Stabilized recombinant SARS-CoV-2 spike antigen enhances vaccine immunogenicity and protective capacity

Abstract

The SARS-CoV-2 spike (S) glycoprotein is synthesized as large precursor protein and must be activated by proteolytic cleavage into S1 and S2. A recombinant modified vaccinia virus Ankara (MVA) expressing native, full-length S protein (MVA-SARS-2-S) is currently under investigation as candidate vaccine in phase I clinical studies. Initial results from immunogenicity monitoring revealed induction of S-specific antibodies binding to S2, but low-level antibody responses to the S1 domain. Follow-up investigations of native S antigen synthesis in MVA-SARS-2-S infected cells revealed limited levels of S1 protein on the cell surface. In contrast, we found superior S1 cell surface presentation upon infection with a recombinant MVA expressing a stabilized version of SARS-CoV-2 S protein with an inactivated S1/2 cleavage site and K986→P and V987→P mutations (MVA-SARS-2-ST). When comparing immunogenicity of MVA vector vaccines, mice vaccinated with MVA-SARS-2-ST mounted substantial levels of S broadly reactive antibodies that effectively neutralized different SARS-CoV-2 variants. Importantly, intramuscular MVA-SARS-2-ST immunization of hamsters and mice resulted in potent immune responses upon challenge infection and protected from disease and severe lung pathology. Our results suggest that MVA-SARS-2-ST represents an improved clinical candidate vaccine and that the presence of plasma membrane-bound S1 is highly beneficial to induce protective antibody levels.

Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles

“…with scalar amounts of different IDLV-CoV2. Data clearly indicated that the prefusion stabilized Spike with 2P substitutions and mutated FCS, preventing S1/S2 cleavage, was a more effective immunogen in terms of level and persistence of autologous Ab and nAb response compared to the wild-type Spike which showed the poorest humoral immunity (IDLV-S-wt vs IDLV-S-2PF), in line with data from other studies ( 81 , 84 ). We also investigated the role of the delivery system in the induction of humoral response, comparing IDLV delivering the purified prefusion stabilized Spike with 2P substitutions and mutated FCS (IDLV-S-2PF) with the same version of the Spike delivered as subunit vaccine administered with a squalene-based adjuvant.…”

“…To further ameliorate the quality of the anti-Spike humoral response, we evaluated mutations aimed at improving exposure of neutralizing epitopes, including the introduction of 2P substitutions (K986P and V987P) for stabilizing Spike in the prefusion conformation ( 76 , 77 ) and the inclusion of the D614G mutation, for enhancing RBM exposure ( 42 ) and Spike protein density ( 78 ) in the IDLV. These modifications have been already adopted, alone or in combination, using different Spike delivery systems, including adenoviral vectors ( 63 , 79 , 80 ), MVA ( 81 ) and mRNA vaccines ( 82 , 83 ).…”

Highly Attenuated Poxvirus-Based Vaccines Against Emerging Viral Diseases

“…In the lethal K18-hACE2 mouse model, vaccinated mice challenged with SARS-CoV-2 were protected from body weight loss and mortality caused by the virus. 103 , 104 , 106 , 107 , 108 , 116 , 117 , 118 In hamsters, SARS-CoV-2 is not lethal, but control of body weight loss was also observed in vaccinated animals, 109 , 112 , 113 , 115 , 116 , 121 , 122 whereas in the NHP model SARS-CoV-2 infection does not induce body weight loss or mortality. 110 , 112 , 119 , 120 The evaluation of the protection exerted showed that most of the MVA-based vaccine candidates induced a significant reduction in the levels of genomic and subgenomic SARS-CoV-2 RNA in the lower (lung) and upper (throat or nasal cavity) respiratory tracts, but with better control of SARS-CoV-2 replication in the lungs than in the upper respiratory tracts, indicating that a sterilizing immunity was not fully obtained.…”

“…Protection against SARS-CoV-2 Wuhan challenge. 116 rMVAs - Prefusion-stabilized full-length S protein, containing 2 amino acid changes to proline (Wuhan strain) - Full-length S protein, non-stabilized (Wuhan strain) - RBD (Wuhan strain) Mouse (BALB/c, C57BL/6, transgenic K18-hACE2) - Binding IgGs (Th1-biased) and NAbs and T cell immune responses (CD8 + T cells, Th1-profile). - Protection against SARS-CoV-2 challenge.…”

“…SARS-CoV-2 full-length S protein is the most common antigen expressed by the different MVA vectors, either in its unmodified native non-stabilized conformation 102 , 103 , 104 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 121 , 122 , 123 , 124 or in an optimized prefusion-stabilized conformation, 106 , 107 , 116 , 117 , 118 , 119 , 120 being the MVA vectors expressing the prefusion-stabilized S protein more immunogenic and efficacious than those expressing the non-stabilized S protein. 106 , 107 , 116 , 117 , 120 Other regions of the SARS-CoV-2 S protein have also been inserted into the MVA vector, such as the receptor binding domain (RBD), the S1 region or a soluble S ectodomain, but their immunogenicity is lower than that induced by MVA vectors expressing the full-length S protein. 117 , 119 , 121 Furthermore, other SARS-CoV-2 antigens, such as the N protein, have been incorporated in combination with the S protein 111 , 112 , 120 , 123 , 124 to broaden the immune response against SARS-CoV-2.…”