Antigen construction and synthesis
COBRA hemagglutinin (HA) proteins corresponding to H1N1 and H3N2 seasonal influenza viruses (IAVs) were design based on the next-generation COBRA methodology as previously described [18]. Briefly, Y2 (H1) COBRA HA was derived from 6232 full length wild-type influenza A(H1N1) HA protein amino acid sequences, residues 1-566 (starting with Methionine as the first amino acid), from human H1N1 virus infections collected from January 1, 2014 to December 31, 2016 were downloaded from the EpiFlu online database and organized by their date of collection [19].
For H3 COBRA HA designated, J4 and TJ5, full length wild-type influenza A(H3N2) HA protein amino acid sequences, residues 1-566 (starting with Methionine as the first amino acid), from 54,041 human H3N2 virus infections collected from May, 1 2008 to September 30, 2019 were downloaded from EpiFlu online database and organized by their date of collection. TJ5 was designed using sequences isolated during May 2008 to September 2012, and May 2013 to April 2016 for J4 [3].
Soluble HA proteins were purified from cells transiently transfected into HEK293T with plasmids expressing a truncated HA gene that was cloned into the pcDNA3.1. The truncated HA genes were generated by replacing the transmembrane domain with a T4 fold-on domain, an Avitag, and a 6× His-tag for purification [23]. The concentration of the soluble HA proteins was determined by conventional bicinchoninic acid assay (BCA) according to the manufacture’s instruction.
Determination of HA content
A high-affinity, 96-well flat bottom ELISA plate was coated with 5–10µg of total protein of VLP and serial dilutions of a recombinant H3 antigen (3006_H3_Vc, Protein Sciences, Meriden, CT) in ELISA carbonate buffer (50mM carbonate buffer, pH 9.5) and the plate was incubated overnight at 4°C on a rocker. The next morning, plates were washed in PBS with 0.05% Tween-20 (PBST), then non-specific epitopes were blocked with 1% bovine serum albumin (BSA) in PBST solution for 1h at RT. Buffer was removed then stalk-specific Group 2 human antibody CR8020 (Sanofi Pasteur, Lyon, France) 1mg/mL, was added to the plate in blocking buffer at a working dilution of 1:4000, and incubated for 1 h at 37°C. Plates were washed, then probed with goat anti-human IgG horseradish-peroxidase-conjugated secondary antibody (1mg/mL) diluted in blocking buffer at a working dilution of 1:4000 (2040-05, Southern Biotech, Birmingham, AL, USA) for 1h at 37°C. Plates were washed then freshly prepared o-phenylenediamine dihydrochloride (OPD) (P8287, Sigma, City, State, USA) substrate in citrate buffer (P4922, Sigma) was added to wells for 3–5 minutes, followed by 1N H2SO4 stopping reagent. Plates were read at 492 nm absorbance using a microplate reader (Powerwave XS, Biotek, Winooski, VT) and background was subtracted from negative wells. Linear regression standard curve analysis was performed using the known concentrations of recombinant standard antigen to estimate HA content. Glycan animo acid motif (NX{S/T}), where X represents any amino acid, was used to identify putative N-linked sites on the COBRA proteins. Y2 HA had 9 of these motifs, J4 had 14 motifs, and TJ5 had 12 potential motifs. These putative N-linked glycosylation sites are consistent with those motifs found in wild-type HA proteins isolated since 2013.
Vaccination and Infection
DBA/2J and BALB/c female mice (n = 66 and n = 30, respectfully; 6–8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and housed in microisolator cages with access to food and water. The mice were cared for by USDA guidelines for laboratory animals and all procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) (no. A2020 03-007-Y1-A0 and LRI2935) and performed in accordance with institutional and ARRIVE guidelines. Mice were randomly divided into six (DBA/2J) or three (BALB/c) groups, with n = 11 or n = 10 mice, respectfully, in each group. Prior to vaccination (day 0), all of the mice were bled via the submandibular to confirm seronegative status against seasonal H1N1 influenza viruses, including: Texas/36/1991, SolomanIsland/03/2006, Brisbane/59/2007, California/07/2009, Michigan/45/2015, Brisbane/02/2018, and H3N2 viruses: Texas/50/2012, Switzerland/9715293/2013, HongKong/4801/2014, Singapore/IFNIMH/2016, Kansas/14/2017, Switzerland/8060/2017, HongKong/2671/2019, and South Astralia/34/2019. On day fourteen, all of the mice were bled and sera were separated from blood cells via centrifugation and then stored at -20°C ± 5°C (Fig. 1B). Mice were vaccinated intranasally (IN) with 1, 0.1, or 0.01µg of each of the COBRA rHA proteins, Y2, J4, and TJ5, plus the addition of 28.4ug of M7-NH2. As controls, some mice were vaccinated without adjuvant (0.9% saline only) or mock vaccinated with M7-NH2 adjuvant only (0.9% saline plus M7-NH2) (Fig. 1A-B). At week 4, the mice were boosted as before following the same vaccination regimen. Following the boost, all of the mice (DBA/2J) were bled on days 42 and 49 as before and the collected sera was separated by centrifugation in microcentrifuge tubes at 10,000 rpm for 10 min, and mixed together equally and then stored at -20°C ± 5°C. All of the BALB/c mice were vaccinated and bled on days 35 and 42 and the collected sera was processed as before, keeping day 35 and day 42 sera separate (Fig. 2A-B). Concurrently, bronchoalveolar lavages were harvested from five BALB/c mice on days 35 and 42 post-vaccination. Vaccinated mice were euthanized and lungs flushed via the trachea with 450uL of cold 1X PBS using 23G needles with 22X1 G polyethylene catheters, attached to 1mL syringes. The samples were placed in sterile microcentrifuge tubes, on ice, followed by centrifugation at 3000 x g for 5 minutes. After centrifugation, the supernatants were tranfered into fresh sterile centrifuge tubes and stored at -20°C ± 5°C. At day 52, all of the DBA/2J mice were anesthetized and infected with 8x106 PFU of A/Brisbane/02/2018 (H1N1) influenza A virus (IAV) via intranasal distillation. Upon recovery, the mice were returned to their cages and monitored daily for 14 days post infection for both morbidity and mortality. At day 55, lungs were harvested from 3 DBA/2J mice in each group, snap-frozen on dry ice, and stored at -80°C for determining viral lung titers.
Hemagglutination Inhibition Assay (HAI)
The HAI assay was performed for the detection of sera antibodies that inhibit binding of influenza viruses from agglutinating red blood cells (RBCs) by preventing binding of viral surface HA to sialic acid residues on RBCs. This protocol was based on the WHO manual for laboratory diagnosis and virological surveillance of influenza [24]. This HAI assay was performed against a panel of H1N1 viruses, including: A/SolomonIsland/3/2006, A/Brisbane/59/2007, A/California/07/2009, A/Michigan/45/2015, A/Brisbane/02/2018, and H3N2 viruses: A/Texas/50/2012, A/Switzerland//2013, A/HongKong/4801/2014, A/Singapore-IFNIMH-16-0019/2016, A/Kansas/14/2017, A/Switzerland/8060/2017, and A/South Australia/34/2019. The HAI assay was performed as previously decribed [25]. Sera from each mouse was initially treated with receptor destroying enzyme (RDE) (Denka Seiken, Co., Tokyo Japan) for eliminating non-specific inhibitors. In a deep-well, 96-well block, sera was diluted to 1/10th final solutions by reconsituting 100µL of serum with 3 volumes of RDE in 1x PBS, followed by overnight incubation at 37°C. The following day, the RDE-treated sera was heat inactivated in a water bath at 56°C for 45 minutes, followed by cooling to room temperature (RT), and the addition of 6 volumes of 1x PBS. At days 35 and 42, lungs from 5 BALB/c mice were harvested and homogenized, in 1mL of cold 1X PBS, using a plunger and 70µm strainer. In a v-bottom 96-well plate, 25µL of PBS was added into each well. RDE treated sera or lung homogenate was added in duplicates and serially diluted across the plate. Following serial dilutions, each virus was tested (1:8 solution). The plates were incubated at RT for 20 min for H1N1 influenza viruses or 30 min for H3N2 influenza viruses. Following incubation, 0.8% turkey red blood cells (TRBCs) for H1N1 viruses or guinea pig red blood cell (GPRBCs) for H3N2 influenza viruses were added to all of the corresponding wells, mixed by agiation, and then incubated at RT for 30 min (H1N1) or 1 h (H3N2). After incubation, the titer of each serum sample was reported as the reciprocal dilution of the last well without agglutination. An HAI titer of 1:40 was considered seroprotective as recommeded by the European Medicines Agency Guidelines on Influenza Vaccines [26].
Enzyme-linked Immunosorbant Assay (ELISA)
To assess total IgG serum antibody reactivity and specificity to COBRA HAs vaccine components or WT IAV HAs, Immulon 4HBX 96-well flat bottom plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 100µL of Y2, J4, TJ5 COBRA or WT Bris/18, Tas/20, or Sing/16 rHAs, at 1µg/mL in carbonate coating buffer (pH 9.4) and incubated overnight in a humidified chamber at 4°C. Following the incubation, the plates were decanted and blocked with 200µL, per well, of 4% FBS + 0.05% Tween 20 blocking buffer (BB) in 1x PBS, for 90 minutes at 37°C. During the blocking incubation, serum samples were prepared at 1:100 ratio and serially diluted (1:3) from an initial 1:500 dilution for sera, or prepared at 1:10 ratio for lung lavages, and serially diluted (1:2) from the initial 1:10. Following blocking completion, 100µL of each sera diluted sample or 50uL of each lung lavage diluted sample was added to the Y2, J4, TJ5 (for sera only) or WT Bris/18, Tas/20, Sing/16 (for sera and lung lavages) coated plates and incubated for 90 min at 37°C. Plates were washed and 100µL of prepared secondary goat anti-mouse IgG HRP (Southern Biotech, Birmingham, AL, USA), diluted 1:4000 in BB, added to each well, followed by incubation for 90 min at 37°C. After incubation with secondary antibody, the plates were washed and received 100µL of 1x ABTS (VWR Corporation) solution and incubated for about 13 min at 37°C. After complete colorimetric development, 50µL 1% SDS solution was added to each well to stop the colorimetric reaction. The optical density (O.D.) of the samples were immediately read at 414nm in a spectrophotometer (PowerWave XS, BioTek) using the Gene05 software to measure the antibody end-point titers, and compared to positive and negative controls. To further assess IgA and specific IgG1, IgG2a, and IgG2b isotype binding antibodies, samples were processed as before on Y2 coated plates (for sera only) or WT Bris/18, Tas/20, Sing/16 rHAs (for sera and lung lavages) and incubated with secondary goat anti-mouse IgA, IgG1, IgG2a or IgG2b antibody solutions and measured as before. Both sera and lung lavages were prepared at an initial 1:10 and serially diluted (1:2) for detection of IgA and all lung lavages were prepared at an initial 1:10 and serilly diluted (1:2) for detection of IgG1, IgG2a, and IgG2b.
Lung Viral Titers
MDCK cells were seeded 1x106 cells per 10cm2 and incubated for 24h and grown to ~ 95% confluency, lungs from each mouse were weighed and homogenized in DMEM supplemented with 1% penicillin-streptomycin (P/S), 10 times their weights. The lung homogenates where then centrifuged at 1500 rpm for 10 min to remove debris and serially diluted, 10-fold. Additionally, a 10-fold serial dilution of Brisbane/02/2018 was used as a positive control. The diluted samples were then added to the MDCK monolayers at 100µL per well, and allowed to infect for one hour, with 15-minute shaking intervals, at RT. Moreover, negative control wells received 100µL of DMEM P/S only. After one hour of infection, the supernatant from each well was aspirated and the wells were washed once with DMEM P/S, with removal of media after the wash. Next, 2mL of a 1:1 solution of 1.6% agarose in 2x cMEM media containing TPCK-Trypsin at 1ug/mL was added to each well and allowed to solidify, followed by incubation for 2–5 days at 37 °C with 5% CO2. Once cytopathic effects were confirmed, the agarose layers were removed from each well and the cells were fixed with 10% formalin solution for 10 minutes at RT. After the 10 minutes, the formalin was removed and the cells were stained with 1% Crystal Violet (Fisher Science Education, Waltham, MA, USA) at RT, for 10–15 minutes. Following completion of staining, the Crystal Violet was removed and the wells were rinsed in water. The plates were allowed to dry and the plaque forming units (PFUs) were counted, followed by calculation of the lung viral titers as PFU/g of tissue.