Introduction of Two Prolines and Removal of the Polybasic Cleavage Site Lead to Higher Efficacy of a Recombinant Spike-Based SARS-CoV-2 Vaccine in the Mouse Model

Abstract

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively unstable, a feature that might be enhanced by the presence of a polybasic cleavage site in SARS-CoV-2 spike. Exchange of K986 and V987 for prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus spike proteins. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin-converting enzyme 2 via adenovirus transduction. Variants tested include spike proteins with a deleted polybasic cleavage site, proline mutations, or a combination thereof, besides the wild-type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the K986P and V987P (PP) mutations completely protected from challenge in this mouse model.IMPORTANCE A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validate the choice of antigens that contain the PP mutations and suggest that deletion of the polybasic cleavage site may lead to a further-optimized design.

Keywords: COVID-19; SARS-CoV-2; spike; vaccine.

FIG 1

Spike construct design and protein characterization. (A to D) Illustration of the wild-type, ΔCS, PP, and ΔCS-PP constructs used in this study. (E) Four antigens on an SDS-PAGE gel stained with Coomassie blue. (F) The same proteins on a Western blot developed with an antibody to the C-terminal hexahistidine tag. While all four proteins are detected as clean, single bands on the SDS-PAGE gel, the Western blot reveals a small fraction of degradation products at approximately 80 kDa for the wild type and PP variants and of approximately 40 kDa for the PP and ΔCS-PP constructs. (G) Binding of MAb CR3022 to the constructs in an ELISA. Data for the negative-control MAb and the blank were combined for the different substrates.

FIG 2

Immunogenicity of different spike variants in the mouse model. (A) Vaccination regimen used for the five groups of mice. (B) Timeline. d-5 and d5, day -5 and day 5. (C and D) Animals were bled 3 weeks after the priming (C) and 4 weeks after the booster (D), and levels of antibody to a mammalian-cell-expressed RBD were measured. Postboost sera were also tested in cell-based ELISAs on cells infected with authentic SARS-CoV-2. Finally, postboost sera were tested in a microneutralization assay against SARS-CoV-2.

FIG 3

Challenge of mice with SARS-CoV-2. Animals sensitized by transient expression of hACE2 via adenovirus transduction were challenged with 10⁵ PFU of SARS-CoV-2, and weight loss was monitored over a period of 14 days (A). (B and C) Day 2 and day 5 lung titers, respectively. (D) Lung immunohistochemistry staining for SARS-CoV-2 nucleoprotein on days 2 and 4 postchallenge. Representative images from two animals each are shown at a 5-fold magnification. Scale bar = 500 μm.

FIG 4

Lung pathology. (A) Histopathological composite scores for animals on day 2 postinfection; (B) representative H&E-stained tissue images from 2 animals per group; (C and D) the same tissues but for day 4 postchallenge. Scale bar = 500 μm.