Ethics statement

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mouse studies were approved by Georgia State University Institutional Animal Care and Use Committee (IACUC) under protocol number A16024. Female BALB/c mice (six- to eight-week old) were purchased from Jackson Laboratory and were housed in the animal facility at Georgia State University. Bleeding, infection, and sampling were performed under light anesthesia via inhalation of isoflurane to reduce mouse suffering.

Design of 4MtG recombinant protein

Tetrameric tandem M2e polypeptides (4 copies) were stabilized by introducing a foreign tetramerization motif (tetra-) GCN4 at the C-terminal of the recombinant 4MtG protein, as diagrammed in Fig.1a58. The encoding gene of 4MtG was generated by primer extension with overlapping Polymerase chain reaction (PCR). A signal peptide encoding sequence from honeybee melittin was employed to facilitate protein expression and secretion in insect Spodoptera frugiperda Sf9 cells (Sf9, ATCC, CRL-1711). A hexa-Histidine tag sequence was added after the GCN4 motif sequence. In each M2e copy of 4MtG, the two site-mutations C17S and C19S were made. The nucleotide sequence of 4MtG is shown in Supplementary Note 1. The four copies of different M2e sequences and their order in 4MtG are listed in Supplementary Table 1 and Fig. 1a. These consensus M2e sequences were made based on 11732 human, 5920 swine, 6267 avian, and 3270 domestic fowl influenza virus M2 sequences deposited in the National Center for Biotechnology Information (NCBI) databank. Molecular evolutionary genetics analysis version 6.0 (MEGA6) was used to align and analyze sequences59. 4MtG was purified from recombinant baculovirus (rBV)-based protein expression in insect cells. In brief, the 4MtG encoding sequence was cloned into the transfer vector pFastBac-1 (Invitrogen) for the rBV generation. 4MtG was purified from the rBV-infected insect cell Sf9 culture supernatants by affinity chromatograph.

Design of head-removed HA recombinant proteins

According to previous results, trimerization motifs facilitate HA oligomerization in the absence of the HA transmembrane domain17,18. A C-terminal sequence containing 6G3S or PGS linker, trimerization motif (tri-) GCN4 and hexa-Histidine tag were added following the hrH1 or hrH3 sequences for oligomerization and purification purposes (diagrammed in Fig. 1b). The coding sequence of the major head domain of H1 HA (GenBank Protein Accession: CAA24272.1, amino acids S53-P321) and the major head domain of H3 HA (GenBank Protein Accession: BAF37221.1, amino acids S61-P322) were replaced with a linker sequence encoding four glycines (4G), which is predicted to be a flexible linker and not disrupt the folding of the remainder of the molecule18,60. To inhibit the conformational shift to the post-fusion form, the residues between F61 and L89 in HA2 of H1 and the residues between F63 and I77 in HA2 of H3 were replaced with flexible non-hydrophobic 4G2S and 5G3S linkers, respectively. The F63 and V73 hydrophobic residues in HA2 are largely responsible for stabilizing the coil of the low pH structure61. In the neutral pH conformation, these deleted residues are a part of loop B connecting helix A and helix C, and participate in the formation a single long helix ABC in the low pH conformation. The deletion of part of loop B can block the formation of the long helix ABC, fixing the protein structure in the pre-fusion state61. The region between R76 and H106 is highly conserved among H3 viruses62. Therefore, the loop B truncation in hrH3 ended at the hydrophobic residue I77 to retain the most conserved regions. To further stabilize the hrH3 construct, an intra-disulfide bond was introduced in H3 by site-mutagenesis at V325C in HA1 and S438C in HA2. The encoding gene was generated from the full-length HA genes by overlapping PCR. Recombinant hrH1 and hrH3 were purified from rBV-based insect cell protein expression as done for the 4MtG purification.

Bis [sulfosuccinimidyl] (BS3) crosslinking

The oligomeric statesof purified 4MtG, hrH1, and hrH3 proteins were determined using the soluble Bis [sulfosuccinimidyl] (BS3) crosslinker (Thermo Scientific, Waltham, MA) in a crosslinking reaction to fix the polymeric structures of proteins followed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Supplementary Fig. 5a, b) and Western blot using anti-His antibody at 1 µg/ml (Cat. No. ab18184, Abcam) (Supplementary Fig. 5c, d), as described previously32. Briefly, 1 μg recombinant protein was incubated at room temperature in the presence of 4 mM BS3 for 30 min. The crosslinking reaction was stopped by the addition of 1 M Tris-HCl pH 8.0 to a final concentration of 50 mM.

PNps fabrication

PNps were made as previously described with modification32. To make the 4MtG core PNps (Uni4MC), the 4MtG protein solution in DPBS (Thermo Scientific, Waltham, MA) was desolvated with a 4:1 volume ratio of absolute ethanol to protein solution. 4.8 ml absolute ethanol was dripped at a constant rate of 1 ml/min into 1.2 ml of 3.2 mg/ml 4MtG protein solution under constant stirring by magnetic stir bar at 600 rpm at room temperature, followed by centrifugation at 15,000 × g for 15 min at room temperature to pellet the PNps. The PNp pellet was resuspended by sonication in either 1 ml 2.8 mg/ml hrH1 in DPBS, 1 ml 3.1 mg/ml hrH3 in DPBS, or 1 ml DPBS to generate hrH1 coated double-layer PNps (Uni4C1), hrH3 coated double-layer PNps (Uni4C3) and uncoated PNps (Uni4MC), respectively. The water-soluble, thiol-cleavable and primary amine-reactive crosslinker 3,3′-dithiobis [sulfosuccinimidylpropionate] (DTSSP; Cat No.21578, Thermo Scientific, Waltham, MA) was used to stabilize the resulting PNps. Crosslinking reactions were performed in 5 mM DTSSP for 1 h while stirring at 4 °C, and were quenched with 30 mM Tris-HCl solution at pH 7.4 for 15 min. Following collection by centrifugation at 20,000 × g for 30 min at 4 °C, PNps were resuspended by sonication in 1 ml DPBS. The fabrication of OVA PNps was described previously33.

PNp characterization

Nanoparticle size distribution was assessed by DLS with a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA). Protein concentration in the PNp solution was assessed with a BCA assay per the manufacturer’s instructions (Thermo Scientific, Waltham, MA). PNps were resuspended in water, air-dried, and sputter-coated with carbon prior to visualization with a Zeiss LEO 1450vp scanning electron microscope (Carl Zeiss, Jena, Germany) at 5.0 kV. Uni4C1, Uni4C3, and Uni4MC PNps were labelled with 1 µg/ml mouse monoclonal antibody C179 (Cat No. M145, TaKaRa), 2 µg/ml 12D1 and the antibody mixture of C179 and 12D1, respectively, and were then labelled with the EM grade gold-conjugated goat-anti-mouse secondary antibody (15 nm, Cat No. 25133, AURION). Ten microliter droplets of stained PNp suspension were adsorbed on a carbon/formvar coated 300-mesh copper grid for 5 min, followed by removal of the remaining liquid with filter paper. Transmission electron microscope LEO 906E (Carl Zeiss) was employed for visualization.

Antigen content of Uni4MC was analyzed in Western blot using anti-M2e antibody at 1 μg/ml (14C2, Cat. No. MA1082, Invitrogen) (Supplementary Fig. 5e), while Uni4C1 and Uni4C3 were analyzed in 10% SDS-PAGE (Supplementary Fig. 5f).

Pull-down assay

The pull-down experiment was performed with Dynabeads Protein G (Cat No. 10003D, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 50 µl of Dynabeads protein G suspension was incubated with rotation with 10 µg of C179, 12D1 or a mixture of these two antibodies in 200 µl DPBS with 0.02% Tween-20 for 10 min at room temperature. Tween-20 was used to avoid aggregation. The tube was placed on a magnet and then the supernatant was removed. After removal of the tube from the magnet, 200 µl DPBS with 0.02% Tween-20 was added to resuspend the beads-Ab complex. To avoid co-elution of antibody, 1 mM BS3 crosslinker reagent was added into the beads-Ab complex and incubated for 1 h at room temperature. After the supernatant was removed and collected (Pre-wash sample), 5 µg PNps Uni4C1 or Uni4C3 were added to separate tubes containing beads-Ab complex and incubated with rotation for 1 h at room temperature to allow PNps to bind to the beads-Ab complex. When the tube was placed on the magnet, the supernatant was transferred to a clean tube for further analysis (Unbound fraction sample). The beads-Ab-PNp complex was washed three times using 200 µl DPBS for each wash, then was resuspended in 100 µl DPBS and transferred into a clean tube. After removal of the supernatant, 20 µl elution buffer (50 mM Glycine at pH 2.8) was added and the beads-Ab-PNp complex was gently resuspended and incubated with rotation for 2 min at room temperature to dissociate the complex. The tube was placed on a magnet and the supernatant containing eluted PNp was transferred to a clean tube (Eluent sample). The collected Pre-wash, unbound fraction and eluent samples were immediately analyzed using Western blot, where 8 µg/ml rabbit anti-His polyclonal antibody (Cat No. PA1-983B, Thermo Fisher Scientific) was used for blotting (Supplementary Fig. 5g).

Enzyme-linked immunosorbent assay

The binding of hrHA to HA stem-specific monoclonal antibodies C179 (Cat No. M145, Clontech Labratories, CA), 12D1 and 9H10 (12D1 and 9H10 monoclonal antibodies are kind gifts from Dr. Peter Palese, Icahn school of medicine at Mount Sinai, New York) was tested using a sandwich-ELISA method described previously19. C179, 12D1, and 9H10 were used as coating antibodies. In 12D1 or 9H10 coated plates, 50 µl of 1:3 serially diluted FL H3 ectodomain protein, hrH3 and 4MtG were added for binding. In C179 coated plates, diluted inactivated PR8 H1N1, hrH1, and 4MtG were added for binding. A monoclonal anti-His-tag antibody conjugated with horseradish peroxidase (HRP) (Cat No. R931-25, Thermo Scientific, Waltham, MA) was used as the detection antibody. The binding of 4MtG to M2e-specific monoclonal antibody 14C2 (Invitrogen) was tested using a sandwich-ELISA method. Monoclonal antibody 14C2 was used as the coating antibody.

Antibody titers were analyzed using Enzyme-linked immunosorbent assay (ELISA) as described previously36. M2e specific antibodies were titrated in immunoplates coated with diverse M2e peptides from human consensus M2e (huM2e), p09, Vtn, and SH. All M2e peptides were synthesized by Synpeptide Co Ltd., Shanghai. PR8 H1, Aic H3, Vtn H5, and SH H7 specific antibodies were titrated in immunoplates coated with different formalin-inactivated viruses, including PR8 H1N1, Aic H3N2, rVn H5N1, and rSH H7N9. The IgG isotype titers were determined by incubating with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 (Cat No. 1071-05, SouthernBiotech, Birmingham, AL) or IgG2a secondary antibodies (Cat No. 1081-05, SouthernBiotech, Birmingham, AL). Antibodies cross-reactive to J57 H2 or GD H10 were titrated in cell-based ELISA. HEK293T cells (ATCC No. CRL-1573) were transfected with engineered pCMV3 plasmid encoding FL H2 (strain: A/Japan/305/1957; Cat No. VG11088-UT, Sino Biological. Inc.) or FL H10 (strain: A/duck/Guangdong/E1/2012; Cat No. VG40351-UT, Sino Biological. Inc.) in Lipofectamine 2000 (Cat No. 11668019, Invitrogen). The transfected HEK293T cells were seeded at a density of 5 × 104 per well in 96-well plate and was fixed by 80% acetone for 10 min at room temperature prior to serum inoculation.

Immunization and influenza A virus challenges

Mice (BALB/c strain, female, 6–8-week-old) received intramuscular (i.m.) immunizations twice, at a 4-week interval, in the hind leg with 50 μl of vaccine mixture in DPBS containing 10.5 μg Uni4MC, 12 μg Uni4C1, 12 μg Uni4C3, 12 μg Uni4C13 (a formulation comprising a mixture of 6 μg Uni4C1 and 6 μg Uni4C3 in DPBS) or 12 μg OVA PNps. Fifty microliter DPBS was used as a placebo. Blood samples were collected at 1 day prior to priming, 3 weeks after priming and boosting and 4 months after boosting to examine the effects on long-term immunity. Four weeks after the boosting immunization, mice were challenged intranasally with 6 × mLD 50 of mouse adapted (m.a.) influenza A virus strains in 50 μl DPBS. The m.a. strains used for these challenges were PR823, p0934, Aic63, A/Philippines/2/1982 (Phi, H3N2)34, rVn (rVtn; HA and neuraminidase (NA) genes were derived from Vtn, and the remaining backbone genes from PR8) or rSH (rSH; HA and NA genes were derived SH, and the remaining backbone genes from PR8).

The rVn H5N1 and rSH H7N9 reassortant viruses were generated and rescued as previously described64,65. The backbone plasmid system for generating reassortant virus was based on PR8 virus and generously provided by Dr. Robert Gordon Webster. The H5 HA and N1 NA genes with non-coding regions derived from H5N1 (A/Vietnam/1203/2004) were chemically synthesized and cloned into the pHW plasmids64. Plasmids pDZ with genes encoding H7 HA and N9 NA derived from A/Shanghai/2/2013 were previously described65 and kindly provided by Dr. Adolfo García-Sastre. The eight plasmids with 6:2 reassortant viral gene segments were co-transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). At day three post transfection, the virus-containing cell culture supernatants were inoculated into the allantoic cavities of ten-day-old embryonated chicken eggs. The rescue of reassortant viruses was determined by hemagglutination of chicken red blood cells. Virus titers were determined by plaque-forming units on Madin-Darby Canine Kidney (MDCK) cells (Cat. No. PAT-6500, ATCC).

Body weight loss and survival rates were monitored daily for 14 days post infection. Weight loss of ≥20% was used as the endpoint at which mice were euthanized per IACUC guidelines.

Interferon gamma (IFNγ) ELISpot procedures

The number of IFNγ secreting cells after restimulation was evaluated using an ELISpot method described previously34. Briefly, 3 weeks after boosting, splenocytes were isolated from all immunization groups. Each well in a 96-well filtration plate (Catalog Number: MSIPS4W10, Fisher Scientific) was loaded with 5 × 105 splenocytes for restimulation and a final concentration of 2 µg/ml of an M2e peptide pool (comprised of equal amounts of huM2e, p09, Vtn, and SH M2e peptides), H1 peptide pool (NR-15433 Peptide Array, H1N1 A/California/4/2009, Beiresources, NIAID), H3 peptide pool (NR-19246 Peptide Array, H3N2 A/Brisbane/10/2007, Beiresources, NIAID), or mock-restimulation. The developed plates were rinsed with purified water and air dried before counting using a Bioreader-6000-E (Biosys, Germany).

Determination of lung virus titers

Three mice per immunization group were euthanized at day 5 post 1 × mLD 50 PR8 or Aic infection. Determination of lung virus titers were described previously29. The presence of virus in the supernatant was assayed by measuring the hemagglutinating activity in the supernatant, using the Reed and Muench method for calculation66.

Histological analysis

Three mice per immunization group were euthanized at day 5 post 1 × mLD 50 PR8 or Aic infection. Lung tissues were isolated and fixed with 10% neutral buffered formalin. Fixed lung tissues were embedded in paraffin and processed for Haemotoxylin and Eosin (H&E) staining. Three sections with 10 μm thickness from three different parts of the lungs were stained with H&E and examined microscopically by three unbiased pathologists. The severity of the inflammation in the examined lung sections was scored on a scale of 0 to 5 (with 0.5 interval). Scores were given as absent (0), subtle (1), mild (2), moderate (3), severe (4), and massive (5).

HAI assay

HAI titers of mouse immune sera were assayed as previously described18 with viruses PR8, Aic H3N2, A/Hong Kong/1/1968 (p68, H3N2), rVn or rSH. The lowest serum dilution able to inhibit virus hemagglutination is shown.

Neutralization assay

The neutralization assay was described previously67. Pooled serum samples were heat-inactivated for 30 min at 56 °C. Mixtures of virus with final concentrations of 100 × TCID 50 virus per mixture and two-fold serial diluted serum samples (final serum dilution from 1:10 to 1:1280) were incubated for 2 h at 376 °C, 5% CO 2 in 50 µl virus medium (DMEM, 100 U/ml penicillin and 100 µg/ml streptomycin), then were subsequently added to the MDCK cells and incubated for 72 h at 376 °C, 5% CO 2 . A standard hemagglutination assay was performed to measure virus inhibition.

Antibody-dependent cellular cytotoxicity (ADCC) surrogate assay

An ADCC surrogate assay was performed according to the kit manufacturer’s protocol (Cat No. M1211, Promega) with modification. Briefly, HEK293T cells were maintained in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine and 1% (w/v) penicillin/streptomycin stock solution at 376 °C, 5% CO 2 . Two days before the experiment, HEK293T cells were transfected with plasmid DNA encoding FL H1 (Cat No. VG11684-UT, Sino Biological. Inc.) or H3 proteins (Cat No. VG1707-UT, Sino Biological. Inc.), using Lipofectamine 2000 (Invitrogen) in Opti-MEM (Invitrogen). The M2-expressing MDCK cell68 culture medium was supplemented with 10 µM Amantadine (Sigma) to support cell growth, 7.5 µg/ml of puromycin (Invitrogen) and 10% FBS at 376 °C, 5% CO 2 . One day before the assay, transfected HEK293T and M2-expressing MDCK cells were harvested and seeded in sterile white 96-well plates (Costar). After 24 h, serum samples were heat inactivated for 30 min at 566 °C and then serially diluted in assay buffer (4% ultra-low IgG FBS [Promega] in RPMI 1640 [Gibco]). Serum dilutions and a stable Jurkat cell line expressing mouse FcγRIV (Cat No. M1211, Promega) were added and incubated for 6 h at 376 °C at a target-effector ratio of 1:5. Cells were equilibrated to room temperature for 15 min before Bio-Glo Luciferase assay substrate (Promega) was added. Luminescence was read out after 10 min on a GloMax (Promega). Data are expressed as luminescence RLU of signal in the absence of serum.

Depletion of AM

Depletion of AM was performed as described previously56. Briefly, BALB/c mice were anesthetized by intraperitoneal (i.p.) injection with ketamine/xylazine and then 100 µl PBS-liposomes or clodronate-liposomes were administered slowly intratracheally (i.t.). Twenty-four hours after liposome administration, mice were injected i.p. with 400 µl pre-immune serum or Uni4C13 serum. After 24 h, mice were bled to determine serum titers by using M2e peptide ELISA, and then were challenged with 3 × mLD 50 Phi H3N2.

Statistical analysis

All data plotted with error bars are expressed as means with standard derivation. The P values were generated by analyzing data with a two-tail unpaired t test using the Prism 5 program (GraphPad software). Survival rate statistical analysis was performed with Kaplan-Meier analysis.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files, or are available from the authors upon request.