Novel Competitive ELISA Utilizing Trimeric Spike Protein of SARS-CoV-2, Could Identify More Than RBD-RBM Specific Neutralizing Antibodies in Hybrid Sera

Author:

Eliadis Petros12ORCID,Mais Annie3ORCID,Papazisis Alexandros1,Loxa Eleni K.1,Dimitriadis Alexios2ORCID,Sarrigeorgiou Ioannis1,Backovic Marija4,Agallou Maria5ORCID,Zouridakis Marios6ORCID,Karagouni Evdokia5,Lazaridis Konstantinos12ORCID,Mamalaki Avgi23,Lymberi Peggy1ORCID

Affiliation:

1. Immunology Laboratory, Immunology Department, Hellenic Pasteur Institute, 11521 Athens, Greece

2. Biotechnology Unit, Hellenic Pasteur Institute, 11521 Athens, Greece

3. Laboratory of Molecular Biology and Immunobiotechnology, Immunology Department, Hellenic Pasteur Institute, 11521 Athens, Greece

4. Institut Pasteur, Unité de Virologie Structurale, Université Paris Cité, CNRS-UMR3569, 75724 Paris, France

5. Immunology of Infection Laboratory, Microbiology Department, Hellenic Pasteur Institute, 11521 Athens, Greece

6. Structural Neurobiology Research Group, Laboratory of Molecular Neurobiology and Immunology, Department of Neurobiology, Hellenic Pasteur Institute, 11521 Athens, Greece

Abstract

Since the initiation of the COVID-19 pandemic, there has been a need for the development of diagnostic methods to determine the factors implicated in mounting an immune response against the virus. The most promising indicator has been suggested to be neutralizing antibodies (nAbs), which mainly block the interaction between the Spike protein (S) of SARS-CoV-2 and the host entry receptor ACE2. In this study, we aimed to develop and optimize conditions of a competitive ELISA to measure serum neutralizing titer, using a recombinant trimeric Spike protein modified to have six additional proline residues (S(6P)-HexaPro) and h-ACE2. The results of our surrogate Virus Neutralizing Assay (sVNA) were compared against the commercial sVNT (cPass, Nanjing GenScript Biotech Co., Nanjing City, China), using serially diluted sera from vaccinees, and a high correlation of ID50–90 titer values was observed between the two assays. Interestingly, when we tested and compared the neutralizing activity of sera from eleven fully vaccinated individuals who subsequently contracted COVID-19 (hybrid sera), we recorded a moderate correlation between the two assays, while higher sera neutralizing titers were measured with sVNA. Our data indicated that the sVNA, as a more biologically relevant model assay that paired the trimeric S(6P) with ACE2, instead of the isolated RBD-ACE2 pairing cPass test, could identify nAbs other than the RBD-RBM specific ones.

Funder

Hellenic Pasteur Institute

Operational Program ‘Competitiveness, Entrepreneurship and Innovation’

Greek Government and the European Union—European Regional Development Fund

Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur

Publisher

MDPI AG

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