A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern.

Shahbaz Ahmed,Rajesh P Ringe,Oyahida Khatun,Nidhi Girish,Raghavan Varadarajan,Poorvi Reddy,Sujeet Jha,Mohammad Suhail Khan,Petrus Jansen Van Vuren,Sarah Goldie,Alexander J Mcauley,Nagendrakumar Balasubramanian Singanallur,Seshadri S Vasan,Unnatiben Rajeshbhai Patel,Samreen Siddiqui,Randhir Singh,Rajesh Pandey,Shashank Tripathi,Akansha Tyagi,Sameer Kumar Malladi,Aditya Upadhyaya,Shane Riddell,S Gayathri ,S Suresh Kumar ,Raju S Rajmani ,S K : Singh Pandey

doi:10.3389/fimmu.2021.765211

Abstract

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.

Highlights

SARS-CoV-2 is the etiological agent of the ongoing pandemic which has so far resulted in over four million deaths worldwide [1]
A schematic of the principle of second-site saturation suppressor mutagenesis (SSSM) to isolate stabilizing mutations is shown in Figures 1A–D; more detailed descriptions can be found in Sahoo et al and Ahmed et al [32, 33]
These mutants were stable to incubation for 2 h at 60°C and for up to 20 h at 50° C, showing superior thermal tolerance to the WT (Supplementary Figure S1). mRBD1-3.3 had an exposed mutation V367F which is found in some circulating SARSCoV-2 virus isolates [44]

Summary

Introduction

SARS-CoV-2 is the etiological agent of the ongoing pandemic which has so far resulted in over four million deaths worldwide [1]. The trimeric spike glycoprotein is the major surface protein of SARS-CoV-2 and the principal target of neutralizing antibodies [2]. The immunogens currently in clinical use or clinical trials are derived from the wildtype (WT) sequence [21] and employ the full-length viral spike as the primary antigen. Current VOC are B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), and B.1.617.2 (delta). Both B.1.351 and B.1.617.2 show substantially decreased neutralization by many existing monoclonal antibodies and by convalescent as well as vaccine sera [22,23,24]. We recently showed that both monomeric and intermolecular disulfide-linked, trimeric RBD derivatives were highly thermotolerant with the latter showing improved immunogenicity [28], albeit with significant antibody titers against the trimerization domain

Methods

Results

Conclusion