key: cord-1055571-mlckuhgf
authors: Li, Tuofan; Kan, Qiuqi; Ge, Jinying; Wan, Zhimin; Yuan, Mengqi; Huang, Yao; Xie, Quan; Yang, Yi; Shao, Hongxia; Li, Xiangdong; Ye, Lilin; Qin, Aijian; Bu, Zhigao; Liu, Pinghuang; Ye, Jianqiang
title: A novel linear and broadly neutralizing peptide in the SARS-CoV-2 S2 protein for universal vaccine development
date: 2021-10-13
journal: Cell Mol Immunol
DOI: 10.1038/s41423-021-00778-6
sha: 919c9ea14d957fd7cdd6cbcf0f2a4b967c585950
doc_id: 1055571
cord_uid: mlckuhgf

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the S and S2 proteins of both SARS-CoV-2 and SARS-CoV, as revealed by immunofluorescence assays and western blotting ( Fig. S1a-c) , confirming their specificity for the S2 protein.

To examine neutralization, these mAbs were tested in assays using hACE2/293T cells. mAbs B10, C10, and G5 efficiently neutralized SARS-CoV-2 S-pseudovirus, with half maximal inhibitory concentrations (IC 50 ) of 18.05, 1.367, and 2.21 μg/mL, respectively (Fig. 1d) . Conversely, mAb E2 did not show detectable neutralization of SARS-CoV-2 S-pseudovirus. mAbs B10, C10, and G5 also effectively neutralized SARS-CoV S-pseudovirus, with IC 50 values of 5.295, 2.325, and 2.695 μg/mL (Fig. 1e) . Antibody G5 neutralized a SARS-CoV-2 pseudovirus bearing the S protein from the B.1.351 variant lineage, with an IC 50 of 2.608 μg/mL (Fig. 1f ). In line with these data, mAbs B10, C10, and G5 (tested with mouse ascites containing mAbs) were able to neutralize wild-type SARS-CoV-2 (Fig. 1g) . Moreover, mAbs B10, C10, and G5, but not mAb E2 and the control mAb, inhibited the spread of a vesicular stomatitis virus expressing SARS-CoV-2 S and green fluorescent proteins (VSV-SARS-CoV2-S-GFP) in Vero E6 cells (Fig. 1h) . Moreover, immunoprecipitation assays revealed that mAbs B10, C10, and G5 efficiently immunoprecipitated both the S and S2 proteins of SARS-CoV-2 but that mAb E2 only immunoprecipitated the S protein of SARS-CoV-2 at low efficiency (Fig. 1i) .

Western blotting using truncated P4 demonstrated that all the mAbs reacted with only S2-P4 and S-P4-1 and not the other truncations (Table S2 and Fig. S1d ,e), which indicates that the epitopes recognized by these mAbs are located in 1147 SFKEELD-KYFKNHTSP 1162 in S2 and that the eight amino acids in 1155 YFKNHTSP 1162 are critical for the epitopes. Whether these mAbs recognize the same epitope or different epitopes needs to be further elucidated. To evaluate whether the epitope in the P4 peptide is of value for vaccine development, a trimeric P4 expression vector, 3×P4, was constructed for protein expression (Table S3 and Fig. S2a , b). Trimeric P4 was readily recognized by mAb G5, highlighting the antigenicity of the expressed trimeric P4 protein (Fig. 1j ). In addition, sera from mice immunized with the purified trimeric P4 protein neutralized SARS-CoV-2 S-pseudovirus Fig. 1 A novel linear and broadly neutralizing peptide in the S2 protein of SARS-CoV-2. a ELISA with the indicated peptides as antigens was used to test sera from recovered COVID-19 patients. LOD, limit of detection. b Sequence alignment showing the location of P4 in SARS-CoV-2, SARS-CoV, BatCoV RaTG13 and SARS-CoV-2 variants. c A 3D structure model diagram of the SARS-CoV-2 S protein generated with PyMOL based on PDB 6VSB_1_1_1 [10] . The P4 peptide is shown in red. d and e Neutralizing activity of B10, C10, E2, and G5 mAbs against SARS-CoV-2 S pseudovirus (d) and SARS-CoV S pseudovirus (e). f Neutralizing activity of mAb G5 against the SARS-CoV-2 S pseudovirus B.1.351 South Africa variant. In (d-f), measurements were performed using purified mAbs and SARS-CoV-2 and SARS-CoV S pseudoviruses in hACE2/293T cells, and the results are shown as the percentage of inhibition (mean ± SD). g Neutralizing activity of B10, C10, E2, and G5 mAbs against wild-type SARS-CoV-2. The assay was performed with mouse ascites and wild-type SARS-CoV-2 in Vero E6 cells, and the data are expressed as the log2 number of dilution. h Inhibition of S protein-mediated virus spread by mAbs. i Affinities of B10, C10, E2, and G5 mAbs for the S protein were analyzed by immunoprecipitation assays. 293T cells were transfected with a plasmid carrying the SARS-CoV-2 S gene, and mAbs B10, C10, E2, and G5 were used as the capture antibody to perform immunoprecipitation, followed by western blot analysis using mAb G5. j Identification of the trimeric P4 protein by western blotting. The supernatants and pellets from lysates of BL21 cells transformed with 3×P4 or the pColdI vector were analyzed by western blotting using mAb G5 as the primary antibody. k Neutralization of SARS-CoV-2 pseudovirus by sera from mice immunized with the trimeric P4 protein. The assay was performed with mouse sera and SARS-CoV-2 pseudovirus in hACE2/293T cells. l Inhibition of S-mediated cell-cell fusion by mAbs. m A schematic diagram showing the potential mechanism by which mAbs B10, C10, E2, and G5 neutralize SARS-CoV-2/SARS-CoV. mAbs against 1147 SFKEELDKYFKNHTSP 1162 inhibit SARS-CoV-2/SARS-CoV viral entry by inhibiting fusion between the virus and cell membrane in a dose-dependent manner (Fig. 1k) . These data demonstrate that trimeric P4 is a potential vaccine candidate for broad protection against SARS-CoV-2.

To further investigate the mechanism underlying inhibition of SARS-CoV-2 by these mAbs, we performed a cell-cell fusion inhibition assay, as previously described [7] . mAb G5, but not E2 or the control mAb, inhibited SARS-CoV-2-S-mediated cell-cell fusion in a dose-dependent manner (Fig. 1l) . Thus, antibodies against P4 might inhibit viral entry by blocking fusion of the virus with the host cell membrane.

In conclusion, we identified a novel linear and broadly neutralizing peptide in the S2 protein of SARS-CoV-2. Different from other crossneutralizing epitopes [4] [5] [6] [7] [8] [9] , this linear peptide ( 1147 SFKEELD-KYFKNHTSP 1162 ) is conserved across SARS-CoV, BatCoV RaTG13, SARS-CoV-2, and SARS-CoV-2 variants. mAbs targeting this peptide efficiently neutralized SARS-CoV-2 and SARS-CoV S-pseudovirus as well as wild-type SARS-CoV-2. These antibodies also inhibited both S-mediated cell-cell membrane fusion and viral spread among cells. Furthermore, the recombinant trimeric P4 carrying the 1147 SFKEELD-KYFKNHTSP 1162 epitope induced neutralizing antibodies against SARS-CoV-2 S-pseudovirus. Taken together, these findings suggest that antibodies targeting the peptide 1147 SFKEELDKYFKNHTSP 1162 may neutralize both SARS-CoV-2 and SARS-CoV by preventing fusion between the virus and cell membrane (Fig. 1m) . In light of the rapid emergence of antigenic variants of SARS-CoV-2, the identification of neutralizing peptides such as 1147 SFKEELDKYFKNHTSP 1162 represents an opportunity for the development of universal vaccines and therapeutic reagents against current pandemic strains as well as future SARS-CoV-2 mutants.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Structural basis for broad coronavirus neutralization

Linear epitopes of SARS-CoV-2 spike protein elicit neutralizing antibodies in COVID-19 patients

Linear epitope landscape of the SARS-CoV-2 Spike protein constructed from 1,051 COVID-19 patients

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies crossreactivity with endemic human coronaviruses

Cross-reactive serum and memory B-cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection

Developing a Fully Glycosylated Full-Length SARS-CoV-2 Spike Protein Model in a Viral Membrane

The authors declare no competing interests.

All animal experiments were performed in accordance with the institutional animal care guidelines approved by the Animal Care Committee of Yangzhou University, China.

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41423-021-00778-6.Correspondence and requests for materials should be addressed to Aijian Qin, Zhigao Bu, Pinghuang Liu or Jianqiang Ye.Reprints and permission information is available at http://www.nature.com/ reprintsOpen 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/.