TY - JOUR
T1 - Quantifying absolute neutralization titers against sars-cov-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of covid-19 sera
AU - COVIDAR Argentina Consortium
AU - Oguntuyo, Kasopefoluwa Y.
AU - Stevens, Christian S.
AU - Hung, Chuan Tien
AU - Ikegame, Satoshi
AU - Acklin, Joshua A.
AU - Kowdle, Shreyas S.
AU - Carmichael, Jillian C.
AU - Chiu, Hsin Ping
AU - Azarm, Kristopher D.
AU - Haas, Griffin D.
AU - Amanat, Fatima
AU - Klingler, Jéromine
AU - Baine, Ian
AU - Arinsburg, Suzanne
AU - Bandres, Juan C.
AU - Siddiquey, Mohammed N.A.
AU - Schilke, Robert M.
AU - Woolard, Matthew D.
AU - Zhang, Hongbo
AU - Duty, Andrew J.
AU - Kraus, Thomas A.
AU - Moran, Thomas M.
AU - Tortorella, Domenico
AU - Lim, Jean K.
AU - Gamarnik, Andrea V.
AU - Hioe, Catarina E.
AU - Zolla-Pazner, Susan
AU - Ivanov, Stanimir S.
AU - Kamil, Jeremy P.
AU - Krammer, Florian
AU - Lee, Benhur
N1 - Funding Information:
We acknowledge the following funding. K.Y.O. and C.S.S. were supported by Viral-Host Pathogenesis Training Grant T32 AI07647. K.Y.O. was additionally supported by grant F31 AI154739. S.I. and C.-T.H. were supported by postdoctoral fellowships from CHOT-SG (Fukuoka University, Japan) and the Ministry of Science and Technology (MOST, Taiwan), respectively. B.L. acknowledges flexible funding support from NIH grants R01 AI123449 and R21 AI1498033; a grant from the Department of Microbiology, Icahn School of Medicine at Mount Sinai; and the Ward-Coleman estate, which endowed the Ward-Coleman Chairs at the ISMMS. J.P.K. and S.S.I. acknowledge funding from an LSUHS COVID-19 intramural grant. J.P.K. and S.S.I. acknowledge additional funding from NIH grants AI116851 and AI143839, respectively. This work was further supported by Microbiology Laboratory Clinical Services of the Mount Sinai Health System and the Mount Sinai Health System Translational Science Hub, and by NIH grant U54TR001433 to the Department of Medicine, ISMMS (S.Z.-P.). We also acknowledge Department of Veterans Affairs Merit Review Grant I01BX003860 (C.E.H., S.Z.-P., and J.P.K.), Research Career Scientist Award 1IK6BX004607 (C.E.H.), and NIH grant AI139290 (C.E.H., S.Z.-P.). Work in the Krammer laboratory was partially supported by NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C (F.K.) and Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051 (F.K.) and generously supported by the JPB foundation, the Open Philanthropy Project (grant 2020-215611), and other philanthropic donations.
Funding Information:
The COVIDAR Argentina Consortium consists of the Fundacion Instituto Leloir-CONICET, Buenos Aires, Argentina; the Universidad Nacional de San Martin, Buenos Aires, Argentina; Laboratorio Lemos SRL, Buenos Aires, Argentina; and INBIRS-CONICET, Facultad de Medicina, Universidad de Buenos Aires, Argentina. Members of the Consortium include D. S. Ojeda, M. M. Gonzales Lopez Ledesma, G. S. Costa Navarro, H. M. Pallar?s, L. N. Sanchez, P. Perez, M. Ostrowski, S. M. Villordo, D. E. Alvarez, J. J. Caramelo, J. Carradori, M. J. Yanovsky, and A. V. Gamarnik. In addition to acknowledging the three labs that provided data for this paper, we acknowledge the generosity of the many labs that independently verified our CoV2pp VNA. We want to especially acknowledge the entirety of the Lee lab, the members of which, during a pandemic, dropped all other work and pooled resources in pursuit of developing this and other tools that are providing support to other labs doing COVID-19 work. Moreover, we acknowledge the generosity of the patients that donated blood for use in convalescent-phase serum studies and for further use in research studies similar to the work presented here. We acknowledge the following funding. K.Y.O. and C.S.S. were supported by Viral-Host Pathogenesis Training Grant T32 AI07647. K.Y.O. was additionally supported by grant F31 AI154739. S.I. and C.-T.H. were supported by postdoctoral fellowships from CHOT-SG (Fukuoka University, Japan) and the Ministry of Science and Technology (MOST, Taiwan), respectively. B.L. acknowledges flexible funding support from NIH grants R01 AI123449 and R21 AI1498033; a grant from the Department of Microbiology, Icahn School of Medicine at Mount Sinai; and the Ward-Coleman estate, which endowed the Ward-Coleman Chairs at the ISMMS. J.P.K. and S.S.I. acknowledge funding from an LSUHS COVID-19 intramural grant. J.P.K. and S.S.I. acknowledge additional funding from NIH grants AI116851 and AI143839, respectively. This work was further supported by Microbiology Laboratory Clinical Services of the Mount Sinai Health System and the Mount Sinai Health System Translational Science Hub, and by NIH grant U54TR001433 to the Department of Medicine, ISMMS (S.Z.-P.). We also acknowledge Department of Veterans Affairs Merit Review Grant I01BX003860 (C.E.H., S.Z.-P., and J.P.K.), Research Career Scientist Award 1IK6BX004607 (C.E.H.), and NIH grant AI139290 (C.E.H., S.Z.-P.). Work in the Krammer laboratory was partially supported by NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C (F.K.) and Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051 (F.K.) and generously supported by the JPB foundation, the Open Philanthropy Project (grant 2020-215611), and other philanthropic donations. We thank Randy A. Albrecht for oversight of the conventional BSL3 biocontainment facility. K.Y.O., C.S.S., and B.L conceived and designed the study. K.Y.O., C.S.S., C.T.H., S.I., J.A.A., S.S.K., J.C.C., H.-P.C., K.D.A., G.D.H., F.A., J.K., I.B., S.A., J.C.B., S.S.I., M.N.A.S., R.M.S., M.D.W., H.Z., and members of the COVIDAR Argentina Consortium collected data. G.D.H., A.J.D., T.A.K., T.M.M., D.T., J.K.L., A.V.G., C.E.H., S.Z.-P., S.S.I., J.P.K., and F.K. contributed valuable reagents, data, and/or tools. K.Y.O. and C.S.S. analyzed the data and wrote the original drafts of the paper. B.L. reviewed the draft, supported data analysis, and provided invaluable direction throughout the conceptualization and execution of the project. All authors had the opportunity to review the manuscript prior to submission, and S.I., J.C.C., K.D.A., D.T., and J.P.K. provided valuable feedback during the editing process.
Publisher Copyright:
© 2021 Oguntuyo et al.
PY - 2021
Y1 - 2021
N2 - The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glyco-protein (VSVDG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n . 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week. IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We there-fore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.
AB - The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glyco-protein (VSVDG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n . 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week. IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We there-fore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.
KW - COVID-19
KW - Convalescent-phase plasma
KW - Neutralizing antibodies
KW - SARS-CoV-2
KW - Viral neutralization assay
UR - http://www.scopus.com/inward/record.url?scp=85101033501&partnerID=8YFLogxK
U2 - 10.1128/mBio.02492-20
DO - 10.1128/mBio.02492-20
M3 - Article
C2 - 33593976
AN - SCOPUS:85101033501
VL - 12
SP - 1
EP - 23
JO - mBio
JF - mBio
SN - 2161-2129
IS - 1
M1 - e02492-20
ER -