Generation of chimeric influenza viruses

We used two strategies of influenza virus modification to deliver SARS-CoV-2 antigens to target cells. The first one involves modification of the HA gene of influenza virus, which includes incorporation of the antigenic fragment of SARS-CoV-2 into the influenza virion as a structural part of the HA protein. In the second case, we modified the NS gene of the LAIV viral vector to ensure independent processing of influenza and SARS-CoV-2 antigens in infected cells.

Generation of recombinant influenza viruses with modified HA genes

We designed SARS-CoV-2 RBD-based cassettes for incorporation into influenza HA molecules because this strategy was successfully used in our previous studies [20, 29]. We inserted the RBD-encoding fragment into the H7N9 influenza virus HA gene between the signal peptide-encoding sequence and the HA1 subunit of the molecule using the GGGGSGGGGS flexible linker. It was shown in previous experiments that cassettes in such constructs are expressed as a part of HA protein and exposed at the surface of the virion [20, 30]. According to our previous studies, the size of the cassette may have a significant impact on the virus growth characteristics and immunogenicity [31]. Therefore, we designed two variants of the SARS-CoV-2 RBD-based immunogenic cassettes of different lengths. The first variant, HA+RBD 194, contained the insertion of the SARS-CoV-2 spike protein’s 333-526 amino acid residues, as described in [32]. Another construct was designed based on the full-length RBD protein of the Wuhan strain which comprises 223 amino acid residues (319-541) of the spike protein [33] (Fig. 1).

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]

We rescued two recombinant influenza viruses expressing chimeric HA proteins as shown in Fig. 1, and carrying intact N9 gene, as well as six remaining genes of A/Leningrad/17 LAIV master donor virus. The chimeric viruses encoding the inserts RBD 194 and RBD 223 were named FluCoVac-19 and FluCoVac-20, respectively (Table 1).

Table 1 Recombinant viruses with SARS-CoV-2 RDB fragments incorporated into HA molecule

The rescued LAIV-RBD viruses were amplified in eggs, and their titers ranged from 8.3 to 8.4 lgEID50/mL (Table 1). Although they were significantly lower than that of the H7N9 LAIV vector, such infectious activity of the recombinant strains is suitable for further manufacturing processes. Ten sequential passages in eggs of the rescued viruses revealed high genetic stability of the RBD 194 (FluCoVac-19) and RBD 223 (FluCoVac-20) inserts, since only a single substitution was found in each virus, both times within the flexible linker: GGGGSGRGGS in FluCoVac-19 virus and GGGGNGGGGS in FluCoVac-20 variant. It is very unlikely that these minor changes will affect the antigenicity of the chimeric HA molecule, since the RBD sequence remained unchanged.

Generation of recombinant influenza viruses with modified NS genes

We also designed a panel of RBD-based constructs for their expression in the target cells as part of the modified NS gene of the LAIV virus. We previously developed recombinant LAIV strains with modified NSs that successfully stimulated T-cell immunity to other respiratory pathogens, such as RSV [35] and human adenovirus [22]. Here, we explored different variants of influenza NS gene modifications, targeting strategies and transgene cassette processing pathways that were found to be prospective for other recombinant vaccines.

We tested two modifications of the NS gene sequence. In the first type, the NS1 coding region was truncated up to 126 residues, followed by the linker and the RBD-based insert. The noncoding fragment of the NS1 ORF was removed from the sequence, except for the regions necessary for splicing for NEP. In another type of construct, we removed all noncoding sequences of NS1 and added necessary sequence after the cassette insertion for full-length NEP ORF recovery, through the T2A self-cleavage site (Fig. 2A). This strategy was previously studied by DiPiazza et al. [36]. In addition to the intact RBD fragment, we designed four variants of the RBD inserts with different targeting sequences that were supposed to enhance the humoral immune response to the transgene.

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)

Two strategies were used for SARS-CoV-2 cassette targeting to lysosomal compartment. A human LAMP-1 transmembrane C-end peptide sequence (35 amino acid residues) was added to the C-end of the RBD cassette (Fig. 2B). Early studies on DNA antiviral vaccines demonstrated that the addition of this peptide to an antigen leads to enhanced immunogenicity [37,38,39]. The mechanism is based on enhanced antigen presentation through the MHC class II pathway. In the second variant, the HLA-II invariant chain with transmembrane anchor domain was added to the N-end of the RBD cassette to serve as a signal of lysosomal targeting [40]. The role of the HLA-II invariant chain in the regulation of Th immune responses [41] suggested using it as a part of the transgene to modulate immune responses to DNA vaccination. In the mouse model, incorporation of CD4+ peptide into the HLA-II invariant chain led to high level of Th immune response after the peptide stimulation [42].

The other two designs included the tissue plasminogen activator (tPA) signal peptide at the N-terminus of the RBD cassette, and one of them also included the HER-2 transmembrane domain (TMD) and the IL2Ra cytoplasmic domain (CPD) fused to the C-end of RBD cassette (Fig. 2B). The tPA signal peptide is a signal sequence often used to transport the attached proteins towards the secretion pathway in mammalian cells. This signal peptide was used in several studies and its addition enhanced the immunogenicity of different constructs (MVA-based vaccine against tuberculosis, DNA vaccine with HIV-1 antigen, DNA vaccine against influenza) [43,44,45]. The idea is based on direct presentation of the target protein to immune cells in the bloodstream. In our case, the RBD structure was further stabilized by additional 55 residues of the spike protein and a single mutation (see below, [19]).

The usage of membrane targeting is a common strategy for enveloped viral vector presentation of foreign antigens. This strategy is widely used in recombinant baculoviruses [46]. The membrane targeting of the antigen in vector vaccines can improve immunogenicity even if the antigen is not originally membrane-bound [47]. The effect of different cytoplasmic domains on the protein’s effective surface presentation and overall vaccine potential was also studied [48]. Here, we tested the erbB-2 (HER-2) transmembrane domain fused with the alpha-subunit of the IL-2 receptor (IL2Ra) cytoplasmic domain, which was described earlier [49] (Fig. 2B). This combination was proven to be useful in previous experiments on cell lines development, e.g., for overexpression of human Fc receptors on the surface of CHO and HEK293 cells (data not shown). Also, there are no tyrosine residues in IL2Ra cytoplasmic domain, and therefore there is no chance of interference with complex signaling pathways in target cells, unlike the biologically active HER2 cytoplasmic domain.

Therefore, we tested several different approaches to trigger and enhance the immune response toward the key receptor-binding domain of the SARS-CoV-2 spike protein, along with the response to the influenza virus.

We also tested three ways to insert the RBD-based cassettes into truncated NS1 ORF (Fig. 2A): (i) using the P2A self-cleavage site previously described for designing T-cell-based vaccines and which ensured independent processing of influenza virus antigens and the inserted transgene [21, 35]; (ii) via the pentanucleotide Stop-Start codon (TAATG) used by influenza B virus to terminate and reinitiate translation, which was tested earlier to express reporter GFP or functional IL-2 [50] or RSV epitopes [51, 52] from the truncated NS1 ORF; and (iii) by fusing the NS126 protein with the RBD fragment through a flexible linker, so that the both proteins are expressed without disintegration. This strategy was shown to be promising in several studies of influenza viruses as viral vectors [53,54,55].

Of note, most of our RBD inserts were designed to have a prolonged region of the spike protein, comprising of residues 319-596 (278 amino acids), since this modification was proven to be highly immunogenic when expressed by an adeno-associated virus (AAV) vector [19].

Overall, we rescued seven recombinant LAIV viruses expressing various RBD-based cassettes from the modified NS1 protein ORF, as listed in Table 2. FluCoVac-35, FluCoVac-41, FluCoVac-59 and FluCoVac-72 encoded different RBD cassettes following the P2A autocleavage site, whereas FluCoVac-78 and FluCoVac-79 were connected to the NS1 protein fragment via the Stop-Start pentanucleotide. The last variant, FluCoVac-83, encoded the RBD antigen in-frame with the NS1 and was separated from the influenza protein by the flexible linker.

Table 2 Recombinant viruses with SARS-CoV-2 RDB fragments incorporated into NS1 ORF

The replicative activity of the recombinant viruses in chicken embryos was compared to that of the modified H7N9 LAIV virus encoding truncated to 126 residues NS1 protein, which was characterized earlier [17]. Most of the chimeric influenza viruses replicated efficiently in eggs, except FluCoVac-79 variant (Table 2). It is likely that this combination of the RBD cassette and the TAATG linking region interfered with the infectious activity of the chimeric virus. Interestingly, the same RBD cassette with SP, TMD and CPD inserted via the P2A self-cleavage site had no negative effect on the growth properties of the recombinant virus; in fact, this variant, FluCoVac-72, replicated better in eggs than the control LAIV NS126 vector virus (Table 2).

Serial passaging of the rescued LAIV/RBD variants revealed high level of genetic stability of all but one recombinant virus (Table 2). Strikingly, the only variant that encoded the full-length NEP following the RBD cassette and the T2A autocleavage site (FluCoVac-41) was unstable and the insert was not detected in the virus after six passages in eggs. However, more research is needed to elucidate the exact genetic mechanisms underlying this phenomenon.

Expression of the RBD protein by the chimeric LAIVsExpression in MDCK cells

The expression of correctly folded RBD protein in MDCK cells infected with experimental vaccine strains was evaluated by sandwich ELISA of cell lysates. The productive infection of the cell with each recombinant virus was confirmed by hemagglutination assay of cell supernatants, as well by the detection of cytopathic effect in each virus-infected well. Unexpectedly, the expression of high levels of RBD protein was detected only in cells infected with two variants with RBD insertions into the influenza HA molecule – FluCoVac-19 and FluCoVac-20 (Fig. 3). No significant expression of the RBD was detected in MDCK cells infected with any of the recombinant viruses with insertions into the NS1 ORF, indicating that synthesis of the RBD protein from the NS1 open reading frame does not result in proper folding of the target antigen within the infected cell (Fig. 3).

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

Western blot

Since RBD fragments inserted into HA molecule of influenza virus are supposed to be exposed on the surface of the virion, we conducted Western blot analyses of the sucrose gradient-purified viruses FluCoVac-19 and FluCoVac-20, using a polyclonal anti-RBD rabbit antibody and a mouse hyperimmune sera raised to the recombinant H7 HA protein expressed in insect cells. The H7N9 LAIV vector, as well as recombinant RBD protein, were used as control antigens in this assay. As shown in Fig. 4A, three apparent bands reacting with anti-RBD antibodies were observed in the FluCoVac-19 virus, suggesting the presence of RBD antigen in complex with the monomeric, dimeric and trimeric influenza HA molecules. The recombinant RBD protein used as a positive control in this study was also detected by anti-RBD antibodies in monomeric and multimeric forms, each monomer with expected size about 35 kDA (rhombus at Fig. 4A). Unexpectedly, no anti-RBD antibody binding was detected in the case of the FluCoVac-20 variant (Fig. 4A), whereas clear RBD expression was noted when MDCK cells were infected with this virus (Fig. 3). The absence of the RBD fragment within the HA molecule of FluCoVac-20 was confirmed by Western blot with anti-H7 antibody: the HA bands in various forms in this variant were identical to the H7N9 LAIV control virus, whereas corresponding bands of the FluCoVac-19 virus appeared at higher molecular weight, confirming the presence of an additional fragment within this antigen (Fig. 4B). Since FluCoVac-20 encoded the RBD fragment within the chimeric HA gene, which was confirmed by Sanger sequencing of the purified virus material, and expressed significant quantities of RBD within infected MDCK cells, most likely that the RBD fragment in this virus is subjected to proteolytic cleavage post-translationally and is not exposed on the surface of the virion.

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

Replication and immunogenicity in BALB/c mice

Despite the lack of RBD protein expression in some of the rescued recombinant LAIV/RBD viruses, all variants were assessed in a mouse model to determine their replicative activity in the respiratory tract and their ability to induce antibody responses to the whole influenza virus, as well as to the target RBD antigen. We hypothesized that the use of a monoclonal antibody in an ELISA expression assay could give a false negative result in the case of incorrect folding of the only epitope to which the antibody is specific, but that an immune response to other epitopes could form correctly.

Groups of mice were i.n. inoculated with 106 EID50 of each virus, and the lungs and nasal turbinates were collected on day 3 post infection. As shown on Fig. 5, very weak replication was detected in all recombinant viruses. The FluCoVac-19 and FluCoVac-20 variants were compared to the classical LAIV virus, and the difference in the replicative activity between chimeric and control viruses suggests that the foreign insert could have interfered with the ability of the LAIV virus to replicate in the mouse URT. For the NS1-modified recombinant viruses, the absence of infectious virus in the mouse respiratory tract is in line with findings that the LAIVs encoding truncated NS1 protein had restricted ability to infect mice [17]. Therefore, the effect of foreign insertions within the NS1 ORF on the infectivity of the virus in mice could not be elucidated.

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

Nevertheless, despite weak virus replication in the mouse respiratory tract, all recombinant vaccine viruses induced high levels of influenza virus-specific serum IgG antibodies three weeks after the second immunization (Fig. 6A). These data indicate that influenza viruses successfully infected target cells and the viral antigens were presented to the mouse immune system. However, rather weak responses were detected to the RBD antigen (Fig. 6B): on day 42 of the study, significant response to RBD was detected only in mice immunized with FluCoVac19 (p=0.027, ANOVA with post-hoc Dunnet’s test) and FluCoVac59 (p=0.0009, Kruskal-Wallis test with post-hoc Dunn’s test). Levels of serum antibodies in the other groups were not significantly different from control LAIV group. Low responses were registered in several animal sera in the LAIV and LAIV NS126 groups, which could be due to the binding of cross-reactive antibodies with low affinity to the RBD protein.

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

Importantly, the sera of immunized mice were unable to neutralize live SARS-CoV-2 infection in vitro (data not shown), suggesting that the levels of induced anti-RBD antibodies were insufficient to inhibit virus replication in Vero cells under our conditions. Of note, the MN method used in our study has lower sensitivity than the PRNT assay which is used in most studies.

Assessment of the selected FluCoVac-19 vaccine candidate in Syrian hamsters

Because the FluCoVac-19 vaccine candidate demonstrated high immunogenic potential against both influenza and SARS-CoV-2 antigens, this variant was selected for further evaluation in Syrian hamsters. For the assessment of infectivity, immunogenicity and protective activity against influenza and SARS-CoV-2 infections, groups of 12 animals were immunized twice with the recombinant virus and the LAIV vector control at a dose of 5×106 EID50, twice with a tree-week interval. A group of control animals received PBS (Fig. 7).

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

Three days after the first immunization, four animals from each group were humanly euthanized to assess influenza virus replication in the respiratory tract. Infectious titers were determined by titration of tissue homogenates in eggs, with the limit of virus detection 1.2 lgEID50. As expected, no virus replication was observed in the lungs of immunized hamsters, confirming the attenuated phenotype of the LAIV virus and the FluCoVac-19 (Fig. 8). In contrast, both viruses replicated efficiently in nasal turbinates, reaching mean titers of 4.4 and 3.2 lgEID50/g for the LAIV and FluCoVac-19 viruses, respectively (Fig. 8).

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

Serum antibody immune responses were measured on day 21 after the second dose by ELISA against whole H7N9 whole influenza virus or recombinant RBD protein. Although the levels of anti-influenza IgG antibodies were slightly lower in the FluCoVac-19 group than in the LAIV control group, these differences were not statistically significant (Fig. 9A). These data indicate that the insertion of the RBD 194 fragment into the HA molecule of the LAIV strain did not impact the overall immunogenicity of the vaccine relative to the influenza virus antigens. Importantly, a significant increase in the anti-RBD IgG antibody levels was found only in animals immunized with the recombinant vaccine candidate (Fig. 9B). Notably, there was a variation in the immunogenicity of the FluCoVac-19 vaccine, as some animals had robust RBD-specific responses, whereas others responded rather weakly to this target antigen (Fig. 9B).

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 2nd 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

Immunized hamsters (n=4) were challenged with Sh/PR8 influenza virus 3 weeks after the second immunization. On day 3 post challenge, a significant reduction in viral pulmonary titers were observed in both LAIV and FluCoVac-19 groups, compared to the hamsters administered with PBS (Fig. 10). These data indicate that the anti-influenza protective immunity was not affected by the modification of the LAIV genome by incorporating a foreign antigen into its HA protein.

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

The remaining four immunized animals in each group were subjected to SARS-CoV-2 challenge. On Day 42 of the study, hamsters were i.n. infected with 105 TCID50 of Wuhan (D614G) SARS-CoV-2 virus. The body weight and clinical symptoms of the disease were monitored for 5 days after challenge. At this time point, the animals were euthanized and respiratory tissues were collected for viral load determination and for the histopathological evaluation. In addition, spleens were harvested, and the numbers of IFNγ-secreting cells in isolated splenocytes were assessed by ELISPOT assay.

The FluCoVac-19 vaccine provided a detectable level of protection against live SARS-CoV-2, as was manifested by reduced weight loss (Fig. 11A), diminished clinical symptoms (Fig. 11B), and reduced viral titers in the URT and LRT of the animals on day 5 post challenge (Fig. 11C), compared to the PBS and H7N9 LAIV groups. Notably, the protection was not even, since one of four animals in the FluCoVac-19 group shed the virus at the same level as control animals.

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 2nd 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

Histopathological evaluation of lung tissues revealed only partial protection of FluCoVac-19-immunized animals against SARS-CoV-2-induced induced alveolar and peribronchiolar inflammation, hemorrhages and endothelial dysfunction (Fig. 12 A-C). In general, the LAIV vector and the chimeric vaccine groups did not differ in the sum of the pathomorphological scale scores, with the exception of a slightly lower severity of vascular changes in the FluCoVac-19 group than in the PBS group (Fig. 12D). It should also be noted that there are no gross histopathological changes in the form of cell rupture or detachment. Overall, based on the clinical, virological and pathomorphological evaluations, the FluCoVac-19 vaccine prototype demonstrated moderate degree of protection against challenge with a high dose of virulent SARS-CoV-2. Further studies with other challenge regimens are needed to fully elucidate the protective potential of this bivalent vaccine candidate.

Fig. 12