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1. Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera

Author

Oguntuyo, Kasopefoluwa, Stevens, Christian S., Hung, Chuan Tien, Ikegame, Satoshi, Acklin, Joshua A., Kowdle, Shreyas S., Carmichael, Jillian C., Chiu, Hsin Ping, Azarm, Kristopher D., Haas, Griffin D., Amanat, Fatima, Klingler, Jéromine, Baine, Ian, Arinsburg, Suzanne, Bandres, Juan C., Siddiquey, Mohammed N. A., Schilke, Robert M., Woolard, Matthew D., Zhang, Hongbo, Duty, Andrew J., Kraus, Thomas A., Moran, Thomas M., Tortorella, Domenico, Lim, Jean K., Gamarnik, Andrea Vanesa, Hioe, Catarina E., Zolla Pazner, Susan, Ivanov, Stanimir S., Kamil, Jeremy, Krammer, Florian, Lee, Benhur, Ojeda, Diego Sebastian, González López Ledesma, María Mora, Costa Navarro, Guadalupe Soledad, Pallarés, H. M., Sanchez, Lautaro Nicolas, Perez, P., Ostrowsk, M., Villordo, S. M., Alvarez, D. E., Caramelo, J. J., Carradori, J., and Yanovsky, M. J.

Subjects
viral neutralization assay, medicine.drug_class, Enzyme-Linked Immunosorbent Assay, SARS-COV-2, Monoclonal antibody, Antibodies, Viral, Microbiology, Virus, Neutralization, Article, NEUTRALIZING ANTIBODIES, purl.org/becyt/ford/1 [https], 03 medical and health sciences, 0302 clinical medicine, Viral entry, Neutralization Tests, Virology, Biosafety level, Potency, Medicine, Humans, neutralizing antibodies, 030212 general & internal medicine, purl.org/becyt/ford/1.6 [https], Neutralizing antibody, 030304 developmental biology, convalescent-phase plasma, 0303 health sciences, biology, business.industry, SARS-CoV-2, fungi, COVID-19, VIRAL NEUTRALIZATION ASSAY, Gold standard (test), biology.organism_classification, Antibodies, Neutralizing, QR1-502, body regions, Titer, Vesicular stomatitis virus, biology.protein, Antibody, business, CONVALESCENT-PHASE PLASMA, Research Article
Abstract

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. Fil: Oguntuyo, Kasopefoluwa. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Stevens, Christian S.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Hung, Chuan Tien. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Ikegame, Satoshi. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Acklin, Joshua A.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Kowdle, Shreyas S.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Carmichael, Jillian C.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Chiu, Hsin Ping. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Azarm, Kristopher D.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Haas, Griffin D.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Amanat, Fatima. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Klingler, Jéromine. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Baine, Ian. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Arinsburg, Suzanne. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Bandres, Juan C.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Siddiquey, Mohammed N. A.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Schilke, Robert M.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Woolard, Matthew D.. State University of Louisiana; Estados Unidos Fil: Zhang, Hongbo. State University of Louisiana; Estados Unidos Fil: Duty, Andrew J.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Kraus, Thomas A.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Moran, Thomas M.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Tortorella, Domenico. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Lim, Jean K.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Hioe, Catarina E.. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Zolla Pazner, Susan. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Ivanov, Stanimir S.. State University of Louisiana; Estados Unidos Fil: Kamil, Jeremy. State University of Louisiana; Estados Unidos Fil: Krammer, Florian. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Lee, Benhur. Icahn School of Medicine at Mount Sinai; Estados Unidos Fil: Ojeda, Diego Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: González López Ledesma, María Mora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Costa Navarro, Guadalupe Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Pallarés, H. M.. No especifíca; Fil: Sanchez, Lautaro Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Perez, P.. No especifíca; Fil: Ostrowsk, M.. No especifíca; Fil: Villordo, S. M.. No especifíca; Fil: Alvarez, D. E.. No especifíca; Fil: Caramelo, J. J.. No especifíca; Fil: Carradori, J.. No especifíca; Fil: Yanovsky, M. J.. No especifíca

Published
2021
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4. Mapping the catalytic pocket of phospholipases A2 and C using a novel set of phosphatidylcholines.

Author

Caramelo JJ, Florín-Christensen J, Florín-Christensen M, and Delfino JM

Subjects
Catalytic Domain, Magnetic Resonance Spectroscopy, Models, Molecular, Phospholipases A chemistry, Substrate Specificity, Type C Phospholipases chemistry, Phosphatidylcholines chemistry, Phospholipases A metabolism, Type C Phospholipases metabolism
Abstract

A set of radioiodinatable phosphatidylcholines (PCs) derivatized with the Bolton-Hunter reagent (BHPCs) was synthesized to probe the substrate recognition and activity of phospholipases. A common feature of this series is the presence of a bulky 4-hydroxyphenyl group at the end of the fatty acyl chain attached to position sn-2. The distance between the end group and the glycerol backbone was varied by changing the length of the intervening fatty acyl chain (3-25 atoms). Except for the shortest, this chain includes at least one amide linkage. The usefulness of this series of substrates as a molecular ruler was tested by measuring the hydrolytic activities of Naja naja naja phospholipase A(2) (PLA(2)) and Bacillus cereus phospholipase C (PLC) in Triton X-100 micelles. The activity of PLA(2) proved to be highly dependent on the length of the fatty acyl chain linker, the shorter compounds (3-10 atoms) being very poor substrates. In contrast, the PLC activity profile exhibited much less discrimination. In both cases, PCs with 16-21 atom chains at position sn-2 yielded optimal activity. We interpret these findings in terms of fatty acyl chain length-related steric hindrance caused by the terminal aromatic group, affecting the activity of PLA(2) and, to a smaller extent, that of PLC. This notion agrees with the more extended recognition of aliphatic chains inside the narrow channel leading to the catalytic site in the former case. Molecular models of these substrates bound to PLA(2) were built on the basis of the crystallographic structure of Naja naja atra PLA(2) complexed with a phospholipid analogue. Docking of these substrates necessarily requires the intrusion of the bulky 4-hydroxyphenyl group inside the binding pocket and also the failure of the amide group to form hydrogen bonds inside the hydrophobic substrate channel.

Published
2000

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