key: cord-0830509-1u96q5ad authors: Donofrio, G.; Franceschi, V.; Macchi, F.; Russo, L.; Rocci, A.; Marchica, V.; Costa, F.; Giuliani, N.; Ferrari, C.; Missale, G. title: A simplified SARS-CoV-2 pseudovirus neutralization assay date: 2021-03-12 journal: nan DOI: 10.1101/2021.03.12.21253435 sha: 23d3ef642aa8f3ff65ba1cb0e96b19177284a28a doc_id: 830509 cord_uid: 1u96q5ad COVID-19 is an ongoing pandemic caused by the highly infectious coronavirus SARS-CoV-2 that is engaging worldwide scientific research to find a timely and effective eradication strategy. Great efforts have been put into anti-COVID-19 vaccine generation in an effort to protect the world population and block SARS-CoV-2 spread. To validate the protective efficacy of the vaccination campaign and effectively control the pandemy, it is necessary to quantify the neutralizing antibodies induction by vaccination, since they have been established to be a correlate of protection. In this work a SARS-CoV-2 pseudovirus neutralization assay, based on a replication incompetent lentivirus expressing an adapted form of CoV-2 S protein and an ACE2/TMPRSS2 stably expressing cell line, have been minimized in term of protocol steps without loss of accuracy. The goal of the present simplified neutralization system is to improve SARS-CoV-2 vaccination campaign by means of an easy and accessible approach to be performed in any medical laboratory, maintaining the sensitivity and quantitative reliability of classical serum neutralization assays. Further this assay can be easily adapted to different coronaviruses variants by simply modifying the pseudotyping vector. 150 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in of the sera where the reduction of the signal is ≥50%. Note of worthy, the titer has to be 196 multiplied per 40 because the initial volume of the sera tested is 0,025mL and it has to be 197 normalized to 1mL. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 225 CoV-2 S is a key factor for SARS-CoV-2 target cell infection [6] [7] [8] . Antibodies from COVID-226 19 convalescent patient sera can block in vitro and in vivo CoV-2 infectivity, thus being 227 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.12.21253435 doi: medRxiv preprint exploitable for CoV-2 infection diagnosis and for the evaluation of CoV-2 vaccination efficacy 228 (the titer of neutralizing antibody present in a serum is a correlate of protection), as well as for 229 preliminary test molecules able to interfere with S, ACE2 and TMPRRS2 interaction. Before perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 276 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 12, 2021. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Luciferase Units (RLUs) were compared and normalized to those derived from wells where 505 pseudovirus were added in the absence of sera (100%). Neutralization titer 50 (NT50) was 506 expressed as the maximal dilution of the sera where the reduction of the signal was ≥50%. In 507 this specific example, the NT50/25µL of the serum 7 is 1:256, that of the serum 8 is 1:128 and 508 that of the serum 9 is 1:64. However, because the titer was measured on 25 µL, the apparent 509 NT50 has to be multiplied per 40 to get the normalized NT50/mL. Therefore, the NT50/mL of the 510 sera 7 is 1:10256, that of the serum 8 is 1:5120 and that of the serum 9 is 1:2560. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.12.21253435 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.12.21253435 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.12.21253435 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.12.21253435 doi: medRxiv preprint Coronavirus 2019-nCoV: A brief perspective from the front line The race for coronavirus vaccines: a graphical guide SARS-CoV-2 immunity: review and applications 376 to phase 3 vaccine candidates SARS-CoV-2 Treatment Approaches: Numerous Options, No Certainty for 378 a Versatile Virus Therapeutic options for the 2019 novel coronavirus (2019-nCoV). Nat Rev 380 Drug Discov preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint B) A topological 1021 and subcellular localization prediction of ACE2 and TMPRSS2 as performed by Protter 1022 preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted