HP avian influenza virus, A/waterfowl/Korea/S57/2016 (H5N6) (clade 2.3.4.4.), containing the polybasic amino acids (RRRK) at the HA cleavage site, was employed in this study. Madin–Darby canine kidney (MDCK) cells (American Type Culture Collection, VA, USA) were used for viral amplification. The MDCK cell line was validated by STR profiling and tested negative for mycoplasma. Cells were all cultured in a humidified incubator at 37 ℃ and 5% CO${}_2$. MDCK cells were maintained in a minimal essential medium (MEM) (Sigma, MO, USA) supplemented with 10% fetal bovine serum and antibiotic–antimycotic solution (Sigma-Aldrich, MO, USA). All studies on HP avian influenza viruses were performed within a Korean government-certified biosafety level 3 laboratory facility.

Synthesized H5 cleavage-site peptides were obtained from Peptron Co. (Daejeon, South Korea) and labeled with keyhole limpet hemocyanin. The synthesized peptides are listed in Table 1.

Fertilized eggs (from white leghorns) were obtained from local farms and hatched in the laboratory. The hatched chicks were grown for 2 weeks before utilization for the vaccine study. Chickens (n = 13 per group) were intramuscularly inoculated with 300 µL containing 5.0 µg of keyhole limpet hemocyanin-labeled H5 cleavage-site peptide and 30% oil (SEPPIC, Courbevoie, France). Booster doses were administered to the immunized chickens 3 weeks after the first dose.

Immunized chicken sera were collected a week after administration of the second dose to measure antibody induction.

Enzyme-linked immunosorbent assay (ELISA) was performed to determine antibody titers with bovine serum albumin (BSA)-labeled peptides: BSA-TGLRNSPLRERRRKR/GLFGAIAGFIEGGWQ, BSA-TGLRNSPLRERRRR/GLFGAIAGFIEGGWQ, BSA-TGLRNSPLRERRR/GLFGAIAGFIEGGQ, BSA-TGLRNSPLRERR/GLFGAIAGFIEGGWQ, BSA-TGLRNSPLRERKR/GLFGAIAGFIEGGWQ, BSA-TGLRNSPLREKR/GLFGAIAGFIEGGWQ, and BSA- TGLRNSPLRER/GLFGAIAGFIEGGWQ.

The wells in the immunoplate were coated with 100 µL of peptide (4 µg/mL) in coating buffer, incubated overnight at 4 °C, and then washed three times with PBS–tween 20 (0.05%). The wells were blocked with 100 µL of 1% BSA in PBS (pH 7.4) for 2 h at room temperature and then 100 µL of diluted chicken sera in PBS (1:100) was added. The plates were incubated for 1 h at room temperature. The wells were washed five times and 100 µL of horseradish peroxidase-conjugated rabbit anti-chicken antibody (Sigma-Aldrich) diluted in PBS (1:5000) was added. The plates were incubated for 1 h at room temperature. The wells were washed five times with PBS–Tween 20 (0.05%) and 100 µL of 3,3${}^{\prime }$,5,5${}^{\prime }$-tetramethylbenzidine substrate solution (Thermo Fisher Scientific, Waltham, MA, USA) was added. The wells were incubated for 30 min at room temperature. The reaction was stopped with 50 µL of 2.5 M sulfuric acid. Optical density (OD) was measured at 450 nm using an ELISA spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).

The vaccinated chickens were intranasally infected with 1 mL (10${}^5$ TCID${}_{50}$/mL) of H5N6 virus. The tracheas and cloacae of infected chickens were swabbed with PBS (pH 7.4) for 10 days post-infection before the viral titers were determined via log${}_{10}$ TCID${}_{50}$/mL (log10 tissue culture infectious dose 50) using MDCK cells. Chickens (three per group) were euthanized 2 days after infection, and tissues (lungs and brains) were collected in PBS (pH 7.4) to measure viral titers.

Confluent MDCK cells were treated with trypsin–EDTA (Sigma-Aldrich) and seeded in a tissue culture plate (96 wells) with MEM containing 10% fetal bovine serum and 1$\times$ Antibiotic–Antimycotic Solution (Sigma-Aldrich). The cells were washed with warm PBS (pH 7.4) before inoculation with collected samples (tracheal and cloacal swabs, and lung and brain tissues). Samples were 10-fold diluted in MEM containing 1.5% BSA prior to inoculation. Prepared samples were inoculated into cells (four wells per diluted sample) for 5 days in a humidified incubator (37 °C, 5% carbon dioxide). Cells were observed for cytopathic effects. The viral titer (log${}_{10}$ TCID${}_{50}$/mL) was determined using the method described by Muench and Reed [18].

Student’s t-test using IBM SPSS Statistics version 20 software (IBM Corp., Armonk, NY, USA) was performed to analyze statistical significance. Statistical significance was based on p-values less than 0.05.

Fig. 1 shows the antibody titers measured using ELISA in sera (n = 10 per group) collected from chickens vaccinated with two doses of H5 cleavage-site peptide containing RRRK. Antibodies were similarly induced in chickens immunized with H5 cleavage-site peptides containing diverse combinations of polybasic amino acids (Table 1), including peptides containing RRRK, RRR, RR, R, RK, K, and RRRK(-), with a mean OD range of 0.51–0.54. The mean OD value for the sera from PBS-mock vaccinated chickens was 0.12.

Fig. 1.

Antibody titers in chickens immunized with H5 cleavage-site peptides containing different combinations of polybasic amino acids. Sera were collected from the intramuscularly immunized chickens (n = 13 per group)—with two doses (5.0 µg per dose) of oil-adjuvanted HLA-labelled peptides—4 weeks after the vaccination. Antibody titers were measured via ELISA using BSA-labelled peptides. **p 0.01. ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin.

We aimed to determine the role of individual amino acids of RRRK in protecting chickens from HP avian influenza viral infections. Chickens immunized with most combinations of polybasic amino acids survived when intranasally challenged with the HP H5N6 virus (Fig. 2). All the immunized chickens with H5 cleavage-site peptides, including RRRK-, RRR-, RR-, R-, and K-containing peptides survived, whereas all chickens immunized with RK-containing peptide died 6 days post-infection. All chickens immunized with RRRK(-) peptide and PBS-mock did not survive (Fig. 2).

Fig. 2.

Mortality of the immunized chickens with H5 cleavage-site peptides containing different combinations of polybasic amino acids. The immunized chickens (Fig. 1) were intranasally challenged with 1 mL (10${}^5$ TCID${}_{50}$/mL) of A/Waterfowl/Korea/S57/2016 (H5N6). The challenged chickens were monitored for survival rate.

No viruses were detected in the tracheal (Fig. 3A) and cloacal swabs (Fig. 3B) of chickens immunized with H5 cleavage-site peptides, including RRRK-, RRR-, RR-, R-, and K-containing peptides. In contrast, high viral titers were detected in tracheal and cloacal swabs in the chickens immunized with RK-containing peptide in the range of 2.0 to 4.5 TCID${}_{50}$/mL (Fig. 3A,B). In addition, similar viral titers were detected in the chickens immunized with either RRRK(-) peptide or PBS-mock in the range of 1.5 to 5.0 TCID${}_{50}$/mL (Fig. 3A,B).

Fig. 3.

Viral titers in swabbed samples in the challenged chickens. Swabs in tracheas (A) and cloacae (B) in the challenged chickens (Fig. 2) were performed in a 2-day interval until 10 days p.i. Viral titers in the swabbed samples were measured using Madin-Darby Canine Kidney (MDCK) cells by log${}_{10}$ TCID${}_{50}$/mL. The limit of detection was 1 TCID${}_{50}$/mL. **p 0.01.

The infected chickens (n = 3 per group) were euthanized to collect the lungs and brains 2 days post-infection to measure tissue viral titers. No virus was detected in either the lung or brain tissues of the chickens immunized with H5 cleavage-site peptides, including RRRK-, RRR-, RR-, R-, and K-containing peptides (Table 2), whereas high viral titers were detected in both the lung and brain tissues of chickens immunized with the RK-containing peptide, in a range of 3.0 to 3.5 TCID${}_{50}$/mL (Table 2). Similar viral titers were detected in the lungs and brain tissues of chickens immunized with either RRRK(-) peptide or PBS-mock in the range of 3.5 to 4.5 TCID${}_{50}$/mL (Table 2).