SARS-CoV-2 Spike Protein Stabilized in the Closed State Induces Potent Neutralizing Responses

Abstract

The majority of SARS-CoV-2 vaccines in use or advanced development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described against both open and closed conformations. The long-term success of vaccination strategies depends upon inducing antibodies that provide long-lasting broad immunity against evolving SARS-CoV-2 strains. Here we have assessed the results of immunization in a mouse model using an S protein trimer stabilized in the closed state to prevent full exposure of the receptor binding site and therefore interaction with receptor. We compared this with other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induced a T cell response and long-lived, strongly neutralizing antibody responses against 2019 SARS-CoV-2 and variants of concern B.1.248 and B.1.351. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralizing responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines. Importance Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next-generation vaccines for use in previously infected or immunized individuals. We assessed the use as an immunogen of a spike protein engineered to be conformationally stabilized in the closed state where the receptor binding site is occluded. Despite occlusion of the receptor binding site, the spike induces potently neutralizing sera against multiple SARS-CoV-2 variants. Antibodies are raised against a different pattern of epitopes to those induced by other spike constructs, preferring conformational epitopes present in the closed conformation. Closed spikes, or mRNA vaccines based on their sequence, can be a valuable component of next generation vaccines.

Analysis of the Neutralizing Activity of Antibodies Targeting Open or Closed SARS-CoV-2 Spike Protein Conformations

“…This result can be considered surprising since nAbs that best mimic ACE2 by binding to a very large portion of the ACE2-RBD interface have been suggested to have the most effective neutralization strategy [29] and are therefore expected to dominate the immune response. Our results as well as others [6,30] rather suggest that the nAb ability to bind the closed conformation of the spike protein, thus blocking its transition to an open state and locking the closed pre-fusion conformation, is a more important feature for having highly neutralizing nAbs dominating the immune response.…”

Comparison of the Immunogenicity of five COVID-19 vaccines in Sri Lanka

“…Since the HAT assay was shown to strongly correlate with Nabs levels 15 , it appears that while those who received Moderna had significantly higher Nabs than those who received other vaccines, those who received the two adenovirus vector vaccines had higher antibody levels to the RBD. While the reasons for these differences are not clear, it could be due to mRNA vaccines having stabilizing substitutions in spike protein to maintain the pre-fusion conformation, whereas AZD1222 and Sputnik may not contain these specific substitutions 26,27 . However, it is possible that these differences are seen due to cross reactive antibody responses to different VOCs elicited by different vaccines.…”

Predicted Epitope Abundance Supports Vaccine-Induced Cytotoxic Protection Against SARS-CoV-2 Variants of Concern

“…For the sake of universality, all assays were conducted with the original Wuhan-1 spike sequence, which have been unmodified by Astra Zeneca AZD1222, Sputnik V and SinoVac (CoronaVac) antigens. In contrast, K986P/V987P mutations were engineered to stabilize the close conformation and enhance the humoral protection (24) in Moderna mRNA-1273, Pfizer BNT162b2 and S3). Representative supertype alleles were selected by prioritizing the first numeric allele one of the same family of the supertype name included in (10) Janssen Ad26.COV2.S antigens, the latter further contained two changes involving the arginines of the furin site to diminish proteolysis (25).…”

“…For the sake of universality, all assays were conducted with the original Wuhan-1 spike sequence, which have been unmodified by Astra Zeneca AZD1222, Sputnik V and SinoVac (CoronaVac) antigens. In contrast, K986P/V987P mutations were engineered to stabilize the close conformation and enhance the humoral protection ( 24 ) in Moderna mRNA-1273, Pfizer BNT162b2 and Janssen Ad26.COV2.S antigens, the latter further contained two changes involving the arginines of the furin site to diminish proteolysis ( 25 ). The epitopes destroyed by these manipulations reached 0 - 3.9% (supertype averages) for Moderna/Pfizer and 0 - 6.1% for Janssen protein versions ( Figure 4 ).…”