Immunization with the receptor-binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement

Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020). CAS Article Google Scholar Amanat, F. & Krammer, F. SARS-CoV-2 vaccines: status report. Immunity 52, 583–589 (2020). CAS Article Google Scholar Chen, W. H., Strych, U., Hotez, P. J. & Bottazzi, M. E. The SARS-CoV-2 vaccine pipeline: an overview. Curr. Trop. Med. Rep. 7, 61–64 (2020). Article Google Scholar Hotez, P. J., Corry, D. B. & Bottazzi, M. E. COVID-19 vaccine design: the Janus face of immune enhancement. Nat. Rev. Immunol. 20, 347–348 (2020). CAS Article Google Scholar Huisman, W., Martina, B. E. E., Rimmelzwaan, G. F., Gruters, R. A. & Osterhaus, A. D. M. E. Vaccine-induced enhancement of viral infections. Vaccine 27, 505–512 (2009). CAS Article Google Scholar Zhou, Y. S., Jiang, S. B. & Du, L. Y. Prospects for a MERS-CoV spike vaccine. Expert Rev. Vaccines 17, 677–686 (2018). CAS Article Google Scholar Jiang, S. B., Lu, L., Liu, Q., Xu, W. & Du, L. Y. Receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines. Emerg. Microbes Infect. 1, 1–8, e13 (2012) . Article Google Scholar Wang, C. et al. A human monoclonal antibody blocking SARS-CoV-2 infection. Nat. Commun. 11, 2251 (2020). CAS Article Google Scholar Block, O. K. et al. A tetravalent recombinant dengue domain III protein vaccine stimulates neutralizing and enhancing antibodies in mice. Vaccine 28, 8085–8094 (2010). CAS Article Google Scholar Sun, J., Li, M., Wang, Y., Hao, P. & Jin, X. Elaboration of tetravalent antibody responses against dengue viruses using a subunit vaccine comprised of a single consensus dengue envelope sequence. Vaccine 35, 6308–6320 (2017). CAS Article Google Scholar Download references We thank Dr. Xiaozhen Liang for providing A20 and K562 cell lines, Dr. Guangxun Meng for THP-1 cell line, Drs. Gary Wong and Jiaming Lan for codon-optimized S gene, Dr. Haikun Wang for assistance in flow cytometry analysis, and Xueyang Zhang, Yu Zhou, Yang Wu and Zhiping Sun for technical assistance. This study was supported by grants from the Chinese Academy of Sciences (XDB29040300) and from the Chinese Ministry of Science and Technology (2020YFC0845900). The BSL-3 lab of Fudan University was supported by Shanghai Science and Technology Committee and Project of Novel Coronavirus Research from Fudan University. These authors contributed equally: Jinkai Zang, Chenjian Gu, Bingjie Zhou, Chao Zhang, Yong Yang. CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Center for Biosafety Mega-Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China Jinkai Zang, Bingjie Zhou, Chao Zhang, Yong Yang, Shiqi Xu, Lulu Bai, Hong Tang, Dimitri Lavillette & Zhong Huang Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200031, China Chenjian Gu, Rong Zhang, Qiang Deng, Zhenghong Yuan & Youhua Xie BSL-3 Laboratory of Fudan University, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China Di Qu You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar Z.H., Y.X., and D.L. conceived and designed the experiments. J.Z., C.G., B.Z., C.Z., Y.Y., S.X., L.B., R.Z., Q.D., Z.Y., H.T., and D.Q. participated in multiple experiments; Z.H., Y.X., D.L., J.Z., C.G., B.Z., C.Z., and Y.Y. analyzed the data. Z.H., Y.X., D.L., and C.Z. wrote the manuscript. Z.H., Y.X., and D.L. provided the final approval of the paper. Correspondence to Dimitri Lavillette or Youhua Xie or Zhong Huang. The authors declare that they have no conflict of interest. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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/. Reprints and Permissions Zang, J., Gu, C., Zhou, B. et al. Immunization with the receptor-binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement. Cell Discov 6, 61 (2020)....


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phenomenon called antibody-dependent enhancement (ADE), which is well documented for flaviviruses 4,5 . So far, all SARS-CoV-2 vaccine candidates entering clinical trials contain or express full-length or near full-length S protein and therefore bear risk of ADE. Thus, it is important to continue the search for a safe and effective SARS-CoV-2 vaccine.
Recombinant RBD proteins of SARS-CoV and MERS-CoV have been shown to potently induce protective neutralizing antibodies and are therefore considered promising vaccine candidates 6,7 . In this study, we evaluated the possibility of developing SARS-CoV-2 RBD (hereafter referred as SARS2-RBD)-based vaccines. Specifically, we investigated whether recombinant SARS2-RBD could elicit in mice neutralizing antibodies and whether such antibodies could promote ADE in vitro.
To rapidly evaluate vaccine potential of SARS2-RBD, a pilot mouse immunization study was performed with recombinant RBD/mouse IgG1-Fc fusion protein (RBD-Fc) as immunogen. The mice received three doses at days 0, 8, and 13. One week after the last immunization, antisera were collected from the three immunized mice for antibody measurement. All three antisera dosedependently reacted with His-tagged SARS2-RBD in ELISA, whereas control sera from a naïve mouse did not show significant reactivity ( Supplementary Fig. S1a). Anti-RBD-Fc sera #1 with the highest RBD-binding titer (2 × 10 5 ) was selected for further analyses. Anti-RBD-Fc sera #1 dose-dependently inhibited binding between recombinant ACE2-Fc fusion protein and His-tagged SARS2-RBD in competition ELISA ( Supplementary Fig. S1b), indicating that the antisera contain antibodies targeting receptor-binding motif (RBM) within RBD. Anti-RBD-Fc sera #1 was then assessed for the ability to neutralize SARS-CoV-2 pseudovirus (hereafter referred as SARS2-PV). The antisera dose-dependently neutralized SARS2-PV entry with a calculated 50% neutralization titer (NT50) value of 10,513 ( Supplementary Fig. S1c). Moreover, anti-RBD-Fc sera #1 were highly effective on neutralizing authentic SARS-CoV-2, based on qRT-PCR and immunofluorescent analyses (Supplementary Fig. S1d and e). These results demonstrate that RBD-Fc is an immunogen capable of efficiently inducing SARS-CoV-2-neutralizing antibodies.
To verify that the RBD part within RBD-Fc fusion protein is indeed responsible for induction of neutralizing antibodies against SARS-CoV-2, we performed a second mouse immunization study with recombinant SARS-CoV-2 RBD as vaccine antigen. A group of BALB/c mice were immunized with recombinant RBD at days 1, 10, and 25 ( Fig. 1a). Another group of mice were injected with an irrelevant protein (HBc, hepatitis B virus core protein), serving as control. Antisera were collected from individual mice at days 20 and 40, and analyzed for SARS2-RBD-specific antibody by ELISA. As shown in Fig.  1b, neither the day-20 nor the day-40 sera in control group exhibited any significant binding activity; in contrast, SARS2-RBD-binding activity was readily detectable for the day-20 anti-RBD sera and a significant increase in SARS2-RBD-binding was observed for the day-40 anti-RBD sera. Equal amount of individual antisera in the same groups were pooled for subsequent analyses. The day-20 and day-40 pooled anti-RBD sera dose-dependently reacted with SARS2-RBD in ELISAs (Fig. 1c) and their binding antibody titers were determined to be 1.6 × 10 5 and 3.2 × 10 6 , respectively. SARS2-RBD-binding activity of anti-RBD sera collected at day 60 (when the mice were euthanized) was comparable to that of the day-40 anti-RBD sera (Supplementary Fig. S2).
SARS2-RBD shares high homology with SARS-CoV RBD (hereafter referred as SARS-RBD) in sequence. This prompted us to evaluate the cross-reactivity of SARS2-RBD-immunized sera towards SARS-RBD. As shown in Fig. 1d, e, both individual and pooled sera from SARS2-RBD-immunized mice showed dose-dependent binding activity with SARS-RBD. SARS-RBD-binding titers of the pooled day-20 and day-40 anti-RBD sera were determined to be 4 × 10 3 and 1.6 × 10 5 , respectively.
The pooled day-40 antisera were assessed for their ability to block interaction between RBDs and human ACE2. ELISA results showed that the day-40 anti-RBD sera, but not control sera, dose-dependently inhibited hACE2-Fc binding to SARS2-RBD (Fig. 1f). Anti-RBD sera also exhibited blockade effect on the SARS-RBD/ hACE2-Fc interaction, albeit with a lower efficiency (Fig. 1g).
SARS-CoV-2 S protein has been shown to bind cellsurface ACE2 and mediate cell-cell fusion, leading to syncytia formation 8 . A cell-cell fusion assay was developed to determine whether anti-RBD sera could prevent S-mediated syncytia formation. Co-culture of 293 T cells expressing S:EGFP fusion protein and 293 T cells expressing human ACE2 fused with mCherry (hACE2: mCherry) led to detection of dual-fluorescent cells, indicating occurrence of cell-cell fusion (Supplementary Fig.  S4). The cells solely emitting green or red fluorescence and the dual-fluorescent cells were quantified by flow cytometry. Treatment with the day-40 anti-RBD antisera but not control sera significantly inhibited cell-cell fusion in an antisera dose-dependent manner ( Fig. 1l and Supplementary Fig. S5). Thus, anti-RBD sera are able to inhibit SARS2-S-mediated cell-cell fusion. Moreover, the day-40 anti-RBD antisera were also able to inhibit SARS-S-mediated cell-cell fusion, albeit with a lower efficiency ( Supplementary Fig. S6).
We then evaluated the ADE potential of the mouse antisera. Several FcR-bearing cell lines were used as target cells in ADE assays, including mouse A20 cells expressing FcγRII, human THP-1 cells expressing both FcγRI and FcγRII, and K562 cells expressing human FcγRII. THP-1 and K562 cells have been shown to support mouse antibody-mediated enhancement of dengue virus infection in previous studies 9, 10 . We found that SARS2-PV entry into the three FcR-expressing cell lines was minimal (< 0.02%) whereas the same amount of SARS2-PV yielded an infection rate of~7% in VeroE6-hACE2 cells. Moreover, treatment with serially diluted control sera or anti-RBD sera did not significantly affect SARS2-PV entry of the three cell lines (Fig. 1m-o), indicating that anti-RBD sera do not promote ADE of SARS2-PV. We selected K562 cells for ADE assay with authentic SARS-CoV-2. No significant increase in viral RNA level was observed for antisera-treated samples as compared to the virus-only control regardless of the antisera dilutions (Fig. 1p). These results demonstrate that anti-RBD antibodies do not promote ADE. Our study demonstrated that anti-RBD sera exhibited potent neutralization effects on SARS-CoV-2. Moreover, anti-RBD sera inhibited SARS2-S-mediated cell-cell fusion. Importantly, anti-RBD antibodies do not promote ADE, at least not in the assay system we used. It remains to be determined whether antibodies targeting other regions of S protein could mediate ADE of SARS-CoV-2. Interestingly, anti-SARS2-RBD sera were found to crossreact with SARS-RBD. Binding titers to SARS-RBD were 20-40 fold lower than that to SARS2-RBD, probably due to the variation in RBD sequence between SARS-CoV and SARS-CoV-2. RBM varies significantly (~47% homology in amino acid sequence) between SARS-CoV and SARS-CoV-2 despite the core subdomain is highly conserved. Therefore, the observed cross-binding towards SARS-RBD and cross-neutralization of SARS-CoV is likely contributed by antibodies targeting the conserved SARS-CoV-2 core subdomain, which contains crossneutralization antibody epitopes. Our work provides important information for further development of RBDbased SARS-CoV-2 or pan-SARS-CoV subunit vaccines.