Developing Pseudovirus-Based Neutralization Assay against Omicron-Included SARS-CoV-2 Variants
<p>Detection of SARS-CoV-2 variants S protein expression in HEK293T cells. (<b>A</b>) Schematic overview of spike protein of SARS-CoV-2 variants, including Alpha (B.1.17), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), and Omicron (B.1.1.529). Amino acid mutations in comparison to the Wuhan-Hu-1 sequence are indicated. RBD, receptor binding domain; NTD, N-terminal domain. (<b>B</b>) Detection of SARS-CoV-2 S protein expression in HEK293T cells by immunofluorescence. The recombinant plasmids containing full-length S genes of SARS-CoV-2 variants were individually transfected into HEK293T cells. Cells transfected with an empty pCAGGS vector with the same procedure were used as the negative control. The cells were fixed after 48 h of incubation and labeled with the corresponding antibodies. Nuclei were stained with DAPI.</p> "> Figure 2
<p>Optimization of SARS-CoV-2 variants pseudovirus production. (<b>A</b>) Schematic representation of the SARS-CoV-2 variants pseudovirus production and neutralization assay. The HIV backbone vector pNL4-3.Luc.R-E- plasmids were cotransfected with pCAGGS-Alpha-S, pCAGGS-Beta-S, pCAGGS-Gamma-S, pCAGGS-Kappa-S, or pCAGGS-Omicron-S, respectively into HEK293T cells to package the pseudotyped lentiviral particles. The supernatants containing SARS-CoV-2 variants pseudovirus with S protein were collected and then ACE2-293T cells were used to measure the pseudoviral titer. (<b>B</b>) Effect of the ratio of the recombinant S protein expression plasmids to the HIV backbone plasmids and the collection time for pseudovirus particles on the production of pseudovirus. Cells without pseudovirus infection were used as background. The data were expressed as mean relative luciferase units (RLU) ± standard deviation (SD) of 3 parallel wells in 96-well culture plates.</p> "> Figure 3
<p>Validation of the neutralization sensitivity of SARS-CoV-2 pseudotyped variants. (<b>A</b>) Neutralizing curves of monoclonal antibodies against pseudotyped SARS-CoV-2 variants. Data are representative of at least two independent experiments. Mean ± SD was shown. (<b>B</b>) The inhibition activity of ten COVID-19 convalescent plasma samples against pseudotyped SARS-CoV-2 variants. Six plasma samples from healthy individuals were tested as negative controls (NC). The initial dilutions for both positive and negative samples were 1:10, followed by a 3-fold serial dilution. Samples were tested in triplicates and the experiments were repeated at least twice. Data from one of at least two independent experiments are presented in Mean ± SD.</p> "> Figure 3 Cont.
<p>Validation of the neutralization sensitivity of SARS-CoV-2 pseudotyped variants. (<b>A</b>) Neutralizing curves of monoclonal antibodies against pseudotyped SARS-CoV-2 variants. Data are representative of at least two independent experiments. Mean ± SD was shown. (<b>B</b>) The inhibition activity of ten COVID-19 convalescent plasma samples against pseudotyped SARS-CoV-2 variants. Six plasma samples from healthy individuals were tested as negative controls (NC). The initial dilutions for both positive and negative samples were 1:10, followed by a 3-fold serial dilution. Samples were tested in triplicates and the experiments were repeated at least twice. Data from one of at least two independent experiments are presented in Mean ± SD.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmids and Cells
2.2. Analysis of SARS-CoV-2 Variant S Protein Expression
2.3. Production and Titration of SARS-CoV-2 Pseudotyped Variants
2.4. Neutralization Assay
3. Results
3.1. Construction of the Recombinant Plasmids Expressing SARS-CoV-2 Variants Spike Proteins
3.2. Optimization of SARS-CoV-2 Variants Pseudovirus Production
3.3. Validation of the Neutralization Sensitivity of SARS-CoV-2 Pseudotyped Variants
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Garcia-Beltran, W.F.; Lam, E.C.; St Denis, K.; Nitido, A.D.; Garcia, Z.H.; Hauser, B.M.; Feldman, J.; Pavlovic, M.N.; Gregory, D.J.; Poznansky, M.C.; et al. Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell 2021, 184, 2372–2383.e9. [Google Scholar] [CrossRef] [PubMed]
- Faria, N.R.; Mellan, T.A.; Whittaker, C.; Claro, I.M.; Candido, D.D.S.; Mishra, S.; Crispim, M.A.E.; Sales, F.C.S.; Hawryluk, I.; McCrone, J.T.; et al. Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil. Science 2021, 372, 815–821. [Google Scholar] [CrossRef] [PubMed]
- Wibmer, C.K.; Ayres, F.; Hermanus, T.; Madzivhandila, M.; Kgagudi, P.; Oosthuysen, B.; Lambson, B.E.; de Oliveira, T.; Vermeulen, M.; van der Berg, K.; et al. SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma. Nat. Med. 2021, 27, 622–625. [Google Scholar] [CrossRef] [PubMed]
- Kuzmina, A.; Khalaila, Y.; Voloshin, O.; Keren-Naus, A.; Boehm-Cohen, L.; Raviv, Y.; Shemer-Avni, Y.; Rosenberg, E.; Taube, R. SARS-CoV-2 spike variants exhibit differential infectivity and neutralization resistance to convalescent or post-vaccination sera. Cell Host Microbe 2021, 29, 522–528.e2. [Google Scholar] [CrossRef]
- Shen, X.; Tang, H.; McDanal, C.; Wagh, K.; Fischer, W.; Theiler, J.; Yoon, H.; Li, D.; Haynes, B.F.; Sanders, K.O.; et al. SARS-CoV-2 variant B.1.1.7 is susceptible to neutralizing antibodies elicited by ancestral spike vaccines. Cell Host Microbe 2021, 29, 529–539.e3. [Google Scholar] [CrossRef]
- Koehler, M.; Ray, A.; Moreira, R.A.; Juniku, B.; Poma, A.B.; Alsteens, D. Molecular insights into receptor binding energetics and neutralization of SARS-CoV-2 variants. Nat. Commun. 2021, 12, 6977. [Google Scholar] [CrossRef]
- Cui, Z.; Liu, P.; Wang, N.; Wang, L.; Fan, K.; Zhu, Q.; Wang, K.; Chen, R.; Feng, R.; Jia, Z. Structural and functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron. Cell 2022, 185, 860–871.e13. [Google Scholar] [CrossRef]
- Hoffmann, M.; Krüger, N.; Schulz, S.; Cossmann, A.; Rocha, C.; Kempf, A.; Nehlmeier, I.; Graichen, L.; Moldenhauer, A.-S.; Winkler, M.S. The Omicron variant is highly resistant against antibody-mediated neutralization: Implications for control of the COVID-19 pandemic. Cell 2022, 185, 447–456.e11. [Google Scholar] [CrossRef]
- Starr, T.N.; Greaney, A.J.; Hilton, S.K.; Ellis, D.; Crawford, K.H.D.; Dingens, A.S.; Navarro, M.J.; Bowen, J.E.; Tortorici, M.A.; Walls, A.C.; et al. Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. Cell 2020, 182, 1295–1310.e20. [Google Scholar] [CrossRef]
- Xie, X.; Liu, Y.; Liu, J.; Zhang, X.; Zou, J.; Fontes-Garfias, C.R.; Xia, H.; Swanson, K.A.; Cutler, M.; Cooper, D.; et al. Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera. Nat. Med. 2021, 27, 620–621. [Google Scholar] [CrossRef]
- Liu, Z.; VanBlargan, L.A.; Bloyet, L.M.; Rothlauf, P.W.; Chen, R.E.; Stumpf, S.; Zhao, H.; Errico, J.M.; Theel, E.S.; Liebeskind, M.J.; et al. Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization. Cell Host Microbe 2021, 29, 477–488.e4. [Google Scholar] [CrossRef] [PubMed]
- Harvey, W.T.; Carabelli, A.M.; Jackson, B.; Gupta, R.K.; Thomson, E.C.; Harrison, E.M.; Ludden, C.; Reeve, R.; Rambaut, A.; Peacock, S.J. SARS-CoV-2 variants, spike mutations and immune escape. Nat. Rev. Microbiol. 2021, 19, 409–424. [Google Scholar] [CrossRef] [PubMed]
- Khan, A.; Zia, T.; Suleman, M.; Khan, T.; Ali, S.S.; Abbasi, A.A.; Mohammad, A.; Wei, D.Q. Higher infectivity of the SARS-CoV-2 new variants is associated with K417N/T, E484K, and N501Y mutants: An insight from structural data. J. Cell. Physiol. 2021, 236, 7045–7057. [Google Scholar] [CrossRef] [PubMed]
- Wang, R.; Zhang, Q.; Ge, J.; Ren, W.; Zhang, R.; Lan, J.; Ju, B.; Su, B.; Yu, F.; Chen, P.; et al. Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species. Immunity 2021, 54, 1611–1621.e5. [Google Scholar] [CrossRef] [PubMed]
- Deng, X.; Garcia-Knight, M.A.; Khalid, M.M.; Servellita, V.; Wang, C.; Morris, M.K.; Sotomayor-González, A.; Glasner, D.R.; Reyes, K.R.; Gliwa, A.S.; et al. Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant. Cell 2021, 184, 3426–3437.e8. [Google Scholar] [CrossRef] [PubMed]
- McCallum, M.; Walls, A.C.; Sprouse, K.R.; Bowen, J.E.; Rosen, L.E.; Dang, H.V.; De Marco, A.; Franko, N.; Tilles, S.W.; Logue, J.; et al. Molecular basis of immune evasion by the Delta and Kappa SARS-CoV-2 variants. Science 2021, 374, 1621–1626. [Google Scholar] [CrossRef]
- Mannar, D.; Saville, J.W.; Zhu, X.; Srivastava, S.S.; Berezuk, A.M.; Tuttle, K.S.; Marquez, A.C.; Sekirov, I.; Subramaniam, S. SARS-CoV-2 Omicron variant: Antibody evasion and cryo-EM structure of spike protein-ACE2 complex. Science 2022, 375, 760–764. [Google Scholar] [CrossRef]
- Sztain, T.; Ahn, S.H.; Bogetti, A.T.; Casalino, L.; Goldsmith, J.A.; Seitz, E.; McCool, R.S.; Kearns, F.L.; Acosta-Reyes, F.; Maji, S.; et al. A glycan gate controls opening of the SARS-CoV-2 spike protein. Nat. Chem. 2021, 13, 963–968. [Google Scholar] [CrossRef]
- Jawad, B.; Adhikari, P.; Podgornik, R.; Ching, W.Y. Binding Interactions between Receptor-Binding Domain of Spike Protein and Human Angiotensin Converting Enzyme-2 in Omicron Variant. J. Phys. Chem. Lett. 2022, 13, 3915–3921. [Google Scholar] [CrossRef]
- Cao, Y.; Wang, J.; Jian, F.; Xiao, T.; Song, W.; Yisimayi, A.; Huang, W.; Li, Q.; Wang, P.; An, R. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature 2022, 602, 657–663. [Google Scholar] [CrossRef]
- Johnson, M.C.; Lyddon, T.D.; Suarez, R.; Salcedo, B.; LePique, M.; Graham, M.; Ricana, C.; Robinson, C.; Ritter, D.G. Optimized pseudotyping conditions for the SARS-COV-2 spike glycoprotein. J. Virol. 2020, 94, e01062-20. [Google Scholar] [CrossRef] [PubMed]
- Giroglou, T.; Cinatl, J., Jr.; Rabenau, H.; Drosten, C.; Schwalbe, H.; Doerr, H.W.; Von Laer, D. Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein. J. Virol. 2004, 78, 9007–9015. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zheng, Y.; Larragoite, E.T.; Williams, E.S.; Lama, J.; Cisneros, I.; Delgado, J.C.; Slev, P.; Rychert, J.; Innis, E.A.; Coiras, M. Neutralization assay with SARS-CoV-1 and SARS-CoV-2 spike pseudotyped murine leukemia virions. Virol. J. 2021, 18, 1. [Google Scholar] [CrossRef] [PubMed]
- Salazar-García, M.; Acosta-Contreras, S.; Rodríguez-Martínez, G.; Cruz-Rangel, A.; Flores-Alanis, A.; Patiño-López, G.; Luna-Pineda, V.M. Pseudotyped vesicular stomatitis virus-severe acute respiratory syndrome-coronavirus-2 spike for the study of variants, vaccines, and therapeutics against coronavirus disease 2019. Front. Microbiol. 2021, 12, 817200. [Google Scholar] [CrossRef] [PubMed]
- Ou, X.; Liu, Y.; Lei, X.; Li, P.; Mi, D.; Ren, L.; Guo, L.; Guo, R.; Chen, T.; Hu, J. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 2020, 11, 1620. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhang, L.; Li, Q.; Liang, Z.; Li, T.; Liu, S.; Cui, Q.; Nie, J.; Wu, Q.; Qu, X.; Huang, W. The significant immune escape of pseudotyped SARS-CoV-2 Variant Omicron. Emerg. Microbes Infect. 2022, 11, 1–5. [Google Scholar] [CrossRef]
- Xiong, H.-L.; Wu, Y.-T.; Cao, J.-L.; Yang, R.; Liu, Y.-X.; Ma, J.; Qiao, X.-Y.; Yao, X.-Y.; Zhang, B.-H.; Zhang, Y.-L. Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells. Emerg. Microbes Infect. 2020, 9, 2105–2113. [Google Scholar] [CrossRef]
- Zhao, G.; Du, L.; Ma, C.; Li, Y.; Li, L.; Poon, V.K.; Wang, L.; Yu, F.; Zheng, B.-J.; Jiang, S. A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV. Virol. J. 2013, 10, 266. [Google Scholar] [CrossRef] [Green Version]
- Du, L.; Zhao, G.; Zhang, X.; Liu, Z.; Yu, H.; Zheng, B.-J.; Zhou, Y.; Jiang, S. Development of a safe and convenient neutralization assay for rapid screening of influenza HA-specific neutralizing monoclonal antibodies. Biochem. Biophys. Res. Commun. 2010, 397, 580–585. [Google Scholar] [CrossRef]
- Han, C.; Johnson, J.; Dong, R.; Kandula, R.; Kort, A.; Wong, M.; Yang, T.; Breheny, P.J.; Brown, G.D.; Haim, H. Key Positions of HIV-1 Env and Signatures of Vaccine Efficacy Show Gradual Reduction of Population Founder Effects at the Clade and Regional Levels. mBio 2020, 11, e00126-20. [Google Scholar] [CrossRef]
- Strebel, K. HIV accessory proteins versus host restriction factors. Curr. Opin. Virol. 2013, 3, 692–699. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lassen, K.G.; Hebbeler, A.M.; Bhattacharyya, D.; Lobritz, M.A.; Greene, W.C. A flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs. PLoS ONE 2012, 7, e30176. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hansen, J.; Baum, A.; Pascal, K.E.; Russo, V.; Giordano, S.; Wloga, E.; Fulton, B.O.; Yan, Y.; Koon, K.; Patel, K.; et al. Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail. Science 2020, 369, 1010–1014. [Google Scholar] [CrossRef] [PubMed]
- Pinto, D.; Park, Y.-J.; Beltramello, M.; Walls, A.C.; Tortorici, M.A.; Bianchi, S.; Jaconi, S.; Culap, K.; Zatta, F.; De Marco, A. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 2020, 583, 290–295. [Google Scholar] [CrossRef]
- Ju, B.; Zhang, Q.; Ge, J.; Wang, R.; Sun, J.; Ge, X.; Yu, J.; Shan, S.; Zhou, B.; Song, S.; et al. Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature 2020, 584, 115–119. [Google Scholar] [CrossRef]
- Westendorf, K.; Žentelis, S.; Wang, L.; Foster, D.; Vaillancourt, P.; Wiggin, M.; Lovett, E.; van der Lee, R.; Hendle, J.; Pustilnik, A.; et al. LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants. Cell Rep. 2022, 39, 110812. [Google Scholar] [CrossRef]
- Rappazzo, C.G.; Tse, L.V.; Kaku, C.I.; Wrapp, D.; Sakharkar, M.; Huang, D.; Deveau, L.M.; Yockachonis, T.J.; Herbert, A.S.; Battles, M.B.; et al. Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody. Science 2021, 371, 823–829. [Google Scholar] [CrossRef]
- Iketani, S.; Liu, L.; Guo, Y.; Liu, L.; Chan, J.F.-W.; Huang, Y.; Wang, M.; Luo, Y.; Yu, J.; Chu, H. Antibody evasion properties of SARS-CoV-2 Omicron sublineages. Nature 2022, 604, 553–556. [Google Scholar] [CrossRef]
- Liu, L.; Iketani, S.; Guo, Y.; Chan, J.F.-W.; Wang, M.; Liu, L.; Luo, Y.; Chu, H.; Huang, Y.; Nair, M.S. Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2. Nature 2022, 602, 676–681. [Google Scholar] [CrossRef]
- Chi, X.; Yan, R.; Zhang, J.; Zhang, G.; Zhang, Y.; Hao, M.; Zhang, Z.; Fan, P.; Dong, Y.; Yang, Y. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science 2020, 369, 650–655. [Google Scholar] [CrossRef]
- Cameroni, E.; Bowen, J.E.; Rosen, L.E.; Saliba, C.; Zepeda, S.K.; Culap, K.; Pinto, D.; VanBlargan, L.A.; De Marco, A.; di Iulio, J. Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift. Nature 2022, 602, 664–670. [Google Scholar] [CrossRef] [PubMed]
- Catanese, M.T.; Dorner, M. Advances in experimental systems to study hepatitis C virus in vitro and in vivo. Virology 2015, 479, 221–233. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yang, Y.; Du, L.; Liu, C.; Wang, L.; Ma, C.; Tang, J.; Baric, R.S.; Jiang, S.; Li, F. Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc. Natl. Acad. Sci. USA 2014, 111, 12516–12521. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Nie, J.; Li, Q.; Wu, J.; Zhao, C.; Hao, H.; Liu, H.; Zhang, L.; Nie, L.; Qin, H.; Wang, M. Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg. Microbes Infect. 2020, 9, 680–686. [Google Scholar] [CrossRef] [Green Version]
- Crawford, K.H.; Eguia, R.; Dingens, A.S.; Loes, A.N.; Malone, K.D.; Wolf, C.R.; Chu, H.Y.; Tortorici, M.A.; Veesler, D.; Murphy, M. Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for neutralization assays. Viruses 2020, 12, 513. [Google Scholar] [CrossRef]
- Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Krüger, N.; Herrler, T.; Erichsen, S.; Schiergens, T.S.; Herrler, G.; Wu, N.-H.; Nitsche, A. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 2020, 181, 271–280.e8. [Google Scholar] [CrossRef]
- Walls, A.C.; Park, Y.J.; Tortorici, M.A.; Wall, A.; McGuire, A.T.; Veesler, D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 2020, 181, 281–292.e6. [Google Scholar] [CrossRef]
- Wang, S.; Qiu, Z.; Hou, Y.; Deng, X.; Xu, W.; Zheng, T.; Wu, P.; Xie, S.; Bian, W.; Zhang, C.; et al. AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells. Cell Res. 2021, 31, 126–140. [Google Scholar] [CrossRef]
- Gu, Y.; Cao, J.; Zhang, X.; Gao, H.; Wang, Y.; Wang, J.; He, J.; Jiang, X.; Zhang, J.; Shen, G.; et al. Receptome profiling identifies KREMEN1 and ASGR1 as alternative functional receptors of SARS-CoV-2. Cell Res. 2022, 32, 24–37. [Google Scholar] [CrossRef]
- Zhang, L.; Li, Q.; Liu, Q.; Huang, W.; Nie, J.; Wang, Y. A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system. Hum. Vaccines Immunother. 2017, 13, 1811–1817. [Google Scholar] [CrossRef] [Green Version]
- Tseng, S.H.; Lam, B.; Kung, Y.J.; Lin, J.; Liu, L.; Tsai, Y.C.; Ferrall, L.; Roden, R.B.S.; Wu, T.C.; Hung, C.F. A novel pseudovirus-based mouse model of SARS-CoV-2 infection to test COVID-19 interventions. J. Biomed. Sci. 2021, 28, 34. [Google Scholar] [CrossRef] [PubMed]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Sun, H.; Xu, J.; Zhang, G.; Han, J.; Hao, M.; Chen, Z.; Fang, T.; Chi, X.; Yu, C. Developing Pseudovirus-Based Neutralization Assay against Omicron-Included SARS-CoV-2 Variants. Viruses 2022, 14, 1332. https://doi.org/10.3390/v14061332
Sun H, Xu J, Zhang G, Han J, Hao M, Chen Z, Fang T, Chi X, Yu C. Developing Pseudovirus-Based Neutralization Assay against Omicron-Included SARS-CoV-2 Variants. Viruses. 2022; 14(6):1332. https://doi.org/10.3390/v14061332
Chicago/Turabian StyleSun, Hancong, Jinghan Xu, Guanying Zhang, Jin Han, Meng Hao, Zhengshan Chen, Ting Fang, Xiangyang Chi, and Changming Yu. 2022. "Developing Pseudovirus-Based Neutralization Assay against Omicron-Included SARS-CoV-2 Variants" Viruses 14, no. 6: 1332. https://doi.org/10.3390/v14061332