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. 2022 Mar 14;7:84. doi: 10.1038/s41392-022-00937-9

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — 3-dose of RBD vaccine effectively boosts long-term memory response. a–g C57BL/6 mice were immunized i.p. with 0.5 μg or 5 μg SARS-CoV-2 recombinant RBD protein (321-591aa) adjuvanted with or without 0.25 mg aluminum hydroxide and 10 μg CpG for 2, 3, or 4 times. Blood, spleen, inguinal LNs, bone marrow, and lung were analyzed 14 days after the last dose immunization. 0.5, 0.5 μg RBD; 5, 5 μg RBD; R, RBD; C, CpG; A, aluminum hydroxide. a SARS-CoV-2 RBD-specific IgG titers were assessed by endpoint dilution ELISA in mice. b, c Frequency of murine GC B cells (B220⁺IgD⁻GL7⁺Fas⁺) and T_FH cells (CXCR5⁺PD-1⁺) in the spleen. d–f Frequency of murine plasmablast cells (B220⁺CD138⁺), plasma cells (B220⁻CD138⁺), and memory B cells (GL7⁻CD38⁺) in the bone marrow. Significance was determined by One-way ANOVA or unpaired t-tests. Black (PBS), green (3-dose RBD), blue (2-dose RCA) and red (3-dose RCA) colored asterisks indicated color representative group as a comparison to other groups if *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All data are representative of at least two independent experiments. g Summary of significances on different immunization manner in mice. Using 3-dose 0.5RBD or 5RBD as control, counted any significant change (including *, **, and ***) as 1 and add up to a total score. h–j Three macaques were immunized i.m. with 12.5 μg SARS-CoV-2 recombinant RBD protein adjuvanted with 0.5 mg aluminum hydroxide and 0.5 mg CpG at 0, 1, and 4 weeks for 3 doses. After 40 weeks apart from the third immunization, three macaques were given a fourth immunization with 50 μg SARS-CoV-2 recombinant RBD protein adjuvanted with 0.5 mg aluminum hydroxide and 0.5 mg CpG. Bloods were collected and analyzed at week 0, 1, 2, 3, 4, 6, 8, 9, 10, 13, 44, 46, 48, 50, 53, and 56. h SARS-CoV-2 RBD-specific IgG titers were assessed by endpoint dilution ELISA in macaques. The dotted line indicated the cut off value (2.1 × the mean titers of the negative control). i The capacity of serum antibodies to inhibit the interaction of RBD and human ACE2. j The capacity of serum antibodies to inhibit the interaction of Omicron mutant protein and human ACE2 was assessed by using inhibition ELISA. The dotted line indicated the cut off value (mean of negative control + 2 × SD)