Preclinical characterization of the Omicron XBB.1.5-adapted BNT162b2 COVID-19 vaccine

Abstract

As SARS-CoV-2 evolves, increasing in potential for greater transmissibility and immune escape, updated vaccines are needed to boost adaptive immunity to protect against COVID-19 caused by circulating strains. Here, we report features of the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine, which contains XBB.1.5-specific sequence changes, relative to the original BNT162b2 backbone, in the encoded prefusion-stabilized SARS-CoV-2 spike protein (S(P2)). Biophysical characterization of Omicron XBB.1.5 S(P2) demonstrated that it maintains a prefusion conformation and adopts a flexible, predominantly open, state, with high affinity for the human ACE-2 receptor. When administered as a 4th dose in BNT162b2-experienced mice, the monovalent Omicron XBB.1.5 vaccine elicited substantially higher serum neutralizing titers against pseudotyped viruses of Omicron XBB.1.5, XBB.1.16, XBB.1.16.1, XBB.2.3, EG.5.1 and HV.1 sublineages and phylogenetically distant BA.2.86 lineage than the bivalent Wild Type + Omicron BA.4/5 vaccine. Similar trends were observed against Omicron XBB sublineage pseudoviruses when the vaccine was administered as a 2-dose series in naive mice. Strong S-specific Th1 CD4⁺ and IFNγ⁺ CD8⁺ T cell responses were also observed. These findings, together with real world performance of the XBB.1.5-adapted vaccine, suggest that preclinical data for the monovalent Omicron XBB.1.5-adapted BNT162b2 was predictive of protective immunity against dominant SARS-CoV-2 strains.

Fig. 1. Biophysical properties and ACE-2 receptor binding affinities SARS-CoV-2 WT and Omicron XBB.1.5 FL S(P2) and RBD.

A Size exclusion chromatography (SEC) profile of the purified WT and Omicron XBB.1.5 FL S(P2) proteins equivalent to the protein antigen encoded by the BNT162b2 vaccines. B Melting temperature (Tm) of DDM-purified S(P2) proteins at 0.35 mg/mL concentration. Assay was run in triplicate. Biolayer interferometry (BLI) sensorgram showing binding of (C) purified S(P2) proteins and (D) RBD to immobilized human angiotensin converting enzyme-2 peptidase domain (ACE-2-PD). Binding data are in black; 1:1 binding model fit to the data is in color. Apparent kinetic parameters are provided in the graph. K_D = equilibrium dissociation constant; k_on = binding rate constant; k_off = dissociation rate constant.

Fig. 2. Cryo-EM structure of SARS-CoV-2 Omicron XBB.1.5 spike protein.

A Representative 2D class averages of full-length prefusion stabilized Omicron XBB.1.5 S(P2). Box size is 40.5 nm in each dimension. B Maps from ab initio reconstruction reveal only one class resembling the S(P2) protein particles with 1-RBD in the ‘up’ position. These particles were used for the final reconstruction. Percentages of the particle population represented in each class are indicated below the models. C The overall structure of Omicron XBB.1.5 S(P2) trimer modeled based on the 2.98 Å density map. Two of the three protomers with RBD in a ‘down’ conformation are represented by a molecular surface colored in white and grey. The remaining protomer with RBD in an ‘up’ conformation is represented by a ribbon diagram; The NTD is colored blue; the RBD is colored yellow; the remaining S1 subunit is colored purple; and the S2 subunit is colored green. Amino acid residues that differ between Omicron XBB.1.5 and the ancestral strain are represented by red spheres.

Fig. 3. Pseudovirus neutralization titers (NT₅₀) elicited by BNT162b2 variant-adapted vaccines administered as a 4th dose in immune-experienced mice.

Female BALB/c mice (10/group) that were previously vaccinated (per schedule described in Supplementary Fig. 4A) with two-doses of monovalent original WT BNT162b2, and one subsequent dose of bivalent WT + Omicron BA.4/5 received a single intramuscular dose of one of these vaccine regimens: monovalent Omicron BA.4/5, bivalent WT+ Omicron BA.4/5, monovalent Omicron XBB.1.5 or bivalent Omicron BA.4/5 + Omicron XBB.1.5. All vaccine formulations contained a total dose of 0.5 µg. Fifty-percent geometric mean serum neutralizing titers were characterized in a pseudovirus neutralization assay at one-month post-4th dose against the WT reference strain, the Omicron sublineages BA.4/5, XBB.1.5, XBB.1.16, XBB.1.16.1, XBB.2.3, EG.5.1, HV.1 and the lineage BA.2.86. A 50% pseudovirus neutralization titers are shown as geometric mean titers (GMT) ± 95% CI of 10 mice per vaccine group. Each point represents an individual animal. B The geometric mean ratio (GMR) is the GMT of individual pseudovirus responses of each vaccine group (monovalent Omicron BA.4/5, monovalent Omicron XBB.1.5 or bivalent Omicron BA.4/5 + Omicron XBB.1.5) divided by the GMT of analogous pseudovirus responses of the bivalent WT + Omicron BA.4/5 group. Statistical differences were analysed by ANOVA with a Dunnett’s multiple comparison test (****p < 0.0001). The limit of detection (LOD) is the lowest serum dilution, 1:20.

Fig. 4. Pseudovirus neutralization titers (NT₅₀) elicited by BNT162b2 variant-adapted vaccines administered as a primary series in naive mice.

Female BALB/c mice (10/group) vaccinated with two-doses of one of the following vaccine regimens at a twenty-one-day interval: monovalent Omicron, BA.4/5, bivalent WT + Omicron BA.4/5, monovalent Omicron XBB.1.5 or bivalent Omicron BA.4/5 + Omicron XBB.1.5. All vaccine formulations contained a total dose of 0.5 µg. Serum neutralizing antibody responses were measured by a pseudovirus neutralization assay at one-month post-second dose against the WT reference strain, the Omicron sublineages BA.4/5, XBB.1.5, XBB.1.16, XBB.1.16.1, XBB.2.3, EG.5.1 and the lineage BA.2.86. A 50% pseudovirus neutralization titers are shown as geometric mean titers (GMT) ± 95% CI of 10 mice per vaccine group. Each point represents an individual animal. B The geometric mean ratio (GMR) is the GMT of individual pseudovirus responses of each vaccine group (monovalent Omicron BA.4/5, monovalent Omicron XBB.1.5 or bivalent Omicron BA.4/5 + Omicron XBB.1.5) divided by GMTs of analogous pseudovirus responses of the bivalent WT + Omicron BA.4/5 group. Statistical differences were analysed by ANOVA with a Dunnett’s multiple comparison test (**p < 0.01; ****p < 0.0001). The limit of detection (LOD) is the lowest serum dilution, 1:20.

Fig. 5. T cell responses elicited by BNT162b2 variant-adapted vaccines administered as a 4th dose in BNT162b2-experienced mice.

One-month after the 4th dose, S-specific CD4⁺ and CD8⁺ splenocytes (n = 5/group) were characterized by a flow cytometry-based intracellular cytokine staining assay. All samples were stimulated separately with S peptide pools from the WT reference strain, Omicron BA.4/5, or XBB.1.5 sublineages. Graphs show the frequency of CD8⁺ T cells expressing IFN-γ (A) and the frequency of CD4⁺ T cells expressing IFN-γ (B), IL-2 (C), TNF-α (D), IL-4 (E) and IL-10 (F) in response to stimulation with each peptide pool across vaccine groups. Bars depict mean frequency ± SEM.

Fig. 6. T cell immune responses elicited by BNT162b2 variant-adapted vaccines administered as a primary series in naive mice.

At one-month post-second dose (completion of primary series), S-specific T cells from fresh spleens (n = 5) were measured by intracellular cytokine staining assay. All samples were stimulated separately with S peptide pools from the WT reference strain and the Omicron BA.4/5 and XBB.1.5 sublineages. Graphs show the frequency of CD8⁺ T cells expressing IFN-γ (A) and the frequency of CD4⁺ T cells expressing IFN-γ (B), IL-2 (C), IL-4 (D), TNF-α (E) and IL-10 (F) in response to stimulation with each peptide pool across vaccine groups. Bars depict mean frequency ± SEM.