key: cord-0775836-3qwhmun5
authors: Zhou, Chuge; Zai, Xiaodong; Zhou, Ziqing; Li, Ruihua; Zhang, Yue; Li, Yaohui; Yin, Ying; Zhang, Jun; Xu, Junjie; Chen, Wei
title: RBD(206)-sc-dimer induced robust cross-neutralization against SARS-CoV-2 and variants of concern
date: 2021-11-10
journal: Signal Transduct Target Ther
DOI: 10.1038/s41392-021-00798-8
sha: 7b1330fdd05b3b92e7400f6416f1792f9e124bf6
doc_id: 775836
cord_uid: 3qwhmun5

nan

To date, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 223 million confirmed cases of coronavirus disease 2019 (COVID-19), including 4.6 million deaths (https://covid19.who.int/). Since 2020, several SARS-CoV-2 variants including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) with immune evasiveness have emerged and fast spread. In a global research effort, scientists proposed multiple effective vaccine strategies to respond to the ongoing COVID-19 pandemic. Most vaccines currently in use or in clinical development target the SARS-CoV-2 Spike (S) glycoprotein, a homotrimer decorates on the viral surface; within it, a distinct receptor-binding domain (RBD, residues 331-524) is responsible for mediating cell entry and interaction with host receptor angiotensin-converting enzyme 2 (ACE2).

Study also shown RBD-directed neutralizing antibodies(nAbs) are less-compromised by SARS-CoV-2 mutations due to their diverse RBD binding modes, 1 therefore it could better protect against circulating variants. To ameliorate the limited immunogenicity of RBD, in early studies on SARS, N-glycan sites on RBD have been found to be promising modification targets. 2 Two N-glycan sites (N331, N343) confirmed lies in the SARS-CoV-2 RBD, which likely play a role in protein folding and immune evasion. 3 Previous studies have also demonstrated a universal dimeric form of CoV RBD (RBD single-chain dimer) that contains two tandem full-length RBD subunits (R319-K537) and boosts immunogenicity in mice. 4 Here, we present the RBD 206 (I332-K537)-dimer, ted immunogea glycan-truncan combining glycosylation modification and structure-guided design. It has been shown to be a remarkable immunogen form that generates more antibodies, higher neutralizing activity and sufficient crossreactive neutralization against SARS-CoV-2 wild-type, B.1.351 (Beta) variant and B.1.617.2 (Delta) variant.

RBD206, based on RBD219 (R319-K537) of wild-type SARS-CoV-2, sequenced from R319 to N331 was deleted, as shown in Fig. 1a . RBD219, RBD206, RBD219-dimer, and RBD206-dimer were expressed in Expi293F cells. The recombinant proteins with different molecular wight were verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis Coomassie-stained gels ( Supplementary Fig. S1b ). We then analyzed the glycosylation of RBD dimers, The truncated RBD 206 monomer and dimers shown less PNGase F activity (Fig. 1b) . The glycopeptides generated by trypsin and chymotrypsin were analyzed by liquid chromatography mass spectrometry, and the N-linked/O-linked glycosylation siteswere determined (Fig. 1b) . Subsequently, the BIAcore assay demonstrated that RBD 206 was bound to hACE2 receptor with similar affinity (1.16 nM) as RBD 219 monomer (1.52 nM) (Supplementary Fig. S2a, b) , while two corresponding dimeric RBDs (2.30 × 10 −2 nM, 4.77 × 10 −2 nM) showed higher receptor affinity ( Supplementary Fig. S2c, d) , suggesting the exposure of two RBMs may account for improved binding affinities. CD spectrum further indicated the similarities in the structure between two monomers and single-chain dimeric RBDs ( Supplementary Fig. S1c) .

In order to evaluate the immunogenicity of the designed RBDs, we assessed IgG titers of BalB/c mice (n = 8) immunized with different antigens and alum according to the two-dose regimen (Fig. 1c) . With increased IgG titer against SARS-CoV 2, RBD 206 was found to be a favorable design (Fig. 1d) . Specifically, we found that the RBD-specific binding antibodies in RBD 206 -dimer groups reached~10 5 as early as 2 weeks post priming, which indicated that RBD 206 -dimer significantly enhanced the immunogenicity of RBD antigens (Fig. 1d ). After boost injection, the IgG titer in RBD 206 -dimer-immunized mice sera increased to~10 6 at day 28, which was 13.8-fold higher than that in the RBD 219 -dimerimmunized group. Then neutralizing titer was further tested. After the prime-boost vaccination, neutralizing antibody(nAb) response elicited by the RBD 206 -dimer against pseudovirus was higher than the RBD 219 -dimer with 50% pseudovirus neutralization titer (NT 50 ) of~10 3 and~10 2 respectively (Fig. 1g) . As illustrated by the neutralizing curve in Fig. 1j , RBD 206 -dimer maintained its high efficacy in live virus neutralization assay with NT 50 > 50, while NT 50 < 30 was elicited by RBD 219 -dimer. We further vaccinated BalB/c mice (n = 8) at 0 and 14 days, using 5, 2, and 1 µg of RBD 219 -dimer and RBD 206 -dimer per dose separately, quantified binding Abs to RBD suggested a dose-dependent response to the vaccine. Sufficient binding antibodies were induced by the low-dose RBD 206 -dimer vaccine after prime vaccination ( Supplementary Fig. S4 ).

To further improve the immunogenicity of RBD 206 -dimer, in this study, CpG2006 (Takara), a 24-mer CpG ODN in the toll-like receptor (TLR9) was formulated along with alum as adjuvants. After the two-dose immunization, RBD 219 induced IgG titer of 10 6 , while that of~10 7 was induced by RBD 206 -dimer (Fig. 1e ). In accordance with IgG results, neutralizing antibodies (nAbs) induced by RBD 206 -dimer, strongly inhibited pseudotyped SARS-CoV-2 infection, with NT 50 up to >10,000 at day 28, which was 10-fold higher than that in the RBD 219 combined with CpG alum group (Fig. 1h) . In the live virus neutralization assay, RBD 206 -dimer with CpG vaccine neutralized over 50% of the live SARS-CoV-2 at the serum dilution ranged from 1:1000 to 1:3000 after two-dose injection, which performing better than other RBD vaccine formulations (Fig. 1j) . As expected, formulation with CpG enhanced nAb titers induced by RBD 206 -sc-dimer compared with immunizations with non-formulated antigens.

In the presence of CpG, we then verified immune response induced by prime-injection of RBD 206 -dimer. At 28 days after prime vaccination, IgG titer of~10 6 was detected in RBD 206 -dimer single-injection group, which was slightly higher than IgG titer induced RBD 219 -monomer boost vaccination (Fig. 1f) . Moreover, the NT 50 of RBD 206 -dimer vaccine prime-injection group ranged from 1:100 to 1:300, of note, immunization with RBD 219 formulated with CpG alum elicited similar levels of neutralizing Abs after two-dose injection. Our results indicated that a primeonly vaccination regimen might be enough to elicit sufficient nAbs against SARS-CoV-2.

Given the efficient antibody response, we next quantified vaccine-specific cytokine responses. Consistent with differences observed in IgG subtypes (Fig. S3) . We verified a Th1-dominant response as displayed by IgG1 subtype-specific titers (~10 5 ) induced by RBD 206 -dimer with alum, which was slightly higher than that induced by RBD 219 . CpG was shown to boost the IgG2a titers. We found that RBD 206 -dimer elicited higher cytokine levels, Letter including IFN-γ+, IL-2+, and TNF-α in RBD peptide-stimulated splenocytes than the RBD 219 antigen (Fig. 1k) . These findings further suggested that RBD 206 -dimer with CpG both induced cellular and boosted humoral immunity.

The emergence of SARS-CoV-2 variants such as Beta and Delta has raised concerns regarding possible reduction in vaccine efficacy. Neutralizing Ab titers are an important correlate of protection against SARS-CoV-2 infection. 5 We therefore assessed the cross-neutralization of RBD 206 -dimer against Beta and Delta variants. Here, we found that nAbs elicited by RBD 206 -dimer formulated with CpG alum could potently neutralize wild type Beta as well as Delta variant. We observed that the Beta variant isolated was 1.6-fold less sensitive to the sera compared with the wild type, and the Delta strain was 1.3-fold loss of neutralization sensitivity compared with the reference wild type (Fig. 1l) , which was in accordance with anti-RBD-binding antibodies. The marginal change of nAb titer provides supportive evidence that RBD 206dimer vaccines could induce RBD-targeted nAbs, maintaing high resistance to SARS-CoV-2 mutations.

At present, multiple RBD-based antigens including RBD-monomer, RBD-dimer, and RBD-trimer, etc. are being developed into diverse recombinant subunit vaccines. In this report, we proposed RBD 206 -dimer subunit vaccine. Further neutralizing assays showed that RBD 206 -dimer created about a 10-fold increase in both binding and neutralizing Abs against wild-type SARS-CoV-2 than RBD 219 -dimer. As shown in Alpha-Fold simulation of protein structure (Fig. 1m) , the deletion of N331-glycan was likely to reduce the glycan masking, and increase the exposure area of antigen binding sites, thereby enhancing the neutralizing antibodies response. 4 Additionally, we observed that the immunization of mice with RBD 206 -dimer formulated with CpG alum elicited comparable cross-reactive nAbs against Beta and Delta variants, showing only minimally reduced vaccine effectiveness compared with wild-type SARS-CoV-2. As recent research has suggested, it is likely that RBD-based recombinant proteins could better cope with the immune evasion by inducing diverse RBDdirected nAbs, other than spike-targeted vaccines. 1 Collectively, our findings highlight RBD 206 single-chain dimeric repeats, as a promising candidate vaccine against COVID-19, which may improve vaccine efficacy against circulating SARS-CoV-2 variants.

The data are available from the corresponding author on reasonable request.

W.C., J.X., X.Z. and C.Z. contributed to study design, project coordination, and manuscript writing. C.Z. and X.Z. contributed to construction of antigens, data analysis, and visualization. C.Z., Z.Z., Y.Z., contributed to construction and purification of antigens. C.Z., X.Z., Y.L. and J.Z. contributed to neutralization assays with pseudovirus and live SARS-CoV-2. C.Z., R.L. and Y.Y. contributed to the animal experiment and immunogenicity evaluation. Fig. 1 SARS-CoV-2 RBD 206 -sc-dimer vaccine elicited a robust cross-reactive neutralizing response in mice. a Expression profiles of SARS-CoV-2 RBD proteins. Wild-type RBD 219 monomer (R319-K537), RBD 206 (I332-K537), 2 RBD 219 -dimer, 4 and RBD 206 -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 RBD 219 was N-glycosylated while RBD 206 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 RBD 219 /RBD 206 -monomer and RBD 219 /RBD 206 -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 RBD 219 , RBD 206 or RBD 219 dimer, or with RBD 206 dimer adjuvanted with alum as shown in d. BalB/c mice (n = 8 per group) were immunized with either RBD 219 , RBD 206 or with RBD 206 dimer in the joint of alum and CpG as shown in e. In f, the group received prime-injection of RBD 206 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 (NT 50 ) (n = 8). Naive mice (BalB/c, n = 8 per group) were immunized with either RBD 219 , RBD 206 or RBD 219 dimer, or with RBD 206 dimer adjuvanted with alum as shown in g. BalB/c (n = 8 per group) were immunized with either RBD 219 , RBD 206 or with RBD 206 dimer adjuvanted with alum and CpG as shown in f. In i the group received prime-injection of RBD 206 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 Log 10 Concentration (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 RBD 206 single-chain dimer structures were simulated by Alpha-Fold. Similar to the RBD 219 -sc-dimer, 4 RBD 206 -sc-dimer contains two tandem repeat domains (I332-K537), otherwise the 13-amino acid-sequence, including N331, has been removed. Two RBD 206 monomers are colored in violet and pale cyan, respectively. The regions of RBM are represented in light yellow ellipses Letter

Humoral immune response to circulating SARS-CoV-2 variants elicited by inactivated and RBD-subunit vaccines

Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate

Site-specific glycan analysis of the SARS-CoV-2 spike

A universal design of betacoronavirus vaccines against COVID-19, MERS, and SARS

Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

The work is supported by the grant from the National Key Research and Development Program of China (2020YFC0841400) and the National Natural Science Foundation of China (31800770, 82041019). We would like to thank Grateful thanks to Meirong Wang, Zhe Zhang, Busen Wang, Zhengshan Chen, Yi Chen, and Xiaohong Song and Yuan Jin for their support in animal experiments. We appreciate the help of Yuan Jin, Junjie Yue, Yuan Jin and Zhenshan Chen for protein structure analysis. Grateful acknowledgment is made to all the patients who participated in this study. animal experiments and contribution to the reagents.

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/.