Pharma Focus Asia

Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies

Marta Alenquer, Filipe Ferreira , Diana Lousa , Mariana Valério, Mónica Medina-Lopes, Marie-Louise Bergman, Juliana Gonçalves, Jocelyne Demengeot, Ricardo B. Leite, Jingtao Lilue, Zemin Ning, Carlos Penha-Gonçalves, Helena Soares, Cláudio M. Soares, Maria João Amorim

Abstract
Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population.

Introduction
Severe acute respiratory syndrome coronavirus–2 (SARS-CoV-2) is the virus responsible for the pandemic of coronavirus disease 2019 (COVID-19) [1] that has caused more than 164 million infections and provoked the death of over 3 million people (as of April 18, 2021).

Materials and methods

Ethics statement
This study was approved by the Ethics committees of Centro Hospitalar Lisboa Ocidental and Centro Hospitalar Lisboa Central, in compliance with the Declaration of Helsinki, and follows international and national guidelines for health data protection. All participants provided informed written consent to take part in the study.

Global variants frequencies
A variant surveillance report was retrieved from GISAID on March 26th, 2021. A total of 864131 submissions were used to establish the world frequency of each variant using specific amino acid mutations (e.g., S_D614G, etc.).

Cloning and spike protein mutations
To facilitate incorporation of spike protein into lentiviral pseudovirions, the C-terminal 19 amino acids encompassing the ER-retention domain of the cytoplasmic tail of spike were deleted.

Discussion
It is critical to understand SARS-CoV-2 signatures that allow escaping neutralizing antibodies, which could pose challenges to vaccination and render therapies ineffective.

We developed spike-pseudotyped lentiviral particles for high-throughput quantitation that express GFP upon entering cells, contributing to the toolkit of neutralizing assay methodologies [73–75]. Our method of reading fluorescent signals in 96 wells plates using the GloMax Explorer System is suitable to assess the neutralization activity of sera/plasma from individuals infected naturally or vaccinated, and to screen for antiviral drugs that block viral entry, such as therapeutic antibodies, in biosafety level 2 settings, which greatly facilitates the procedure and broadens its usage.

Acknowledgments:
The authors acknowledge Sean Whelan (Washington University School of Medicine St Louis) for providing reagents, João F. Viana and his team for organizing sera collection at Centro Hospitalar Lisboa Ocidental in Portugal, and the IGC COVID-19 team (Patrícia C. Borges, Christian Diwo, Paula Matoso, Vanessa Malheiro, Lígia A. Gonçalves, Nádia Duarte, Ana Brennand, Lindsay Kosack and Onome Akpogheneta) for processing and ranking samples, for this paper and during early settings.

Citation: Alenquer M, Ferreira F, Lousa D, Valério M, Medina-Lopes M, Bergman M-L, et al. (2021) Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies. PLoS Pathog 17(8): e1009772. https://doi.org/10.1371/journal.ppat.1009772

Editor: Michael S. Diamond, Washington University School of Medicine, UNITED STATES

Received: May 20, 2021; Accepted: June 30, 2021; Published: August 5, 2021.

Copyright: © 2021 Alenquer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This project is supported by the grants PTDC/CCI-BIO/28200/2017 awarded to DF; FCT RESEARCH4COVID 19 (Refs 580 and 644) awarded by the Fundação para a Ciência e a Tecnologia (FCT, Portugal, https://www.fct.pt/index.phtml.en) to MJA and also by COVID-19 emergency funds 2020 from Fundação Calouste Gulbenkian - Instituto Gulbenkian de Ciência (FCG-IGC, Portugal, https://gulbenkian.pt/ciencia/) to MJA.

Competing interests: The authors have declared that no competing interests exist.

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