Flu-COVID Combo Recombinant Protein Vaccines Elicited Protective Immune Responses Against Both Influenza and SARS-CoV-2 Viruses Infection in Mice

2.1. Cells and Viruses

Madin-Darby canine kidney (MDCK) cells and Vero-E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (10,000 U/mL) at 37°C with 5% CO2. Influenza viruses used in this study include 4 H1N1 influenza viruses [A/Brisbane/59/2007 (Bris/07), A/California/07/2009 (CA/09), A/Brisbane/02/2018 (Bris/18), and A/Guangdong-Maonan/SWL 1536/2019 (GD/19)] and 6 H3N2 viruses [A/Switzerland/9715293/2013 (Switz/13), A/Hongkong/4801/2014 (HK/14), A/Singapore/INFIMH-16–0019/2016 (Sing/16), A/Kansas/14/2017 (KS/17), A/Hongkong/2671/2019 (HK/19), A/South Australia/34/2019 (SA/19)]. Influenza virus stocks were obtained from BEI Resources and then propagated in embryonated chicken eggs. SARS-CoV-2 USA-WA/1/2020 (WA-01) strain was also obtained from BEI and propagated in Vero-E6 cells. All the experiments involving live SARS-CoV-2 were performed in the biosafety level-3 (BSL-3) facility at the University of Georgia following the approved standard operating procedures.

2.2. Vaccine design and protein production

COBRA H1 and H3 Haemagglutinin (HA) sequences were designed and generated as previously described [11]. WA-01strain Spike (S) protein sequence was used to generate SARS-CoV-2 recombinant protein. Soluble HA and S proteins were obtained by transfecting truncated influenza virus HA and SARS-CoV-2 S protein genes that were cloned into the pcDNA3.1+ plasmid into HEK293F suspension cells as previously described [17]. The truncated HA (aa 1–528) and S (aa 1–1213) protein genes were generated by replacing the transmembrane domain with a T4 fold-on domain, an Avitag, and a 6 His-tag for purification [17]. The concentration of the soluble HA and S proteins was determined by conventional bicinchoninic acid assay (BCA) according to the manufacturer’s instructions.

2.3. Animals, vaccination, and infection

6–8 weeks old female hK18-hACE-2 transgenic mice were purchased from Jackson laboratory (Bar Harbor, ME, USA), housed in microisolator units with free access to food and water. They were cared under the University of Georgia Research Animal Resources Guidelines for laboratory animals. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). hK18-hACE-2 mice (n=12/group) were vaccinated with COBRA H1, COBRA H3 HA proteins, and S proteins (3μg/protein/mouse) alone or mixed those three proteins together, or PBS alone as mock controls. All proteins and PBS were formulated with AddaVax (water-in-oil emulsion) in a 1:1 ratio. Vaccines were administered intramuscularly at days 0, 28 and 56 respectively, blood were collected after fourteen or twenty-one days of each vaccination, and sera were separated for further assays. On day 80 after the first vaccination, mice were challenged with either 8.7 × 10⁶ plaque forming unit (PFU) of A/Brisbane/02/2018 (H1N1) or 2 ×10³ PFU of SARS-CoV-2 (WA-01). Three mouse lungs from each group were collected on day 4 post-infection, and the rest of the mice were observed for up to 14 days post-infection for morbidity and mortality, body weights and clinical scores were recorded daily. Terminal blood for all the survived mice were collected on day 14 post-infection, sera were separated and stored at − 80°C in a BSL-3 facility.

2.4. Hemagglutination Inhibition Assay (HAI)

The HAI assay was used to evaluate functional antibodies binding to HA protein that are capable of inhibiting red blood cell agglutination. This protocol was adapted from the WHO laboratory influenza surveillance manual [29]. In this study, HAI assays were performed against a panel of 4 H1N1 influenza viruses, including: Bris/07, CA/09, Bris/18, GD/19 and 6 H3N2 viruses including Switz/13, HK/14, Sing/16, KS/17, HK/19, SA/19. The HAI assay was performed as previously described [18]. Briefly, sera were treated with receptor destroying enzyme (RDE) (Denka Seiken, Co., Tokyo, Japan) prior to being tested to remove nonspecific inhibitors by incubating overnight at 37°C, the RDE was then further inactivated at 56 °C for 45 min. An amount of 25 μL of PBS was added to a 96-well V-bottom plate in row 2–12, 50 μL RDE-treated sera was added into row 1, and then a 2-fold serial dilution was performed across the plate. For H1N1 viruses, an equal volume of H1N1 virus with approximately 8 hemagglutination units (HAU)/50 μL was added into each well. The plates were incubated at RT for 30 min, and then a solution of 0.8% turkey erythrocytes in PBS were added in a volume of 50 μL to each well. The plate was mixed by agitation and incubated at RT for another 30 min. For H3N2 viruses, an equal volume of H3N2 virus with approximately 8 HAU/50 μL in the presence of 20 nM Oseltamivir carboxylate was added into each well. The plates were incubated at RT for 30 min, and then 0.8% guinea pig erythrocytes (Lampire Biologicals, Pipersville, PA, USA) in PBS were added into each well, and the plates were mixed by agitation, and incubate for 1 hour at RT. The HAI titer was determined as the reciprocal dilution of the last well that contained non-agglutinated RBCs. An HAI titer greater than 1:40 was defined as seroprotective [19].

2.5. Plaque assay

MDCK or Vero-E6 cells were seeded in each well of a twelve-well plate at a concentration of 5 × 10⁵ cells/well one day prior to performing the plaque assay. Frozen Lung tissues were thawed on ice and homogenized in 1 mL of DMEM. Homogenate was centrifuged at 2000 rpm for 5 min to remove tissue debris, and the supernatant was collected and subjected a serial 10-fold dilution in DMEM supplemented with 1% penicillin-streptomycin. MDCK/Vero-E6 cells with a 90% confluency in each well were infected with 100 μL of each dilution of homogenate supernatant. The plates were then shaken every 15 min for 1 h. After 1 h incubation, the supernatant was removed and cells were washed twice with fresh DMEM. Finally, 2 mL of 2 × MEM and 2.4% Avicel overlay was added into each well, and the plates were incubated at 37 °C with 5% CO2 for another 72 h. After that, overlay was removed from each well, and the cells were fixed with 10% buffered formalin for 20 min and stained with 1% crystal violet (Fisher Science Education, Waltham, MA, USA) for 15 min at RT. Plates were then rinsed thoroughly using tap water to remove excess crystal violet. The plaques were enumerated, and the lung viral titers were calculated and presented as PFU/mL.

2.6. Focus Forming reduction assay (FRA) for influenza virus

The FRA used in this study was initially developed by the World Health Organization collaborating center in London, UK [20, 21], and modified by U.S. Centers for Disease Control and Prevention (CDC). Sera samples were treated with RDE as described above. Initially, 100 μL of MDCK cells at a concentration of 3×10⁵ cells/mL were seeded into each well in a 96-well flat-bottom plate. After 24 h, the cells were allowed to reach 95% to 100% confluency and were then washed with PBS. Next, 50 μL of 2-fold serial diluted pooled sera samples were added to each well staring with a 1:20 dilution in virus growth medium (VGM) supplemented with 1 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (VGM-T) (Sigma, St. Louis, MO, USA). Afterwards, influenza virus was diluted in VGM-T, and 50 μL of virus at a concentration of 1.2×10⁴ FFU/mL was added to each well; VGM-T alone was also added to cell control wells. Plates were incubated at 37 °C for 2 h, and then 100 μL of overlay was added into each well. The overlay consists of equal volumes of 1.2% Avicel RC/CL (FMC Health and Nutrition, Philadelphia, PA, USA) and 2×MEM supplemented with 1 μg/mL TPCK-treated trypsin, 0.1% BSA, and 1% penicillin-streptomycin [20]. After 18~22 h of incubation at 37 °C, the overlay was removed and the cells were washed twice using PBS. Lastly, cells were fixed with ice-cold 4% formalin at 4°C for 30 min, followed by washing once with PBS and permeabilizing with 0.5% Triton X-100 at RT for 20 min. Monolayers were washed three times with wash buffer (PBS containing 0.1% Tween 20) and incubated with a mouse-anti-IAV nucleoprotein monoclonal antibody [22] at 37°C for 1 h. After washing three time with wash buffer, the cells were incubated with a secondary antibody, goat anti-mouse peroxidase labeled IgG (SeraCare, Inc., Milford, MA, USA), for 1 h at RT. Cells were then washed three times with wash buffer, and TrueBlue substrate (SeraCare, Inc., Milford, MA, USA) containing 0.03% H₂O₂ was added and incubated for 10 to 15 min at RT. The reaction was terminated by washing plates with distilled water five times. Plates were air-dried and foci were counted using a CTL BioSpot Analyzer with ImmunoCapture 6.4.87 software (CTL, Cleveland, OH, USA). The FRA titer was presented as the reciprocal of the highest dilution of sera corresponding to 50% focus reduction compared to the virus control wells minus the cell control wells.

2.7. Neutralizing antibody titration for SARS-CoV-2

Viral neutralization assay was performed on mouse sera using the SARS-CoV-2 (WA-01) (100 TCID50/50 μL; NCBI accession number: PRJNA717311) as previously described [23]. Briefly, mouse sera were heat inactivated for 30 mins at 56°C to inactivate complements. Sera were then subjected to 2-fold serial dilution starting at 1:25, 50 uL mouse serum was serially diluted and mixed with an equal volume of SARS-CoV-2 (100 TCID50/50μl) for 1 hour at 37 °C in a BSL-3 laboratory in triplicates. The serum-virus mixture was then transferred to Vero E6 cells monolayer in 96-well plates, and then plates were incubated for 72 hours in a 37°C 5% CO2 incubator. After incubation, cells were fixed with 10% formalin, and stained with 1% crystal violet. The endpoint titer was represented as the average reciprocals of the last dilutions where no CPE were detected.

2.8. ELISA

Mouse sera were subjected to 3-fold serial dilution starting at 1:100 dilution. Plates were coated with different proteins to assess antibody binding reactivity which was performed as previously described [24]. In brief, Immulon 4HBX plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 4 °C overnight with 100 μL per well with a solution of carbonate buffer (pH 9.4) containing 1 μg/mL of the different proteins including Bris/18 HA protein, full-length S protein, S protein RBD or a chimeric protein cH6/1 (HA head from H6, HA stem from H1) and 5 μg/mL of bovine serum albumin (BSA) in a humidified chamber. An amount of 5 μg/mL BSA (50 μL per well) was coated alone as a negative control. Plates were blocked with 200 μL/well of ELISA blocking buffer for 1 h at 37°C. Serum samples were 3-fold serially diluted starting from 1:100, and then added into recombinant protein coated plates. After incubation at 4 °C overnight, the 1:2000 diluted goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA) secondary antibody, was added into each well in a volume of 100 μL, and incubated at 37 °C for 1 h. Finally, 50 μL of ABTS substrate (VWR Corporation) was added into each well and further incubated at 37 °C for 15 min. Colorimetric conversion was terminated by adding 50 μL of 1% SDS into each well. The O.D. values (OD 414) were measured by a spectrophotometer (PowerWave XS, BioTek) at 414 nm. The area under curve (AUC) was calculated using Prism 9.

2.9. Statistical Analysis

All data are presented as absolute mean values ± standard errors of the means (SEM) or standard deviation (SD). One-way ANOVA was used to analyze the statistical differences among groups, Two-way ANOVA was used to analyze the difference of body weight among different vaccinated groups (GraphPad Prism 9 software, GraphPad, San Diego, CA, USA). A “p” value less than 0.05 was defined as statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

3.1. Combo vaccine elicited comparable level of HAI+ antibody responses compared to the mono vaccines

To assess whether the HAI+ antibodies elicited by the combo vaccine were equivalent to the mono vaccines, K18-hACE-2 transgenic mice (n=12/group) were intramuscularly vaccinated with either the combo recombinant protein vaccine containing COBRA Y2 (H1), NG2 (H3) rHA proteins and S protein formulated with Addavax (an oil-in-water-based adjuvant), or were singly vaccinated with each adjuvanted component or PBS in a prime-boost-boost regimen with 4-week intervals. The antisera from week 10 post-initial vaccination were collected for the haemagglutinin inhibition (HAI) assay against a panel of H1N1 and H3N2 viruses spanning from 2007 to 2019. Only Y2 and combo vaccinated mice had HAI+ antibodies against H1N1 viruses (Fig. 1A, C), while NG2, S and mock vaccinated mice had no HAI+ antibodies detected as expected (Fig. 1B, D, E). The combo vaccine elicited significantly higher HAI+ antibodies against CA/09 and Bris/18 than GD/19 (Fig. 1C), and Y2 shared the same pattern of HAI+ antibodies as the combo vaccine (Fig. 1A).

For H3N2 viruses, NG2 and combo vaccinated mice had HAI+ antibodies detected (Fig. 1F, G), while Y2, S and mock vaccinated mice had no HAI+ antibodies against H3N2 viruses (Fig. 1E, H, I). The combo vaccine elicited higher HAI+ antibodies against Sing/16 and SA/19, but very low titers against KS/17 and HK/19 (Fig. 1G), which had the same pattern of HAI+ antibodies as the NG2 vaccine (Fig. 1F). The HAI+ antibody results suggested that the combo vaccine elicited antibodies had the same pattern of HAI+ activities as those elicited by mono vaccine against H1N1 and H3N2 viruses.

3.2. Combo vaccine possessed the same kinetics and magnitude of antibody response with mono vaccine against Influenza viruses

To evaluate the dynamic of antibody responses after vaccination, antisera collected from weeks 3, 6, and 10 post-initial vaccination were subjected to HAI assay against the same panel of viruses mentioned above. As shown in Figure 2, no detectable HAI+ antibodies were detected at week 3 post-vaccination in mice no matter what vaccines were given (Fig. 2). HAI+ antibodies became detectable at week 6 post-vaccination, but the titers were under 1:40 expect for one mouse vaccinated with Y2 (Fig. 2A) and one mouse vaccinated with NG2 (Fig. 2C) had titer over 1:40. By week 10 post-vaccination, combo and Y2 all had average HAI titer above 1:80 against CA/09 and Bris/18 (Fig. 2A, B), and combo and NG2 had the average HAI titer against Switz/13, Sing/16 and SA/19 between 1:40 to 1:80 (Fig. 2C, D), while HAI titer against other H3N2 stains were below 1:40, suggesting that combo vaccine shared the same dynamic of HAI+ antibody responses against H1N1 and H3N2 viruses.

To further assess the magnitude of HAI+ antibodies elicited by combo and mono vaccine, HAI titers against H1N1 and H3N2 viruses were compared between combo vaccine and mono vaccine at different time points post vaccination. The combo vaccine elicited slightly lower HAI+ antibody titers than the mono vaccine against H1N1 and H3N2 viruses after the 2^nd and 3^rd vaccination, but the differences of HAI titers were not statistically different (Suppl. Fig1).

3.3. Combo vaccine elicited equivalent or higher total IgG antibodies than the mono vaccines

To assess whether the combo vaccine elicited IgG antibody levels were equivalent to those of mono vaccines against Bris/18 and SARS-CoV-2, antisera collected from mice vaccinated with different vaccines were evaluated their binding abilities to Bris/18 HA, S, S-RBD, and cH6/1 proteins (Fig. 3). For influenza specific antibodies, combo and mono vaccines elicited significantly high IgG antibody titers against Bris/18 HA in vaccinated mice, while no antibodies were detected in mock-vaccinated mice (Fig. 3A). However, Y2-vaccinated mice had significantly lower IgG titers than combo vaccinated mice, as well as NG2- and S-vaccinated mice against Bris/18 HA. Therefore, we further test the antibodies against chimeric H6/1 to access the HA stem binding antibodies. As Shown in Figure 4B, combo-, NG2-, and S-vaccinated mice had significantly higher stem-binding IgG than Y2-vaccinated mice, indicating the higher level of stem-binding IgG may contribute to the high level of total IgG in the combo, NG-2 and S-vaccinated mice.

For SARS-CoV-2 (WA-01) specific antibodies, the combo vaccine elicited equivalent total IgG against the full-length S protein (Fig. 3C), while it elicited slightly lower IgG against RBD of the S protein which is not statistically different (Fig. 3D). However, we noticed that Y2- and NG2-vaccianted mice also elicited IgG antibodies against full length S protein, while they all have significantly lower IgG compared to S and combo vaccine, indicating that influenza HA proteins may induce cross-reactive antibodies that bind to the SARS-CoV-2 S protein, but not to the S-RBD region (Fig. 3D).

3.4. Combo vaccine elicited a comparable neutralizing antibody level as mono vaccines against both influenza and SARS-CoV-2 viruses

To evaluate the neutralizing activity of antibodies elicited by combo vaccine, a focus reduction assay was used to assess neutralizing titers against challenge viruses (Bris/18 and WA-01) in vitro (Fig. 4). For Bris/18 virus infection, mice vaccinated with Y2, NG2 and PBS (mock) did not have detectable neutralizing antibodies against Bris/18 virus (Fig. 4A), In contrast, mice vaccinated with the combo vaccine had a log 2 titer of 11 (80% inhibition) and 12.32 (50% inhibition), and mice vaccinated with Y2 had a log2 titer of 10.32 (80% inhibition) and 11.32 (50% inhibition). The neutralizing antibodies titers elicited by the combo vaccine were slightly higher than Y2 (Fig. 4A).

For SARS-CoV-2 (WA-01) infection, no neutralizing antibodies were detected in the antisera collected from Y2, NG2 and mock vaccinated mice (Fig. 4B) However, statistically similar high neutralizing antibodies were detected in mice vaccinated with the combo and S vaccines (Fig. 4B), suggesting that the combo vaccine is as efficient as the S vaccine in eliciting neutralizing antibodies against SARS-CoV-2.

3.5. Combo vaccine protected mice from influenza A virus and SARS-CoV-2 infection as efficiently as the homologous mono vaccines

To assess the protective efficacy of Flu-COVID combo vaccine, K18-hACE-2 transgenic mice (n=12/group) were intramuscularly vaccinated with either the combo vaccine formulated with Addavax, or were singly vaccinated with each adjuvanted component or PBS in a prime-boost-boost regimen with 4-week intervals (Figure 5A). On week 12 post the initial vaccination, mice were intranasally challenged with 8.7×10⁶ PFU of A/Brisbane/02/2018 H1N1 (Bris/18) influenza virus. Mice that received the combo vaccine and Y2 mono vaccine barely lost any body weight during the entire observation period (Fig. 1B), and 100% of mice survived from Bris/18 challenge (Fig. 5C). However, NG2- or mock-vaccinated mice started to lose their body weight from 2 dpi and lost around 25% of their original body weight by 7 dpi (Fig. 5B), and 83% of them succumbed to disease, with only one mouse in each group survived from the infection (Fig. 5C). However, mice vaccinated with S protein lost around 25% body weight between 6 and 7 dpi (Fig. 5B). 50% of those mice succumbed to the disease by 7 dpi (Fig. 5C), while the other 50% of the mice started to gain weight and survived from Bris/18 infection (Fig. 5C).

Another set of K18-hACE-2 transgenic mice was vaccinated with the same vaccines using the same regimen mentioned above. On week 12, mice were intranasally inoculated with 2×10³ PFU of SARS-CoV-2 (WA-01) in a volume of 50 μL. Mice were then monitored for body weight loss and clinical scores for 2 weeks post-infection. As shown in Fig. 5D, mice vaccinated with the combo vaccine and S protein vaccine did not lose any body weight and no clinical signs were observed (Fig. 5E) throughout the entire observation time, and 100% of mice survived from lethal dose infection (Fig. 5F). As expected, mock and Y2 vaccinated mice gradually lost their body weights from 3 dpi, reaching to the endpoint between 6 and 7 dpi (Fig. 5D). Clinical signs started from 5 dpi and reached the peak by 7 dpi (Fig. 1E), no mice survived from SARS-CoV-2 (WA-01) infection (Fig. 5F). Surprisingly, there was a 2-day delay for NG2 vaccinated mice to reach the humane endpoint compared to mock-vaccinated group (Fig. 5D). the clinical scores of NG2 vaccinated mice were significantly lower than those in the mock control group from 5 to 7 dpi (Fig. 5D), and no clinical signs were observed after 10 dpi (Fig. 5E). Moreover, 33.3% of NG2 vaccinated mice survived from SARS-CoV-2 (WA-01) infection, which is significantly higher than that in the mock and Y2 vaccinated group (Fig. 5F).

3.6. Combo vaccine decreased lung viral loads as efficiently as the mono vaccines

In order to assess the viral titers in lung tissues after infection, three mice from each group were sacrificed on day 4 dpi, and the virus titers of Bris/18 and SARS-CoV-2 (WA-01) in the lung tissues were evaluated (Fig. 6). For Bris/18 virus infection, mock-vaccinated mice had the highest lung viral titers (9.33 × 10⁶ PFU) on 4 dpi, and mice vaccinated with NG2 and S proteins had statistically similar lung viral titers compared to the mock-vaccinated mice (Fig. 6A). However, Combo- and Y2 vaccinated mice barely had detectable viral titers in the lungs (Fig. 6A), suggesting that combo vaccine eliminated the Bris/18 virus in the lung tissues as efficiently as the H1 mono vaccine.

For SARS-CoV-2 (WA-01) infection, mock-vaccinated mice had 2.82×10³ PFU of viral load detected at 4dpi, and the Y2 (0.55×10³ PFU) and NG2 (1.1×10³ PFU) vaccinated mice had statistically similar viral loads with mock-vaccinated mice (Fig. 6B). As expected, S protein and combo vaccinated mice had no detectable viral titers in lung tissues at 4 dpi, the viruses had been (Fig. 6B), demonstrating combo vaccine cleared the SARS-CoV-2 in lung tissues as efficiently as the S protein mono vaccine.