Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative

Abstract

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80–100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25–250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

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

“…The protein concentration was estimated using NanoDrop™ 2000c with ε 280 calculated using the ProtParam tool (ExPASy). A polyclonal Flp-In CHO stable line was generated as described previously ( 28 ), the mRBD1-3.2 RBD protein expressed at yield of ~60 mg/l.…”

“…Pseudoviral neutralization assays using spike variants displayed on an pHIV-1 NL4.3Δenv-Luc backbone were performed as described ( 28 ). Pseudovirus neutralization titers (ID 50 ) were determined as the serum dilution at which infectivity was blocked by 50%.…”

“…Replicative virus culture and neutralization assays were carried out essentially as described previously ( 28 ). Briefly, each serum sample was diluted 1:80 in DMEM-D in a deep-well plate, followed by a twofold serial dilution up to 1:163840.…”

“…The hamsters were housed and maintained at the Central Animal Facility at IISC, Bangalore, with feed and water ad libitum and a 12-h light and dark cycle. Hamster immunization was performed as described ( 28 ). Briefly, animals were acclimatized for 2 weeks and were randomly grouped (n = 6/group).…”

“…Thus, there is still a need for inexpensive, rapidly producible, and highly efficacious vaccines against VOC, which preferably do not require low-temperature storage. 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.…”