent experiments. Data represented as mean ± SD. Adjusted p values were calculated using two-way ANOVA with Tukey′s multiple comparisons test. e. ELISA showing the BA.2 RBD-specific IgG in the Siglece−/− or WT mice vaccinated subcutaneously with one dose of 10 ug of BA.2 or BA.2(S375) RBD nanoparticles at day 14. n = 5 from 2 independent experiments. Data represented as mean ± SD. Adjusted p values were calculated using two-way ANOVA with Tukey′s multiple comparisons test. f. Heat map showing differentially expressed genes in XBB.1 or XBB.1(S375) RBD nanoparticles-treated WT or Siglece−/− BMDMs. n = 3 for each group. See Supplementary Data 1 for a full list of genes and their normalized expression. g. qRT-PCR showing IL1b, IL6, Cd74 and B2m expression in the LPS-stimulated BMDMs for 48 hours, which were pretreated with XBB.1 or XBB.1(S375) nanoparticles for 24 hours, Gapdh expression as reference. n = 3 from 3 independent experiments. Adjusted p values were calculated using two-way ANOVA with Tukey′s multiple comparisons test. h. GSEA showing the enrichen pathways between Siglece−/− and WT BMDMs treated by XBB.1 RBD nanoparticle using the MSigDB Hallmark collection. n = 3 for each group and the dot represented mean value.
Extended Data Fig. 6 BA.5(SR) RBD nanoparticle elicited potent immune responses.
a. Neutralization assay showing the antisera efficacy in the BALB/c mice vaccinated with one dose of 10 ug of BA.5, BA.5(S375), BA.5(F486) or BA.5(R493) RBD nanoparticles at day 28. n = 5 from 2 independent experiments. Data were presented as mean ± SEM. Adjusted p values were calculated using one-way ANOVA with Tukey′s multiple comparisons test (*p < 0.05, **p < 0.01, ****p < 0.0001). b. The uptake assay by flow showing RFP percentages of BMDMs, which were treated with RFP-BA.5, RFP-BA.5(S375), RFP-BA.5(F486) or RFP-BA.5(R493) RBD nanoparticles for four hours. n = 5 from 2 independent experiments. Data were presented as mean ± SEM. Adjusted p values were calculated using one-way ANOVA with Tukey′s multiple comparisons test (***p < 0.001, ns, not significant). c. Representative BIAcore plots of BA.5(SR) RBD monomer or nanoparticle bound to hACE2. The KD value shown as a mean of three independent experiments. d. Schematic of bivalent RBD nanoparticle construction and vaccination of K18-hACE2 mice. e. Viral RNA copies in the lungs of K18-ACE2 mice infected with BA.5 or Delta were determined by qRT-PCR and plotted as log10 copies per microgram total RNA. The dotted line indicates the limit of detection (LOD). n = 6 for each group. Data are represented as mean ± SD. Adjusted p values were calculated using one-way ANOVA with Tukey′s multiple comparisons test (∗∗∗∗p < 0.0001). f. Representative BIAcore plots of the antisera against BA.5 or XBB.1 RBD in the rabbit vaccinated with 2 does of indicated nanoparticles at day 14 post-dose 2. the indicated RBD nanoparticle immunizations bound to BA.5 RBD. g. Schematic of rhesus macaque vaccination.
Supplementary information
Supplementary Information
Supplementary Figs. 1–5 and Tables 1–3.
Supplementary Data 1
Normalized counts of the RNA-seq analysis by DESeq2.
Supplementary Data 2
Statistical source data for Supplementary Fig. 2.
Source data
Source Data
Statistical source data for Figs. 1–6 and Extended Data Figs. 1–6.
Source Data
Unprocessed western blots for Fig. 4 and Extended Data Fig. 5.
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He, X., Zhang, X., Wu, B. et al. The receptor binding domain of SARS-CoV-2 Omicron subvariants targets Siglec-9 to decrease its immunogenicity by preventing macrophage phagocytosis. Nat Immunol 25, 622–632 (2024). https://doi.org/10.1038/s41590-024-01776-2
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DOI: https://doi.org/10.1038/s41590-024-01776-2
