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Comment
. 2021 Nov 10;6(1):390.
doi: 10.1038/s41392-021-00798-8.

RBD206-sc-dimer induced robust cross-neutralization against SARS-CoV-2 and variants of concern

Chuge Zhou #  1 , Xiaodong Zai #  1 , Ziqing Zhou  1 , Ruihua Li  1 , Yue Zhang  1 , Yaohui Li  1 , Ying Yin  1 , Jun Zhang  1 , Junjie Xu  2 , Wei Chen  3
Affiliations
Comment

RBD206-sc-dimer induced robust cross-neutralization against SARS-CoV-2 and variants of concern

Chuge Zhou et al. Signal Transduct Target Ther. .
No abstract available

PubMed Disclaimer

Conflict of interest statement

Beijing Institute of Biotechnology has submitted provisional patent applications related to use of RBD206-dimer to COVID-19 vaccines.

Figures

Fig. 1
Fig. 1
SARS-CoV-2 RBD206-sc-dimer vaccine elicited a robust cross-reactive neutralizing response in mice. a Expression profiles of SARS-CoV-2 RBD proteins. Wild-type RBD219 monomer (R319-K537), RBD206 (I332-K537), RBD219-dimer, and RBD206-dimer were expressed in Expi293F. b The tPA-tagged proteins were harvested from supernatant and then purified, as verified by gel electrophoresis and HPLC, see also Supplementary Fig. S1a. RBD-based proteins were identified under reducing and unreducing conditions (Supplementary Fig. S1b). Five micrograms of of SARS-CoV-2 RBD-based antigens before and after PNGase-F treatment were loaded on a 4–12% Tris-glycine gel in a reduced condition. The size reduced after PNGase-F treatment suggested that RBD219 was N-glycosylated while RBD206 was less N-glycosylated. The graph below summarized quantitative mass spectrometric analysis of the glycan population present at individual N-linked/O-linked glycosylation sites, principal glycan types were simplified into two colors, O-linked glycan series were colored blue, N-linked glycans were red, and the circular shapes summarized the relative intensities of these glycans. c Schematic diagram of immunization and serum sample collection. In the pilot study, BalB/c mice (6–8 weeks, n = 8) were immunized at day 0 and day 14 with 5-µg doses of RBD219/RBD206-monomer and RBD219/RBD206-dimer with 50-µg aluminum hydroxide (alum). PB was administered as control group. In the following study, BalB/c mice (6–8 weeks, n = 8) were immunized with antigen in the combination of alum and CpG2006 (25-µg) following the same procedure. Serum samples were collected at days 14, 28, and 42 as indicated. Spleen samples were collected at day 56. d–f RBD-specific IgG titers were tested by ELISA. Naive mice (BalB/c, n = 8 per group) were immunized with either RBD219, RBD206 or RBD219 dimer, or with RBD206 dimer adjuvanted with alum as shown in d. BalB/c mice (n = 8 per group) were immunized with either RBD219, RBD206 or with RBD206 dimer in the joint of alum and CpG as shown in e. In f, the group received prime-injection of RBD206 dimer with alum and CpG was highlighted. Data represented antibody titers on Day 28 post prime-injection. The experiments were further repeated twice, and similar results were obtained. All the data were presented as mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.1. ns: No significant difference. g–i Pseudovirus-based neutralization assays were performed to detect neutralizing antibody (NAb) titer against SARS-CoV-2, Neutralizing antibodies of wild type SARS-CoV-2 pseudoviruses were assessed in 293T-ACE2 cells (n = 8), and the neutralizing level was shown as 50% neutralizing titer (NT50) (n = 8). Naive mice (BalB/c, n = 8 per group) were immunized with either RBD219, RBD206 or RBD219 dimer, or with RBD206 dimer adjuvanted with alum as shown in g. BalB/c (n = 8 per group) were immunized with either RBD219, RBD206 or with RBD206 dimer adjuvanted with alum and CpG as shown in f. In i the group received prime-injection of RBD206 dimer with alum and CpG was highlighted. Data represent neutralizing antibody titer at day 28 after prime-injection. The experiments were further repeated twice, and similar results were obtained. All the data were presented as mean ± SEM. ***P < 0.001; **P < 0.01, *P < 0.1. ns: No significant difference. j Neutralization titer of sera collected at day 28 against live SARS-CoV-2 were shown as serial dilution curves (n = 8). All the data were presented as mean ± SEM. The experiments were performed in duplicate, and similar results were obtained. k The level of cytokines secreted by stimulated splenocytes of mice (n = 4) vaccinated was shown by Log10Concentration (pg/ml) in the heat map. The RBD peptide pool was used as a mock antigen to investigate the effect of splenocytes. In all, 2 µg/ml peptide mixture was then co-incubated with splenocytes for at least 48 h at 37 °C. Both stimulated and unstimulated splenocyte supernatants were collected, and cytokines in the supernatants were detected by ELISA using Bio-Plex Pro Mouse Cytokine Crp I Panel 19-plex. l Cross-neutralization of serum of immunized BALB/c mice was detected with live wild-type SARS-CoV-2, Beta variant and Delta variant (n = 8). Neutralizations at original dilution of 1:30 of serum are shown. Neutralization titers against live wild-type SARS-CoV-2/Beta/Delta variant are shown as individual values (n = 8). ***P < 0.001; **P < 0.01, *P < 0.1. ns: No significant difference. m RBD206 single-chain dimer structures were simulated by Alpha-Fold. Similar to the RBD219-sc-dimer, RBD206-sc-dimer contains two tandem repeat domains (I332-K537), otherwise the 13-amino acid–sequence, including N331, has been removed. Two RBD206 monomers are colored in violet and pale cyan, respectively. The regions of RBM are represented in light yellow ellipses
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