Study design

This study was designed to evaluate the immunogenicity and in vivo protection of recombinant mHA constructs as universal influenza B inactivated virus vaccines in the mouse model. Different inactivated vaccine preparations and adjuvants were tested. Humoral immune responses measuring the level of antibodies targeting the conserved regions of the HA, cross-reactivity against a panel of HAs, HI antibodies, Fc mediated effector functions and antibody subclasses were evaluated in mice sera post-vaccination. Germinal center activation and T cell immunity was evaluated post-vaccination in inguinal lymph nodes and spleens, respectively. In vivo protection was tested in direct and serum passive transfer challenge experiments using three different influenza B viruses. Randomization was achieved by randomly distributing mice into different cages upon arrival. No animal subjects were excluded from the sample collection or analysis unless the sample was exhausted. This study was performed in preparation for phase I clinical trials.

Cell culture

Madin-Darby canine kidney (MDCK) cells were maintained in minimum essential medium (MEM; Gibco) supplemented with 10% (vol/vol) of heat inactivated fetal bovine serum (FBS), 100 units/mL of penicillin, 100 µg/mL of streptomycin (P/S; Gibco), 2 mM of L-glutamine (Gibco), 0.15% (w/vol) of sodium bicarbonate (Corning) and 20 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES; (Gibco). Cell lines were maintained at 37 °C with 5% CO2.

Rescue of recombinant wildtype and mosaic HA influenza B viruses

Wildtype (WT) recombinant viruses contained the HA gene sequences of the B/Phuket/3073/2013, B/Yamagata/16/1988 or B/Brisbane/60/2008 strains. The mosaic HA (mHA) gene segments were designed by replacing the major antigenic sites of the HA gene sequences of B/Phuket/3073/2013, B/Yamagata/16/1988 and B/Brisbane/60/2008 with the corresponding sequences of H5 (A/Vietnam/1203/2004 H5N1-PR8-IBCDC-RG/GLP), H8 (A/mallard/Sweden/24/2002 H8N4) and H13 (A/black-headed gull/Sweden/1/1999 H13N6) to generate mH5/BPhu, mH8/BYam, and mH13/BBris, respectively. Sequences were reported in previous works17,18. The HA gene fragments were ordered as synthetic DNA gBlocks from Integrated DNA Technologies (IDT) and cloned into the pDZ plasmid17,18, and mHA and WT viruses were rescued using a reverse genetics protocol, as previously described5,17,18.

Hemagglutination inhibition (HI) assay

Mouse sera were treated with receptor destroying enzyme (RDE; Denka Seiken) to eliminate non-specific inhibitors in the sera. Briefly, serum was mixed with RDE in a 1:3 ratio (vol/vol). RDE-treated samples were incubated at 37 °C for 18–20 h and the reaction was stopped by the addition of 2.5% sodium citrate solution in a 1:3 ratio (vol/vol). The samples were then heat-treated at 56 °C for 30 min. Serum was finally diluted with sterile phosphate-buffered saline (PBS) to reach a final dilution of 1:10. To perform HI assays, virus stocks were diluted in PBS to a final HA titer of 8 HA units (4 wells of HA) per 50 μL sample. Two-fold dilutions (25 μL) of RDE-treated serum in PBS prepared in 96-well V-bottom microtiter plates (Thermo Fisher Scientific) were then combined with 25 μL of the diluted virus. The plates were incubated for 30 min at room temperature (RT) to allow HA-specific antibodies to bind to virus. Then 50 μL of a 0.5% suspension of turkey red blood cells (Lampire) were added to each well. HI titers were defined as the reciprocal of the highest dilution of serum that inhibited hemagglutination of red blood cells.

Preparation of inactivated viruses for vaccination

Production of mHA and WT influenza B virus vaccine preparations was performed in specific pathogen free embryonated chicken eggs (Charles River Laboratories) incubated at 33 °C. After a 3-day incubation, eggs were cooled to 4 °C overnight, and allantoic fluid was harvested and clarified by low-speed centrifugation using a Sorvall Legend RT Plus Refrigerated Benchtop Centrifuge (Thermo Fisher Scientific). The presence of virus in the allantoic fluid was measured by hemagglutination (HA) assay and the HA sequence was confirmed by Sanger sequencing (Genewiz). Clarified allantoic fluids were treated with formaldehyde (FA) or with beta-propiolactone (BPL). FA inactivation was performed with 0.03% (vol/vol) FA at 4 °C under continuous shaking for 72 h18. BPL inactivation was performed with the addition of 13 mM disodium phosphate (DSP) to stabilize the pH followed by 0.05% (vol/vol) BPL and rocked at 4 °C under continuous shaking for 30 min57. The BPL mixture was then placed in a 37 °C water bath for 2 h shaken every 15 min. The inactivated allantoic fluid was clarified by centrifugation at 4000 rpm for 30 min using a Sorvall Legend RT Plus Refrigerated Benchtop Centrifuge (Thermo Fisher Scientific). Clarified allantoic fluids were laid on top of a 30% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM Ethylenediamine tetraacetic acid (EDTA), pH 7.4). Ultracentrifugation in a Beckman L7-65 ultracentrifuge at 25,000 rpm for 2 h at 4 °C using a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA) was performed to pellet the viruses through the sucrose cushion while soluble egg proteins were removed. The virus pellets were re-suspended in PBS (pH 7.4). The splitting process was performed with 1% (vol/vol) Triton X-100 (Fisher Bioreagents) at room temperature (18–20 °C) under continuous shaking for 1 h. Triton X-100 was removed by mixing the split virus with Bio-beads SM-2 (BioRad) at 4 °C under continuous shaking overnight. The total protein content was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific) according to the manufacturer’s protocol. HA content was measured using an in-house ELISA and densitometry in protein gels of the different vaccine preparations19,58,59.

Immunization studies

For animal immunizations, 8- to 10-week-old female BALB/c mice (Jackson Laboratories) were used for all experiments. Experiments were performed in accordance with protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC). Vaccination with QIV (Flulaval Quadrivalent, GSK) vaccinated group and an unvaccinated group (PBS) were included as controls. Vaccines were administered intramuscularly (IM) at a dose of 0.1 or 1 μg HA per mouse diluted in a total volume of 100 μL with or without 10 or 30 μg of CpG 1018 adjuvant (DynaVax), 10 μg of CpG 1018 + 50 μg of aluminum hydroxide gel (Invivogen), or 50 μL of AddaVax adjuvant (1:1 v:v, Invivogen), respectively. Vaccines were diluted in sterile PBS (GIBCO) except for the combination of the CpG 1018 and aluminum hydroxide gel 2% (alum) adjuvants, when vaccines were prepared in a saline solution composed of 20 mM Tris and 100 mM NaCl (pH = 7.5). Intranasal administration of mH8/BYam virus was performed in a total volume of 30 μL in a drop-by-drop manner distributing equal volumes between each nostril of the mice after anesthesia with a ketamine/xylazine cocktail administered intraperitoneally. PBS-vaccinated mice were included as negative controls. Vaccinations were given in 3- or 4-week intervals, unless otherwise indicated. After the final immunization, mice were euthanized at defined time points. Terminal bleed was collected using cardiac puncture method, in which mice were administered a terminal anesthesia cocktail of ketamine/xylazine at a dose of 80–100 mg/kg of ketamine and 5–10 mg/kg of xylazine intraperitoneally. Euthanasia was performed following approved IACUC protocols with CO2 followed by cervical dislocation. After euthanasia, spleen, lymph nodes and blood were collected. Spleen and lymph nodes were stored in PBS (Gibco) on ice until further processing. Blood was obtained by cardiac puncture or cheek bleeding and sera were isolated by low-speed centrifugation and were stored at −20 °C until use.

Passive transfer and challenge studies

The challenge viruses B/New York/PV01181/2018 and B/New York/PV00094/2017 were isolated by the Personalized Virology Initiative at the Icahn School of Medicine at Mount Sinai (ISMMS) and were mouse adapted in a previous work18. Mice were infected intranasally with 5 murine 50% lethal doses (5× LD50) of the mouse-adapted B/New York/PV01181/2018 virus, mouse-adapted B/New York/PV00094/2017 virus or B/Lee/1940 virus in a volume of 30 μL of sterile PBS after anesthesia with a ketamine/xylazine cocktail administered intraperitoneally. In direct challenge, mice were challenged 3-4-weeks after the second boost. For passive transfer experiments, 8- to 10-week-old female BALB/c mice (Jackson Laboratories) received 100 μL of pooled sera via intraperitoneal injection 2 h before the challenge. Animals were monitored for survival and weight loss for 14 days post-challenge and were scored dead and humanely euthanized if they lost more than 25% of their initial body weight.

Enzyme-linked immunosorbent assay (ELISA)

Recombinant HA proteins were produced using the baculovirus expression system as described previously59,60. Proteins were coated onto Immulon® 4 HBX 96-well microtiter plates (Thermo Fisher Scientific) at 2 μg/mL in 1× coating buffer (SeraCare Life Sciences Inc.) at 50 μL/well overnight at 4 °C. All plates were washed 3 times with 225 μL PBS containing 0.1% (vol/vol) Tween-20 (PBST) and 220 μL blocking solution (3% v/v goat serum, 0.5% w/v non-fat dried milk powder in PBST) was added to each well and incubated for 1 h at RT. Individual serum samples or pooled sera were serially diluted 3-fold in blocking solution followed by a 2-h incubation at RT. ELISA plates were washed 3 times with PBST and 50 μL of secondary antibody conjugated with horseradish peroxidase (HRP) was added. For total IgG quantification, a 1:3000 dilution of anti-mouse IgG (H&L) peroxidase conjugated (Cytiva) in blocking solution was added. For IgG1 and IgG2a quantification, a 1:2000 dilution in blocking solution of anti-mouse IgG1 or anti-mouse IgG2a (Abcam) was added, respectively. After 1 h, plates were washed 3 times with PBST and developed using SigmaFast o-Phenylenediamine dihydrochloride (Sigma-Aldrich) for 10 min. Reactions were stopped by adding 50 μL 3 M hydrochloric acid (HCl) and absorbance at 492 nm was determined on a plate reader. For each ELISA plate, the average plus 3 standard deviations of absorbance values of blank wells was used as a cutoff to determine endpoint titers and the area under the curve (AUC) using GraphPad Prism version 10.0.0 for Windows, (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com).

Antibody dependent cell-mediated cytotoxicity (ADCC) reporter assay

White flat-bottom 96-well plates (Corning) were seeded with 2× 104 MDCK cells per well. After 24 h at 37 °C, the MDCK cells were washed once with PBS and then infected with influenza B viruses at a multiplicity of infection (MOI) of 5 for a single cycle of virus replication for one hour, then the infection media was removed and infected MDCK cells were incubated overnight at 33 °C. The next day, the culture medium was removed and 25 μL of assay buffer (Roswell Park Memorial Institute (RPMI) 1640 supplemented with 4% low-IgG FBS [Gibco]) was added to each well. Pooled mouse sera were diluted 1:2 (from a starting dilution of 1:30) in RPMI 1640 medium (Gibco) and added (25 μL per well) to the virus infected MDCK cells in duplicates for 30 min. Genetically modified Jurkat cells expressing the mouse FcγRIV with a luciferase reporter gene under transcriptional control of the nuclear factor-activated T (NFAT) cell promoter were then added to the plate at 7.5× 104 cells in 25 μL/well (Promega) and incubated for 6 hours at 37 °C. FcγRIV receptor will bind to anti-influenza antibodies and the genetically modified Jurkat cells will express luciferase through activation of NFAT promoter, At the end of the incubation time, a volume of 75 μL of Bio-Glo Luciferase assay reagent (Promega) was added to each well and luminescence was measured using a Synergy 4 microplate reader (BioTek) using Gen5 2.09 software. Fold induction was calculated as follows: (RLUinduced−RLUbackground)/(RLUuninduced−RLUbackground) and the geometric mean of AUC was calculated using GraphPad Prism version 10.0.0 for Windows, (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com).

Multiplex ELISA

A Th1/Th2 cytokine panel was used with simultaneous measurements of different cytokines in serum samples taken 4 h after vaccine administration adapted from61,62 using the Th1/Th2 Cytokine 11-Plex Mouse ProcartaPlex™ Panel (Cat#EP100-20820-901, Thermo Fisher Scientic). The following cytokines were quantified: GM-CSF, IFN-γ, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-12p70, IL-13, IL-18 and TNF-α. Serum samples were serially diluted 1:2. Cytokine levels were measured using a MAGPIX device (Luminex xMAP detection system, TX, USA) following the manufacturer’s instructions. Fold change (log 2) values over the PBS vaccination group were reported.

Spleen processing and intracellular cytokine staining

Splenocytes were resuspended in complete RPMI 1640 media (cRPMI, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% w/v FBS (Gibco), 100 U/mL of P/S (Gibco), and 2 mM L-Glutamine (Gibco). Cells were seeded in V-bottom 96-well plates (CELLSTAR, Greiner Bio-One North America Inc., Monroe, NC, USA) at an average of 2 ×106 cells/well in cRPMI media containing anti-mouse CD28 (1:500, BD), brefeldin A (1:1000, GolgiPlug™, BD Biosciences), and monensin (1:1,000, GolgiStop™, BD Biosciences). Splenocytes were stimulated with B/Florida/4/2006 Nucleoprotein and Matrix Protein 1 (NP, NR-36045 and M1, NR-36046, BEI Resources) pooled peptides at a final individual peptide concentration of 5 µg/mL at 37 °C with 5% CO2 10 h. Negative control cells were stimulated with an equivalent volume of dimethyl sulfoxide (DMSO). Positive control cells were stimulated with a cocktail containing phorbol 12-myristate 13-acetate (0.5 µg/mL, Sigma-Aldrich) and ionomycin (1 µg/mL, Sigma-Aldrich). The unstimulated control cells were only treated with the cRPMI media. After stimulation, cells were washed with PBS containing 2% FBS and centrifuged at 500 × g for 5 min and then stained with Zombie Red™ diluted in PBS (1:500, BioLegend) for 15 min at RT in the dark. Cells were washed in PBS containing 2% FBS (500 × g for 5 min) and incubated with surface staining cocktail containing Fc Block CD16/CD32 1:50 (BD) and the anti-mouse antibodies BV 711 CD3 (1:300, clone 17A2 BioLegend Cat#100241, AB_2563945), Pacific Blue CD4 (1:400, clone GK1.5 BioLegend Cat#100428 RRID: AB_493647), PerCP/Cy5.5 CD8 (1:200, clone 53-6.7 BioLegend Cat#100734 RRID: AB_2075238) for 30 min at 4 °C in FACS buffer. Cells were washed in FACS buffer and then incubated in fixation/permeabilization buffer (CytoFix, BD Biosciences) for 5 min at 4 °C. After fixation, cells were washed in 1× permeabilization buffer (CytoPerm, BD Biosciences), then incubated with the intracellular staining cocktail containing anti-mouse antibodies Alexa Fluor 647 IFN-γ (1:400, clone XMG1.2 BioLegend Cat#505814, RRID:AB_493314), Alexa Fluor 488 TNF-α (1:300, clone MP6-XT22 BioLegend Cat#506313 RRID:AB_493328), PE/Cy7 IL-2 (1:300, clone JES6-5H4, BioLegend Cat#503832 RRID:AB_2561750) in 1× permeabilization buffer for 1 h at 4 °C. Samples were then washed in 1× permeabilization buffer and resuspended in PBS buffer for acquisition. Samples were acquired on an Aurora spectral cytometer (Cytek, Fremont, CA, USA) using SpectroFlo® software (Cytek), with the relevant single fluorochrome compensation controls set by the daily acquisition of Cytometer Setup and Compensation beads (Ultracomp beads, Invitrogen). Analysis was performed with FCS Express 7 (DeNovo Software) and analyzed using the GraphPad Prism version 10.0.0 for Windows, (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com). Briefly, the percentage of cytokine CD4+T or CD8+T cells was calculated, and the value of stimulated groups was subtracted from that of the unstimulated condition.

Lymph node processing and B cell studies

Inguinal lymph nodes were dispersed into single-cell suspensions by mechanical disruption through 40 μm cell strainer (Fisher Scientific) using syringe plungers into cold PBS. Cells were pelleted by centrifugation (500 × g, 5 min) and re-suspended in FACS buffer (PBS containing 1% bovine serum albumin [BSA] and 2 mM EDTA) containing Fc block anti-mouse CD16/CD32 (1:50, eBioscience) in 50 μL for 10 min at 4 °C and then stained with primary antibodies cocktail (30 min, 4 °C). The primary antibody cocktail contained (50 μL): Zombie Red (1:400, BioLegend, Cat# 423109) as the viability dye, Pacific Blue anti-mouse CD3 (1:200, clone 17A2, BioLegend, Cat# 100214, RRID: AB_493645), Alexa Fluor 700 anti-mouse/human CD45R/B220 (1:200, clone RA3-6B2, BioLegend, Cat# 103232, RRID: AB_493717), Brilliant Violet 785 anti-mouse CD19 (1:100, clone 6D5, BioLegend, Cat# 115543, RRID: AB_11218994), Brilliant Violet 605 anti-mouse IgD (1:200, clone 11–26 c.2a, BioLegend, Cat# 405727, RRID: AB_2562887), APC/Cyanie7 anti-mouse IgM (1:400, clone RMM-1, BioLegend, Cat# 406516, RRID: AB_10660305), PerCP/Cyanine 5.5 anti-MU/HU GL7 antigen (1:50, clone GL7, BioLegend, Cat# 144610, RRID: AB_2562979), and PE/Cyanine 7 anti-mouse CD38 (1:400, clone 90, BioLegend, Cat# 102718, RRID: AB_2275531). Cells were washed in FACS buffer (500 × g, 5 minutes) and then incubated in 4% methanol-free paraformaldehyde (PFA) fixation buffer for 30 min at 4 °C. Cells were then washed and resuspended in FACS buffer and kept in the dark at 4 °C before acquisition. Samples were measured on an Aurora spectral cytometer (Cytek, Fremont, CA, USA) using SpectroFlo® software (Cytek), with the relevant single fluorochrome unmixing reference controls set by the daily acquisition of Cytometer Setup and Tracking beads. Analysis was performed with FCS Express 7 (DeNovo Software) and GraphPad Prism version 10.0.0 for Windows, (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com). Germinal center B cells were gated as live CD3-B220+CD19+IgD-IgM-GL7+CD38low population. The frequency of germinal center B cells out of the B220+CD19+ was calculated and graphed.

Viral lung titers

Virus titers in the lungs of mice challenged with 50× LD50 of B/Lee/1940 harvested at day 3 after challenge were analyzed by the plaque assay method in 12-well plates. Harvested lungs were homogenized in 2 disruption cycles (10 s/cycle) using tubes that contained high impact zirconium beads (Andwin Scientific) and 1 mL of PBS. MDCK cells were seeded onto 12-well plates in growth media at 4× 105 cells per well and cultured for one day. Tissue homogenates were 10-fold serially diluted in infection medium (PBS with 0.21% (w/v) BSA (MP Biomedicals), 100 unit/mL of penicillin, 100 µg/mL of streptomycin (P/S; Gibco) and 0.83 mM CaCl2 and 0.1 mM MgCl2). Two hundred microliters of each dilution were inoculated onto each well. The plates were incubated at 33 °C for 1 h with occasional rocking every 10 min. The inoculum in each well was then removed and 1 mL of agar overlay containing 0.7% of agar in 2× MEM was placed onto each well. Once the agar was solidified, the plates were incubated at 33 °C with 5% CO2. Three days later, the plates were fixed with 4% (v/v) formaldehyde in PBS for 2 h and cells were later permeabilized with 0.5% Triton X-100 (v/v) for 15 min. The plaques were immuno-stained with a mice polyclonal serum vaccinated with a Yamagata-like virus at a 1:1000 dilution in 5% (w/v) dried milk in PBS. An HRP-conjugated goat anti-mouse secondary antibody was used at 1:2000 dilution in 5% (w/v) dried milk in PBS and the plaques were visualized using TrueBlueTM Peroxidase Substrate (SeraCare Life Sciences Inc.). Virus titers are presented as the log10 plaque forming units (PFU) per mL.

Phylogenetic tree

Phylogenetic tree of HA sequences was performed from the historical annual formulation sequences for IBV vaccine strains from 1999 to 2023 (Data taken from Global Influenza Programme (who.int) as of 1st Aug 2023 and reported in Supplementary Fig. S1). The phylogenetic tree was constructed using the Maximum Likelihood method and Tamura-Nei model and was visualized through Mega1163,64. Sequences were searched in the Influenza Research Database. The phylogenetic tree was constructed using raxML treeAlgorithm (https://www.fludb.org/)65 and was visualized through FigTree66.

Statistics

Normality/lognormality test, unpaired one-tailed t test, Kruskal–Wallis test corrected using Dunn’s test and two-way ANOVA test corrected using Dunnet’s test for group comparison were performed using GraphPad Prism version 10.0.0 for Windows (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com). T-test data passed a normality/lognormality.