Expression of the SARS-CoV-2 receptor-binding domain by live attenuated influenza vaccine virus as a strategy for designing a bivalent vaccine against COVID-19 and influenza

Abstract

Influenza and SARS-CoV-2 are two major respiratory pathogens that cocirculate in humans and cause serious illness with the potential to exacerbate disease in the event of co-infection. To develop a bivalent vaccine, capable of protecting against both infections, we inserted the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein into hemagglutinin (HA) molecule or into the open reading frame of the truncated nonstructural protein 1 (NS1) of live attenuated influenza vaccine (LAIV) virus and assessed phenotypic characteristics of the rescued LAIV-RBD viruses, as well as their immunogenicity in mouse and Syrian hamster animal models. A panel of 9 recombinant LAIV-RBD viruses was rescued using the A/Leningrad/17 backbone. Notably, only two variants with RBD insertions into the HA molecule could express sufficient quantities of RBD protein in infected MDCK cells. Intranasal immunization of mice induced high levels of anti-influenza antibody responses in all chimeric LAIV-RBD viruses, which was comparable to the LAIV virus vector. The RBD-specific antibody responses were most pronounced in the variant expressing RBD194 fragment as a chimeric HA protein. This candidate was further tested in Syrian hamsters and was shown to be immunogenic and capable of protecting animals against both infections.

Keywords: Bivalent vaccine; COVID-19; Immunogenicity; Influenza; Recombinant influenza virus; SARS-CoV-2; Syrian hamsters; Virus vectored vaccine.

Fig. 1

Schematic representation of the chimeric HA constructions. A schematic representation of the chimeric HA genes encoding RBD fragments of the SARS-CoV-2 spike protein. B-E schematic visualization of SARS-CoV-2 RBD-based cassettes inserted into HA. B RBD 194 cassette (based on PDB 6vxx); C RBD 223 cassette (based on PDB 6vxx); D H7 HA trimer with RBD 194 cassette connected to one of the three HA monomers. The linker is colored in black; E H7 HA trimer with the RBD223 cassette connected to one of the three HA monomers. The linker is colored in black. Figures were prepared using UCSF Chimera 1.11.2 [34]

Fig. 2

Schematic representation of the chimeric NS1 genes encoding RBD fragments of the SARS-CoV-2 spike protein, along with different targeting signals. A Types of modifications of the influenza NS1 gene; B Types of RBD cassettes inserted into NS1 ORF. SP: signal peptide. TMD: transmembrane domain, erbB-2 (HER-2). CPD: cytoplasmic domain (alpha-subunit of the IL-2 receptor CPD)

Fig. 3

Expression of RBD protein by recombinant LAIV/RBD viruses in infected MDCK cells. The cells were infected with each virus in triplicates and the concentration of RBD in cell lysates was measured 60 hpi by sandwich ELISA

Fig. 4

Western blot analysis of sucrose gradient-purified influenza viruses and a recombinant RBD protein using: A anti-RBD rabbit polyclonal antibody (*) – influenza HA monomer with RBD insertion; (♦) – monomeric recombinant RBD (♦♦) – dimeric form of RBD; the higher bands are oligomers of these forms; B anti-H7 HA mouse hyperimmune sera. (*) – influenza H7 HA monomer with RBD insertion is higher than H7 HAs without insertions (triangle); Cov19: FluCoVac-19. Cov20: FluCoVac-20. The H7N9 LAIV vector (H7N9) and recombinant RBD protein (RBD) were used as control antigens in this assay

Fig. 5

Replication of experimental viruses in BALB/c mouse nasal turbinates (A) and lung tissue (B). BALB/c mice were immunized with experimental vaccine strains at a dose of 10⁶ EID₅₀ and tissues were collected on day 3 post immunization. Influenza viral titers were determined in eggs

Fig. 6

Serum IgG antibody response to H7N9 influenza virus (A) and to SARS-CoV-2 RBD (B) in BALB/c mice immunized with experimental vaccine strains on day 21 post second immunization (day 42 total). Data from 3 experiments are summarized on the graph. A titers of IgG anti-influenza antibodies in sera of immunized animals significantly differ from titers of anti-influenza IgG antibodies from PBS group (statistically significant for all groups, p<0.05, Kruskal-Wallis test, post-hoc Dunn’s test, not shown on the graph). B (*) p<0.05 ANOVA with post-hoc Dunnet’s test, (***) p<0.005, Kruskal-Wallis test with post-hoc Dunn’s test

Fig. 7

The scheme of the experiment on assessment of safety, immunogenicity and protective potential of the FluCoVac-19 in Syrian hamsters. D – days of the experiment

Fig. 8

Replication of FluCoVac-19 and control H7N9 LAIV virus in the respiratory tract of Syrian hamsters on Day 3 after immunization. Animals were i.n. immunized with 5×10⁶ EID₅₀ of each virus and viral titers in the lungs and in the nasal turbinates (n=4) were determined on day 3 post inoculation. Data were analyzed by one-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; **—p < 0.01; ***—p < 0.001

Fig. 9

Serum antibody immune responses in Syrian hamsters immunized with FluCoVac-19 experimental vaccine. Syrian hamsters were twice immunized with 5×10⁶ EID₅₀ of H7N9 LAIV or FluCoVac-19 at 3-week intervals; sterile PBS was used as a control. Sera were collected 3 weeks after the 2^nd dose and assessed by ELISA against whole influenza virus antigen (A) or against recombinant RBD protein (B). Data were analyzed by one-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; ***—p < 0.001; ****—p < 0.0001

Fig. 10

Replication of Sh/PR8 influenza virus in the respiratory tract of Syrian hamsters on day 3 after challenge with influenza virus. Animals were twice immunized with each virus and the challenge influenza virus Sh/PR8 was intranasally inoculated on day 21 after the second dose. Three days post challenge, viral pulmonary titers were determined by titration of tissue homogenates on MDCK cells. Data were analyzed by one-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; **—p < 0.01; ***—p < 0.001

Fig. 11

Protective activity of the FluCoVac-19 experimental vaccine in Syrian hamster model of SARS-CoV-2 infection. Syrian hamsters were immunized twice with 5×10⁶ EID₅₀ of H7N9 LAIV or FluCoVac-19 at 3-week intervals; sterile PBS was used as a control. Three weeks after the 2^nd dose animals were challenged with 10⁵ TCID₅₀ of Wuhan (D614G) SARS-CoV-2 virus. A Body weight monitoring during five days post challenge. B Sum of pathology scores over the challenge phase. C SARS-CoV-2 virus titer in lung tissue at day 5 after challenge, assessed by titration in Vero cells. Data were analyzed by one-way or two-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; **—p < 0.01

Fig. 12

Pathomorphological evaluation of lung tissues of hamsters immunized with FluCoVac-19, or control LAIV, as well as non-immunized animals (PBS group) on day 5 after challenge with SARS-CoV-2. A Representative macrographs of the lungs of hamster from each study group. B Representative micrographs of the hematoxylin-eosin stained lung sections (magnification ×50): asterisk – foci and/or diffuse mix cell infiltrate C Representative micrographs of the hematoxylin-eosin stained lung sections (magnification ×20): AS – alveolar sept, EH – epithelial hyperplasia, ET – endothelial cells, ER – erythrocytes, IN – infiltration cells, MP – macrophages. D Semi-quantitative analyses of the airway, lung/alveolar and vascular damage. Data were analyzed by one-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05

Fig. 13

Cell-mediated immune response to influenza and SARS-CoV-2 antigens on day 5 after challenge with SARS-CoV-2 in the splenocytes of immunized Syrian hamsters. Isolated splenocytes were stimulated overnight with A H7N9 LAIV purified virus, B live SARS-CoV-2 purified virus or with C peptide mixture (PepTivator N + S). IFNγ-secreting cells were detected with a Hamster IFN-γ ELISpot Plus kit. Data were analyzed by one-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; **—p <0.01