Cell lines, viruses, and mice
Human embryonic kidney cells (293 T) kept by our laboratory were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Thermo Scientific), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C with 5% CO2.
Influenza virus strains A/swine/Guangdong/01/1998(H3N2), A/chicken/Guangdong/32/2007(H9N2) and A/Puerto Rico/8/1934(H1N1) were kept by our laboratory.
BALB/c mice were bred at the Laboratory Animal Center of Sun Yat-Sen University under the pathogen-free conditions. Six-eight-week-old female mice were randomly assigned to treatment or control groups in the experiment.
RNA of H3N2 viruses (GenBank accession no. FJ830855.1) and 293 T cells were extracted using HiPure Viral RNA/DNA Kit (Magen) and TRIzol (Invitrogen) respectively. cDNA was prepared using PrimeScript™ II 1st Strand cDNA Synthesis Kit (TAKARA). H3 HA was amplified from H3N2 viruses using the following primers: H3 HA-Forward (Kpn I) 5′-GGGGTACCTAATTCTATCAACCATGAAGACTAT-3′ and H3 HA-Reverse (Xho I) 5′-CCGCTCGAGAGGGTGTTTTTAATTACTAATATACTCA-3′. PDI and ERp57 segments were amplified from 293 T cells using the following primers: PDI-Forward (Hind III) 5′-CCCAAGCTTCCAGGATTTATAAAGGCGAGGC-3′, PDI-Reverse (Not I) 5′-CGGAATTCCGGGTCTGGCTTTGCGTAT-3′, ERp57-Forward (Hind III) 5′-CCCAAGCTTCGCAAGCAGCGGGTTAGT-3′ and ERp57-Reverse (BamH I) 5′-CGGGATCCTCCTAGTCCTCCCCAATGGTT-3′. H3 HA, PDI and ERp57 PCR products were cloned into pcDNA3.1(+) (Invitrogen) respectively. All clones were verified by sequencing (Thermo).
Expression of HA proteins with PDIs overexpression in 293 T cells
To investigate whether PDIs can improve the stability of HA, cells were transfected with 2 μg of H3 HA and PDI (or ERp57) plasmids in a 6-well plate, using PEI-MAX (Polyscience) according to the manufacturer’s protocol. Briefly, 293 T cells were seeded in a 6-well plate at a density of 1 × 105 cells/mL and cultured until the cells reached approximately 80–90% confluence. Purified plasmids and PEI-MAX (Polyscience) were incubated in the opti-MEM at the concentration ratio of 1:4 respectively at room temperature for 5 min and then mixed to incubate at room temperature for 20 min. The mixture was added to 293 T cells to incubate at 37 °C for 8 h with 5% CO2 before the replacement with the fresh medium. After 48 h incubation at 37 °C with 5% CO2, 293 T cells were collected.
Expression of HA proteins with ERp57 RNA depletion by small interfering RNA (siRNA)
To downregulate ERp57 expression, 293 T cells were transfected with 200 nM siRNA against ERp57 (5′-GGACAAGACTGTGGCATAT-3′, RIBOBIO) using lipo2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 293 T cells were seeded in a 12-well plate at a density of 1 × 105 cells/mL and cultured until the cells reached approximately 50% confluence. siRNA and lipo2000 at the concentration ratio of 1:2 were incubated in the opti-MEM respectively at room temperature for 5 min and then mixed to incubate at room temperature for 20 min. After the mixture was added to 293 T cells to incubate at 37 °C for 24 h with 5% CO2, 293 T cells were transfected with 1 μg H3 HA plasmids in a 12-well plate using PEI-MAX. After incubation for 48 h at 37 °C with 5% CO2, 293 T cells were collected.
Characterization of H3 HA and PDIs expression
To confirm and characterize H3 HA and PDIs expression, real-time qPCR, western-blot, immunofluorescence assay, and flow cytometry were carried out.
For real-time qPCR, total RNA was extracted from 293 T cells with TRIzol, and treated with DNase I. cDNA was synthesized by reverse transcription using RT-PCR kit (TaKaRa, Dalian). Real-time qPCR was performed with the following primers (ERp57-Forward 5′- TCACGGACGACAACTTCGAG-3′, ERp57-Reverse 5′- GTTGGCAGTGCAATCAACCT-3′, GAPDH-Forward 5′- AACGGATTTGGTCGTATTG-3′, GAPDH-Reverse 5′- GGAAGATGGTGATGGGATT-3′) using LightCycler 480 SYBR Green I Master (Roche) according to the manufacturer’s protocol. Briefly, PCR was performed in a 20 μL volume containing 100 ng of cDNA, 10 μL of 2 × LightCycler 480 SYBR Green I Master, and a 0.2 μM of each gene-specific primer. The thermal cycling parameters were referring to the manufacturer’s protocol: 95 °C for 5 min, 40–45 cycles of 95 °C for 10 s, 58 °C for 20 s, 72 °C for 30 s, 1 cycles of 95 °C for 5 s, 65 °C for 1 min, 97 °C for continuous, and 40 °C for 10 s. The final cycle was set to obtain a melting curve for the PCR products to determine the specificity of the amplification. The GAPDH gene was utilized as the reference gene. Expression levels of genes were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples.
For western-blot, cells were collected and lysed with RIPA lysis buffer. The protein concentration in samples was quantified by BCA assay (Thermo Fisher Scientific). And then samples were heated at 100 °C for 10 min in the loading buffer with (for reducing SDS-PAGE) or without (for non-reducing SDS-PAGE) 1% β-mercaptoethanol. Total proteins were separated by 10% SDS polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membrane (Millipore, 45 μm), and then detected with mouse polyclonal anti-H3 HA serum (kept by our lab), rabbit polyclonal anti-PDI antibody (Abcam, ab3672), mouse monoclonal anti-ERp57 antibody (Abcam, ab13506) respectively, followed with the corresponding secondary antibody and the commercial ECL kit (Fdbio science). Protein loads were estimated using murine anti-β-actin mAb (Abmart, M20010). The band intensity was quantified using ImageJ software.
For immunofluorescence assay, after 48 h transfection, cells were collected and fixed in 4% phosphate-buffered paraformaldehyde at room temperature for 10–15 min, permeabilized, blocked, and reacted with primary antibodies (same as those used in western-blot) and mouse-specific Cy3-conjugated secondary antibody/rabbit-specific FITC-conjugated secondary antibody (PTG). Cell nuclei were stained with 4′-6′-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and visually inspected by epifluorescence light microscopy before the acquisition of representative areas by confocal microscopy. Stained sections were analyzed by a scanning confocal microscopy (Leica TCS-SP5).
For flow cytometry, cells were collected in a single-cell suspension with the 0.05% Trypsin-EDTA digestion, fixed in 70% ethanol in the ice for 30 min, permeabilized, blocked, and reacted with primary antibodies (same as those used in western-blot) and secondary antibodies (same as those used in immunofluorescence assay). Flow cytometric analyses were performed using FACS Calibur (BD biosciences), and data were analyzed using FlowJo software.
Purification of H3 HA proteins
H3 HA proteins were purified by using ion-exchange chromatography as described previously . Briefly, samples were lysed in Buffer A (20 mM sodium phosphate, 1.0 mM EDTA, 0.01% Tergitol-NP9, 5% glycerol, pH 5.89) in the ice for 30 min and centrifugated at 10,000×g for 25 min. The supernatant was loaded on tandem Q/SP columns and the columns were disconnected after washed by buffer A. HA was eluted from the SP column with buffer B (20 mM sodium phosphate, 0.03% Tergitol-NP9, 5% glycerol, pH 7.02) and buffer C (20 mM sodium phosphate, 150 mM NaCl, 0.03% Tergitol-NP9, 5% glycerol, pH 7.02) consecutively, but mostly in the buffer C. The eluted HA proteins in buffer C was further purified and concentrated by ultrafiltration with a stirred cell using a 100 kDa MWCO regenerated cellulose membrane (Millipore).
Mouse immunization and challenge
Six- to eight-week-old female BALB/c mice (n = 5) were vaccinated subcutaneously twice at the interval of 2 weeks with 10 μg of H3 HA expressed in ERp57-overexpressed 293 T cells (named as H3 HA/ERp57) or in the conventional 293 T cells (named as H3 HA) in a 100 μl volume with oil emulsion containing 2% aluminum stearate, 2% Tween-80 and 4% span 80. Blood samples were collected from the tail vein of each mouse 2 weeks after the second immunization. After one more week, mice were challenged intranasally with 40 μL of mouse-adapted wild-type H1N1 viruses at a dose of 3 × 50% mice lethal dose (MLD50). After the challenge, the mice were monitored for 14 days for survival and weight loss daily. The mice that lost 25% or more of their initial body weight were scored dead and euthanized.
Hemagglutination inhibition (HI) assay
HI assay was performed as described before . Briefly, four HA units of inactivated H1N1, H3N2, and H9N2 viruses were used. Each serum sample was treated with receptor destroying enzyme (RDE, Denka Seiken) at 37 °C overnight, then incubated at 56 °C for 1 h immediately for the hydrolysis of RDE. Serum samples were diluted 10-fold initially then 2-fold continuously for ten times. The highest dilution of serum samples that inhibits hemagglutination was defined as the HI titers.
Enzyme-linked immunosorbent assay (ELISA) for testing HA-specific IgG antibodies
HA specific IgG antibody titers were tested by ELISA as previously described . Briefly, inactivated H1N1, H3N2, and H9N2 purified viruses at the concentration of 1 μg/mL of HA1 were coated at 4 °C overnight, incubated with serial dilutions of each serum sample at 37 °C for 1 h, and detected by HRP-conjugated goat anti-mouse IgG polyclonal antibodies (Proteintech). The optical densities were detected at 450 nm (OD450) using ELISA plate reader (Bio-Tek Instruments). The antibody titer was determined as the reciprocal of the highest dilution of three-fold OD450 reading of negative control.
All statistical analyses were performed using Graphpad Prism software (Graphpad). Statistical analyses were performed using one or two-way ANOVA. Data are shown as mean ± SD or mean ± SEM as indicated in the figure legends. Significance was assumed with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.