All experimental protocols involving animals were approved by the ethics committee of Southwest Jiaotong University (Approval number: 7792). All animal procedures were carried out in accordance with the Guidelines for Use and Care of Experimental Animals in Southwest Jiaotong University. All methods are reported in accordance with ARRIVE guidelines.
Specific pathogen-free (SPF) white Leghorn chickens (7 days old) were purchased from SLC Laboratory Center (Shanghai, China) and housed as 5 chicks per cage (50 cm × 45 cm × 45 cm) in an environmentally controlled house. The chickens were fed a pathogen-free diet and water.
All the virus challenge experiments were performed in enhanced animal biosafety level-3 (BSL-3) facilities. Body weight loss of greater than 25% was used as the criterion for euthanasia. All the surviving chickens were euthanized using CO2 inhalation for 5 min at 14 days post-infection.
Vaccine preparedness, oral immunization and sample collection
HA gene (1650 bp) of A/Vietnam/1203/2004 (H5N1) (clade 1) (GenBank accession No. EU122404) without the signal and transmembrane region was subcloned into surface expression plasmid pYD1, EBY100/pYD1-HA was generated as previously described15, except that EBY100/pYD1-HA expressing HA was not quantified.
The induction of EBY100/pYD1-HA was expressed in yeast nitrogen base (YNB)—casamino acids (CAA) medium (20 g/L galacotose, 6.7 g/L YNB without amino acids, 13.61 g/L Na2HPO4, 7.48 g/L NaH2PO4 and 5 g/L casamino acids) at 20 °C for 72 h.
The expression of HA protein in S. cerevisiae was determined by Western blot analysis as described previously15. Briefly, 5 OD600nm of EBY100/pYD1-HA pellets were boiled for 10 min with 100 µL of 6× sodium dodecyl sulfate (SDS) loading buffer and then run on a 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Bio-Rad, Hercules, CA, USA). The gel was transferred to a 0.45 μm nitrocellulose membrane. After blocking with 5% nonfat milk at room temperature for 2 h, the membrane was incubated with a monoclonal chicken anti-HA antibody (1:500 diluted) (R&D Systems, USA) overnight at 4 °C, and followed by 1:5000 diluted horseradish peroxidase (HRP)-conjugated goat anti-chicken IgG (R&D Systems, USA) at room temperature for 1 h. Lastly, the membrane was reacted with the West Pico Chemiluminescent Substrate (Bio-Rad, Hercules, CA, USA) in the dark for 5 min and the blot signal was imaged using Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA). Meanwhile, Precision Plus Protein™ WesternC™ (Bio-Rad, Hercules, CA, USA) was used as a protein marker.
EBY100/pYD1-HA was inactivated at 60 °C for 1 h and then used for subsequent oral immunization. The final concentration of EBY100/pYD1-HA was adjusted to 0.5 optical density (OD)600 nm/μL. Phosphate-buffer saline (PBS) and EBY100/pYD1 served as controls.
Three groups of chickens (n = 26 per group) were orally immunized with 200 μL of PBS, 100 OD600nm of EBY100/pYD1 or 100 OD600nm of EBY100/pYD1-HA on day 1 (prime immunization) and day 14 (boost immunization).
Sera (n = 10 per group), intestine washes (n = 5 per group) and spleen (n = 5 per group) were collected from the vaccinated chickens on days 13 and 28 after the initial immunization.
Quantification of EBY100/pYD1-HA expressing HA protein
Quantification of the HA protein was performed by enzyme-link immunosorbent assay (ELISA) as previously described20. In brief, 5 OD600nm of EBY100/pYD1-HA were resuspended in 100 μL of a solution of monoclonal mouse anti-HA antibody (0, 15, 30, 45, 60, 75, 90, 105, 120 μg/mL) (kindly provided by NIH Biodefense and Emerging Infections Research Resources Repository, Manassas, VA, USA) in PBS containing 2% bovine serum albumin (BSA) and incubated at room temperature for 2 h. This was followed by incubation with goat anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (1 mg/mL) (R&D Systems, Minneapolis, MN, USA) at room temperature for 1 h. After washing with sterile PBS, the cells were resuspended in 100 µL of the HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (R&D Systems, Minneapolis, MN, USA) in the dark for 25 min, and then, 100 μL of 2 mol/l H2SO4 was added to stop the reaction. The OD450nm value of the supernatant was measured using a microplate reader. EBY100/pYD1was used a negative control. A solution of purified HA protein (60 µg/mL) (kindly provided by NIH Biodefense and Emerging Infections Research Resources Repository, Manassas, VA, USA) was used a positive control.
Measurement of antibody responses
HA-specific serum IgG and mucosal IgA antibody levels were separately determined by ELISA as previously described15. Briefly, 2 µg of recombinant HA protein of A/Vietnam/1203/2004 (H5N1) (clade 1) (kindly provided by NIH Biodefense and Emerging Infections Research Resources Repository, Manassas, VA, USA) was used as the antigen to coat 96-well ELISA plates overnight at 4 °C. The wells were washed three times with Tris-buffered PBS containing 0.05% Tween 20 (TBS-T) and blocked with TBS-T containing 1% BSA at room temperature for 2 h. Serially diluted chickens sera or 1:50 intestine washes were added to the plates and incubated at 37 °C for 1 h, followed by incubation with biotinylated goat anti-chicken IgG (R&D Systems, Minneapolis, MN, USA) or biotinylated goat anti-chicken IgA (R&D Systems, Minneapolis, MN, USA) and alkaline phosphatase (AP)-labeled streptavidin (R&D Systems, Minneapolis, MN, USA) at 37 °C for 1 h, respectively. The plates were washed three times with TBS-T and then incubated with 100 μL of p-nitrophenyl phosphate (PNPP) substrate (R&D Systems, Minneapolis, MN, USA). The reaction was developed at room temperature for 25 min and then was stopped with 50 μL of 2 M sodium hydroxide (NaOH). The OD value was measured at 405 nm using an ELISA plate reader (Bio-Tek instrument Inc., Winooski, USA). The IgG and IgA titers were determined based on the lowest dilution with an OD405nm greater than the mean OD405nm of naïve controls plus 2 standard deviations.
ELISpot to determine T cell responses
Splenocytes (n = 5 per group) were isolated from the vaccinated chickens on days 13 and 28 after the initial vaccination. HA-specific cells secreting Interferon gamma (IFN-γ) and Interleukin-4 (IL-4) were analyzed using commercial ELISpot assay kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, splenocytes (1.0 × 106 cells/well) isolated from the vaccinated chickens were cultured in 96-well plates containing chicken IFN-γ or IL-4 and stimulated with 10 µg/mL of HA–specific peptide (ISVGTSTLNQRLVP) for 36 h in a humidified incubator at 37 °C with 5% CO2. The plates were washed with sterile PBS and incubated with biotinylated goat anti-chicken IFN-γ or IL-4 antibodies overnight at 4 °C, followed by AP-conjugated streptavidin at room temperature for 2 h. The plates were washed, developed with BCIP/NBT, and counted using an ImmunoSpot ELISpot reader (Bio-Tek instrument Inc., Winooski, USA).
Hemagglutination inhibition (HI) assay
The HI titer was determined as previously described15. Briefly, receptor-destroying enzyme (RDE)-treated sera were serially diluted (twofold) in v-bottom 96-well microtiter plates and 4 hemagglutination units (HAU) of A/Vietnam/1203/2004 (H5N1) (clade 1) or A/Chicken/Henan/12/2004 (H5N1) (clade 8) whole inactivated virus were added. Then, 1% (v/v) chicken red blood cells (RBCs) were added and incubated for 30 min at room temperature. HI titer was determined based on the reciprocal value of the last dilution of the sera that completely inhibited hemagglutination of the chicken RBCs. A negative HI titer was defined as less than 10.
H5N1 viruses challenge
Two weeks after the final vaccination, chickens (n = 16 per group) (Fig. 1) were intranasally infected with 25 μL of 104 50% egg infective dose (EID50) of A/Vietnam/1203/2004 (H5N1) (clade 1) or A/chicken/Henan/12/2004 (H5N1) (clade 8). The challenged chickens were monitored daily for 14 days to observe changes in body weight and survival rate. Lungs (n = 3 chickens/group) were isolated at day 3 post-challenge to determine viral titers.
All data were represented as the mean ± standard deviation (SD). To determine the statistical significance, Kaplan–Meier survival analysis was performed using GraphPad Prism. Two tailed Student’s t test and one-way analysis of variance (ANOVA) were used to determine differences between groups. Values of P < 0.05 were considered statistically significant.