Wuhan Sequence-Based Recombinant Antigens Expressed in E. coli Elicit Antibodies Capable of Binding with Omicron S-Protein

Abstract

The development of cross-reactive vaccines is one of the central aims of modern vaccinology. Continuous mutation and the emergence of new SARS-CoV-2 variants and subvariants create the problem of universal coronavirus vaccine design. Previously, the authors devised three recombinant coronavirus antigens, which were based on the sequence collected in 2019 (the Wuhan variant) and produced in an E. coli bacterial expression system. The present work has shown, for the first time, that these recombinant antigens induce the production of antibodies that clearly interact with produced in CHO full-length S-protein of the Omicron variant. The immunogenicity of these recombinant antigens was studied in formulations with different adjuvants: Freund's adjuvant, Al(OH)₃ and an adjuvant based on spherical particles (SPs), which are structurally modified plant virus. All adjuvanted formulations effectively stimulated Omicron-specific IgG production in mice. These universal coronavirus antigens could be considered the main component for the further development of broad-spectrum coronavirus vaccines for the prevention of SARS-CoV-2 infection. The present work also provides evidence that the synthetic biology approach is a promising strategy for the development of highly cross-reactive vaccines. Moreover, it is important to note that the bacterial expression system might be appropriate for the production of antigenically active universal antigens.

Keywords: Omicron variant; SARS-CoV-2; plant virus-based adjuvant; recombinant protein vaccine.

Figure 1

Linear diagram of the full-length SARS-CoV-2 spike protein domain structure (A) and coronavirus recombinant antigens Co1 (B), PE (C) and CoF (D). Coordinates of amino acid residues included in the recombinant antigens are indicated. Domains are coloured according to the coordinates indicated in Sun et al. (2021) [11]. Diagrams are not to scale. NTD—N-terminal domain; RBD—receptor-binding domain; CTD1 and CTD2—C-terminal domains 1 and 2; FP—fusion protein; HR1 and HR2—heptad repeats 1 and 2; CH—central helix; CD—connector domain; TM—transmembrane domain; CT—cytoplasmic tail.

Figure 2

Interaction of recombinant S-protein of the Omicron variant of SARS-CoV-2 with mice polyclonal antisera specific to the RBD-domain and/or S2-subunit fragments of the Wuhan variant of SARS-CoV-2. L—protein molecular weight markers ladder (molecular weights, in kDa, are indicated on the left). S and N—SARS-CoV-2 B.1.1.529/Omicron recombinant S-protein and N-protein, respectively. Co1, PE, CoF—recombinant coronavirus antigens, which are expressed in the E. coli system. Co1 and PE represent SARS-CoV-2 isolate Wuhan-Hu-1 RBD-domain and conserved among betacoronaviruses S2-subunit fragments, respectively. CoF is a fusion of Co1-protein with a highly conserved epitope of S2-subunit. An 8–20% SDS-PAGE electrophoresis analysis, staining by Coomassie G-250 (A). Western blot analysis with primary polyclonal antisera specific to Co1 (1:3000) (B), PE (1:3000) (C) or CoF (D) (1:3000). Anti-mouse IgG HRP-conjugate was used as secondary antibodies in a dilution of 1:10,000.

Figure 3

Immunisation schedule and description of mice groups involved in the experiment. Mice from groups 2–5 were immunised intramuscularly with a corresponding formulation containing 60 µg of 3AG (20 µg each). All samples administered were prepared in PBS; the final volume was 0.26 mL. In the case of the formulation with Freund’s adjuvant, the 3AG solution was prepared in half of the final volume and mixed with an equal amount of Freund’s adjuvant immediately prior to the immunisation. 3AG—the mixture of the three coronavirus recombinant antigens Co1, PE and CoF in equal mass ratio.

Figure 4

Comparison of the titres of total IgG specific to the S-protein of SARS-CoV-2 variant Omicron in mice sera obtained from intact animals and animals immunised with a mixture of recombinant antigens Co1, PE and CoF (3AG), either individually or in composition with various adjuvants. Mice were immunised intramuscularly twice, with a 21-day interval between immunisations. The scheme of the study is presented in Figure 3. Titres were evaluated by indirect ELISA. For all groups, titres of sera pools were detected (A) and, for groups with 3AG and 3AG + SPs-based adjuvant, titres of individual mice sera were subjected to additional analysis (B). Antigen concentration on the microplate was 5 µg/mL for evaluation of titres in pooled sera, and 2.5 µg/mL for evaluation of titres in individual mice sera. ●—titres of sera pool or individual mice serum; ▬—median; I—interquartile range. The Kruskal–Wallis test with a post hoc Dunn’s test was used for multiple comparisons of titres from each group with the titres from the group of intact animals. * p < 0.05, *** p < 0.001. The complete data on sera pool titres and individual mice sera titres are presented in Table S2 and Table S3, respectively.