Optimization and validation of a virus-like particle pseudotyped virus neutralization assay for SARS-CoV-2

Abstract

Spike-protein-based pseudotyped viruses were used to evaluate vaccines during the COVID-19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus-like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS-COV-1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild-type VLP pseudotyped virus over 100-fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS-CoV-2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze–thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra- and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r = 0.9). Suitable for high-throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS-CoV-2 based on VLP pseudotyped virus.

Keywords

neutralizing antibody / pseudotyped virus / SARS-CoV-2 / virus-like particle (VLP)

References

1	Q Li, X Guan, P Wu, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia. N Engl J Med. 2020;382(13):1199-1207.
2	W Tan, X Zhao, X Ma, et al. A novel coronavirus genome identified in a cluster of pneumonia cases—Wuhan, China 2019–2020. China CDC Wkly. 2020;4(2):61-62.
3	C Wang, PW Horby, FG Hayden, GF Gao. A novel coronavirus outbreak of global health concern. Lancet. 2020;395(10223):470-473.
4	Coronaviridae Study Group of the International Committee on Taxonomy of V. The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol. 2020;5(4):536-544.
5	R Lu, X Zhao, J Li, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020;395(10224):565-574.
6	H Zhou, X Chen, T Hu, et al. A novel bat coronavirus closely related to SARS-CoV-2 contains natural insertions at the S1/S2 cleavage site of the spike protein. Curr Biol. 2020;30(19):3896.
7	Y Luo, S Liu, J Xue, et al. High-throughput screening of spike variants uncovers the key residues that alter the affinity and antigenicity of SARS-CoV-2. Cell Discov. 2023;9(1):40.
8	M Hussain, N Jabeen, F Raza, et al. Structural variations in human ACE2 may influence its binding with SARS-CoV-2 spike protein. J Med Virol. 2020;92(9):1580-1586.
9	G Salvatori, L Luberto, M Maffei, et al. SARS-CoV-2 SPIKE PROTEIN: an optimal immunological target for vaccines. J Transl Med. 2020;18(1):222.
10	L Du, Y He, Y Zhou, S Liu, BJ Zheng, S Jiang. The spike protein of SARS-CoV—a target for vaccine and therapeutic development. Nat Rev Microbiol. 2009;7(3):226-236.
11	KL Flanagan, CR MacIntyre, PB McIntyre, MR Nelson. SARS-CoV-2 vaccines: where are we now? J Allergy Clin Immunol Pract. 2021;9(10):3535-3543.
12	L Enjuanes, S Zuniga, C Castano-Rodriguez, J Gutierrez-Alvarez, J Canton, I Sola. Molecular basis of coronavirus virulence and vaccine development. Adv Virus Res. 2016;96:245-286.
13	JH Beigel, KM Tomashek, LE Dodd, et al. Remdesivir for the treatment of Covid-19 – final report. N Engl J Med. 2020;383(19):1813-1826.
14	R Najjar-Debbiny, N Gronich, G Weber, et al. Effectiveness of paxlovid in reducing severe coronavirus disease 2019 and mortality in high-risk patients. Clin Infect Dis. 2023;76(3):e342-e349.
15	W Wen, C Chen, J Tang, et al. Efficacy and safety of three new oral antiviral treatment (molnupiravir, fluvoxamine and paxlovid) for COVID-19: a meta-analysis. Ann Med. 2022;54(1):516-523.
16	LA Jackson, EJ Anderson, NG Rouphael, et al. An mRNA vaccine against SARS-CoV-2 – preliminary report. N Engl J Med. 2020;383(20):1920-1931.
17	FP Polack, SJ Thomas, N Kitchin, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med. 2020;383(27):2603-2615.
18	MN Ramasamy, AM Minassian, KJ Ewer, et al. Safety and immunogenicity of ChAdOx1 nCoV-19 vaccine administered in a prime-boost regimen in young and old adults (COV002): a single-blind, randomised, controlled, phase 2/3 trial. Lancet. 2021;396(10267):1979-1993.
19	M Voysey, SAC Clemens, SA Madhi, et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet. 2021;397(10269):99-111.
20	ME Dieterle, D Haslwanter. A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition. Cell Host Microbe. 2020;28(3):486-496. e486.
21	J Nie, Q Li, J Wu, et al. Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg Microbes Infect. 2020;9(1):680-686.
22	R Yang, B Huang, R Ruhan, et al. Development and effectiveness of pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro. Biosaf Health. 2020;2(4):226-231.
23	S He, AA Waheed, B Hetrick, et al. PSGL-1 inhibits the incorporation of SARS-CoV and SARS-CoV-2 spike glycoproteins into pseudovirions and impairs pseudovirus attachment and infectivity. Viruses. 2020;13(1):46.
24	Q Li, Q Liu, W Huang, X Li, Y Wang. Current status on the development of pseudoviruses for enveloped viruses. Rev Med Virol. 2018;28(1):e1963.
25	JB Case, PW Rothlauf, RE Chen, et al. Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2. Cell Host Microbe. 2020;28(3):475-485. e475.
26	AM Syed, TY Taha, T Tabata, et al. Rapid assessment of SARS-CoV-2-evolved variants using virus-like particles. Science. 2021;374(6575):1626-1632.
27	H Liu, R Wu, L Yuan, et al. Introducing a cleavable signal peptide enhances the packaging efficiency of lentiviral vectors pseudotyped with Japanese encephalitis virus envelope proteins. Virus Res. 2017;229:9-16.
28	R Yadav, JK Chaudhary, N Jain, et al. Role of structural and non-structural proteins and therapeutic targets of SARS-CoV-2 for COVID-19. Cells. 2021;10(4):821.
29	Z Liang, J Tong, Z Sun, et al. Rational prediction of immunogenicity clustering through cross-reactivity analysis of thirteen SARS-CoV-2 variants. J Med Virol. 2024;96(1):e29314.
30	J Nie, W Huang, Q Liu, Y Wang. HIV-1 pseudoviruses constructed in China regulatory laboratory. Emerg Microbes Infect. 2020;9(1):32-41.
31	S Umeki, R Suzuki, Y Ema, et al. Anti-adhesive property of P-selectin glycoprotein ligand-1 (PSGL-1) due to steric hindrance effect. J Cell Biochem. 2013;114(6):1271-1285.
32	H Pang, Y Liu, X Han, et al. Protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine. J Gen Virol. 2004;85(10):3109-3113. Pt.
33	SQ Shi, JP Peng, YC Li, et al. The expression of membrane protein augments the specific responses induced by SARS-CoV nucleocapsid DNA immunization. Mol Immunol. 2006;43(11):1791-1798.
34	J Malicoat, S Manivasagam, S Zuniga, et al. Development of a single-cycle infectious SARS-CoV-2 virus replicon particle system for use in biosafety level 2 laboratories. J Virol. 2022;96(3):e0183721.
35	I Ricardo-Lax, JM Luna, TTN Thao, et al. Replication and single-cycle delivery of SARS-CoV-2 replicons. Science. 2021;374(6571):1099-1106.
36	W Wang, X Peng, Y Jin, JA Pan, D Guo. Reverse genetics systems for SARS-CoV-2. J Med Virol. 2022;94(7):3017-3031.
37	T Tanaka, A Saito, T Suzuki, et al. Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening. Antiviral Res. 2022;199:105268.
38	Y Li, X Tan, J Deng, et al. An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening in vitro and in vivo. Virol Sin. 2024;26:S1995-820X(24)00035-X
39	Y Zhang, L Zhang, J Wu, et al. A second functional furin site in the SARS-CoV-2 spike protein. Emerg Microbes Infect. 2022;11(1):182-194.
40	S Liu, Z Jia, J Nie, et al. A broader neutralizing antibody against all the current VOCs and VOIs targets unique epitope of SARS-CoV-2 RBD. Cell Discov. 2022;8(1):81.