SARS-CoV-2 Neutralization Assay System using Pseudo-lentivirus

Anastasia Armimi, Afina Firdaus Syuaib, Katherine Vanya, Marselina Irasonia Tan, Dessy Natalia, David Virya Chen, Chikako Ono, Yoshiharu Matsuura, Anita Artarini, Ernawati Arifin Giri-Rachman


BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects humans' lower respiratory tracts and causes coronavirus disease-2019 (COVID-19). Neutralizing antibodies is one of the adaptive immune system responses that can reduce SARS-CoV-2 infection. This study aimed to develop a SARS-CoV-2 neutralization assay system using pseudo-lentivirus.

METHODS: The plasmid used for pseudo-lentivirus production was characterized using restriction analysis. The gene encoding for SARS-CoV-2 spike protein was confirmed using sequencing. The transfection pseudo-lentivirus optimal condition was determined by choosing the transfection reagents and adding centrifugation step. Optimal pseudo-lentivirus infection was analysed using fluorescent assay and luciferase assay. The optimal condition of pseudo-lentivirus infection was determined by the target cell type and the number of pseudo-lentiviruses used for neutralization test. SARS-CoV-2 pseudo-lentivirus was used to detect neutralizing antibodies from serum samples.

RESULTS: The plasmid used for pseudo-lentivirus production was characterized and confirmed to have no mutations. Lipofectamine 2000 reagent generated pseudo-lentivirus with a higher ability to infect target cells, as indicated by a percentage green fluorescent protein (GFP) of 12.68%. Pseudo-lentivirus centrifuged obtained more stable results in luciferase expression. Optimal pseudo-lentivirus infection conditions were obtained using puromycin-selected HEK 293T-ACE2 cells as target cells. The number of pseudo-lentiviruses used in the neutralization assay system was multiplicity of infection (MOI) 0.075. Serum A samples with a 1:10 dilution had the highest neutralizing antibody activity.

CONCLUSION: This study shows that SARS-CoV-2 neutralization assay system using pseudo-lentivirus successfully detected neutralizing antibodies in human serum, which were indicated by a decrease in the percentage of pseudo-lentivirus infections.

KEYWORDS: COVID-19, neutralizing antibody, neutralization assay, pseudo-lentivirus, SARS-COV-2

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Rhein C, Scholz T, Henss L, Wiedemann R, Schwarz T, Rodionov R, et al. Comparison of potency assays to assess SARS-CoV-2 neutralizing antibody capacity in COVID-19 convalescent plasma J Virol Methods. 2021; 288: 114031, CrossRef.

Yuliawuri H, Christian EJ, Steven N. Non-synonymous mutation analsis of SARS-CoV-2 ORF3a in Indonesia. Mol Cell Biomed Science. 2022; 6 (1): 20-7, CrossRef.

Nie J, Li Q, Wu J, Zhao C, Hao H, Liu H, et al. Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay. Nat Protoc. 2020; 15(11): 3699-715, CrossRef.

Lu Y, Wang J, Li Q, Hu H, Lu J, Chen Z. Advances in Neutralization Assays for SARS-CoV-2. Scand J Immunol. 2021; 94(3): e13088, CrossRef.

Jiang S, Hilyer C, and Du L. Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses. Trends Immunol. 2020; 41(5): 355-9, CrossRef.

Chen M, and Zhang X. Construction and applications of SARS-CoV-2 pseudoviruses: a mini review. Int J Biol Sci. 2021; 17: 1574-80, CrossRef.

Xiang Q, Li L, Wu J, Tian M, Fu Y. Application of pseudovirus system in the development of vaccine, antiviral-drugs, and neutralizing antibodies. Microbiol Res. 2022; 258: 126993, CrossRef.

Hyseni I, Molesti E, Benincasa L, Piu P, Casa E, Temperton NJ, et al. Characterisation of SARS-CoV-2 lentiviral pseudotypes and correlation between pseudotype-based neutralisation assays and live virus-based micro neutralisation assays. Viruses. 2020; 12(9): 1011, CrossRef.

Tolah A, Sohrab S, Tolah K, Hassan A, Kafrawy S, Azhar E. Evaluation of a pseudovirus neutralization assay for SARS-CoV-2 and correlation with live virus-based micro neutralization assay. Diagnostics. 2021; 11(6): 994, CrossRef.

Xiong H, Wu Y, Cao J, Yang R, Liu Y, Ma J, et al. 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(1): 2105-13, CrossRef.

Crawford K, Eguia R, Dingens A, Loes A, Malone K, Wolf C, et al. Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for neutralization assays. Viruses. 2020; 12(5): 513, CrossRef.

Zhang J, Cai Y, Xiao T, Lu J, Peng H, Sterling SM, et al. Structural impact on SARS-CoV-2 spike protein by D614G substitution. Science. 2021; 372(6541): 525-30, CrossRef.

Korber B, Fischer W, Gnanakaran, S, Yoon H, Theiler H, Abfalterer W, et al. Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell. 2020; 182(4): 812-27.e19, CrossRef.

Riepler L, Rössler A, Falch A, Volland A, Borena W, von Laer D, et al. Comparison of four SARS-CoV-2 neutralization assays. Vaccines. 2020; 9(1): 13, CrossRef.

Dalby B, Cates S, Harris A, Ohki EC, Tilkins ML, Price PJ, et al. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods. 2004; 33(2): 95-103, CrossRef.

Wang T, Larcher L, Ma L, Veedu R. Systematic screening of commonly used commercial transfection reagents towards efficient transfection of single-stranded oligonucleotides. Molecules. 2018; 23(10): 2564, CrossRef.

Douzandegan Y, Gray Z, Mohebbi A, Moradi A, Tabarraei A. Optimization of kyse-30 esophagus cancer cell line transfection using lipofectamine 2000. J Clin Basic Res. 2017; 1(1): 16-20, article.

Capcha J, Lambert G, Dykxhoorn D, Salerno A, Hare J, Whitt M, et al. Generation of SARS-CoV-2 spike pseudotyped virus for viral entry and neutralization assays: a 1-week protocol. Front Cardiovasc Med. 2021; 7: 618651, CrossRef.

Moore MJ, Dorfman T, Li W, Wong SK, Li Y, Kuhn JH, et al. Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2. J Virol. 2004; 78(19): 10628-35, CrossRef.

Donofrio G, Franceshi V, Macchi F, Russo L, Rocci A, Marchica V, et al. A simplified SARS-CoV-2 pseudovirus neutralization assay. Vaccines. 2021; 9(4): 389, CrossRef.

Case J, Rothlauf P, Chen R, Liu Z, Zhao H, Kim A, 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): 475-85.e5, CrossRef.

Cantoni D, Mayora-Neto M, Temperton N. The role of pseudotype neutralization assays in understanding SARS CoV-2. Oxf Open Immunol. 2021; 2(1): iqab005, CrossRef.


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