The avian influenza virusA/waterfowl/Korea/S57/2016 (H5N6) (clade 2.3.4.4.), which was isolated in our laboratory, contains RRRK at the hemagglutinin (HA) cleavage site. The avian influenza virus, A/Hong Kong/213/2003 (H5N1) (clade 1) containing RRRKK at the HA cleavage site was kindly provided by Dr. Malik Peiris (University of Hong Kong). Madin–Darby canine kidney (MDCK) cells (American Type Culture Collection, Manassas, VA, USA) were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1$\times$ antibiotic–antimycotic solution (Sigma-Aldrich, St. Louis, MO, USA). MDCK cell lines were validated by STR profiling and tested negative for mycoplasma. Cells were all cultured in a humidified incubator at 37 °C and 5% CO${}_2$.

All experiments on HP avian influenza viruses were conducted in a biosafety level 3 facility in the animal facility building at Chungnam National University (Daejeon, South Korea).

H5 cleavage-site peptides labeled with keyhole limpet hemocyanin (KLH) were synthesized by Peptron Co. (Daejeon, South Korea). The synthesized peptides were KLH-TGLRNSPLRERRRKR/GLFGAIAGFIEGGWQ (clade 2.3.4.4.) and KLH-CTGLRNSPQRERRRKKR/GLFGAIAGFIEGGW (clade 1).

Two-week-old chickens (n = 13 per group) hatched in our laboratory were intramuscularly vaccinated with 300 µL of KLH-labeled bivalent peptide antigens (5 µg per antigen) diluted in phosphate-buffered saline (PBS; pH 7.4) and 30% oil (SEPPIC, Courbevoie, France); after three weeks, the immunized chickens were vaccinated with the second dose. The schedule of the vaccine study is shown in Fig. 1.

Fig. 1.

Vaccination and challenge diagram of bivalent vaccines in chickens.

Sera were collected from the immunized chickens 1 week after they were immunized with a second dose of peptide antigens. An enzyme-linked immunosorbent assay (ELISA) was used to measure the antibody titers. Immunoplate wells were coated with bovine serum albumin (BSA)-labeled peptides: BSA-TGLRNSPLRERRRKR/GLFGAIAGFIEGGWQ (clade 2.3.4.4.) and BSA-CTGLRNSPQRERRRKKR/GLFGAIAGFIEGGW (clade 1).

The ELISA microplate was coated with 100 µL of peptide (4 µg/mL) diluted in coating buffer and then incubated at 4 °C for 12 h. The peptide-coated plates were washed three times with PBS containing Tween 20 (0.05%), and 1% BSA (100 µL) diluted in PBS (pH 7.4) was added to the wells of the plate to block the wells for 2 h at 25 °C. Subsequently, 100 µL of chicken sera diluted in PBS (1:100) was added to the blocked wells following incubation for 1 h at 25 °C. The wells were washed five times with PBS–Tween 20 (0.05%) following the addition of horseradish peroxidase-conjugated rabbit anti-chicken antibody (Sigma-Aldrich; 100 µL) diluted in PBS (1:5000) prior to incubation for 1 h at 25 °C. After the wells were washed five times with PBS–Tween 20 (0.05%), 100 µL of 3,3${}^{\prime }$,5,5${}^{\prime }$-tetramethylbenzidine substrate solution (Thermo Fisher Scientific, Waltham, MA, USA) was added prior to incubation for 30 min at 25 °C. Finally, 2.5 M sulfuric acid (50 µL) was added to stop the reaction. Optical density (OD) was measured at 450 nm using an ELISA spectrophotometer (Thermofisher, MA, USA).

The bivalent peptide-immunized chickens were intranasally infected with 1 mL (1 $\times$ 10${}^5$ median tissue culture infectious dose [TCID${}_{50}$]/mL) of H5N1 (A/Hong Kong/213/2003, clade 1) or H5N6 HP (A/waterfowl/Korea/S57/2016, clade 2.3.4.4.) avian influenza viruses. The tracheae and cloacae of challenged chickens were swabbed with PBS (pH 7.4) for 10 d. In addition, three chickens per group were euthanized 2 d after the challenge to collect the lung and brain tissues for determining viral titers. The collected tissue samples were homogenized in PBS (0.1 g/1 mL PBS) using BeadBlaster 24 (Benchmark Scientific, Sayreville, NJ, USA). The swabbed samples were vortexed before they were used for viral titers.

MDCK cells were cultured in MEM containing 10% FBS and 1$\times$ antibiotic–antimycotic solution (Sigma-Aldrich), and the cells were then detached using 1$\times$ trypsin–EDTA (Sigma-Aldrich) before seeding in 96-well culture plates. Confluent MDCK cells were washed with 400 µL of PBS (pH 7.4), and the wells were inoculated with swab and tissue samples that were 10-fold serially diluted in PBS containing 1.5% BSA. The inoculated wells were incubated for 5 d at 37 °C in a humidified incubator containing 5% CO${}_2$. Cytopathic effects were observed to determine viral titers in log${}_{10}$TCID${}_{50}$/mL as described by Muench and Reed [15].

Lung tissues from immunized chickens (n = 3 per group) challenged with H5N1 or H5N6 avian influenza viruses were collected at 2 days post-infection and fixed in 10% phosphate-buffered formalin (Triangle Biomedical Sciences, General Data Healthcare, Cincinnati, OH, USA) for 6 h before being embedded in paraffin. Tissue sections (5 µm) were prepared and stained with hematoxylin and eosin (H&E). Stained tissues were observed using an Olympus DP70 microscope (Olympus, Tokyo, Japan).

Statistical analysis was performed via Student’s t-test using IBM SPSS Statistics version 20 (IBM, Armonk, NY, USA). p-values 0.05 were considered significant.

Sera were collected from chickens (n = 13 per group) immunized with bivalent peptide antigens comprising 5.0 µg of KLH-TGLRNSPLRERRRKR/GLFGAIAGFIEGGWQ (clade 2.3.4.4.) or 5.0 µg of KLH-CTGLRNSPQRERRRKKR/GLFGAIAGFIEGGW (clade 1). The collected sera were used to determine antibody titers via ELISA. Strong antibody responses against each peptide were detected. The mean OD values for RRRK-containing (clade 2.3.4.4) and RRRKK-containing (clade 1) peptides were 0.57 and 0.58, respectively (Fig. 2).

Fig. 2.

Serum antibody titers from chickens vaccinated with dual H5 cleavage-site peptides. Chickens (n = 13 per group) were intramuscularly (i.m.) immunized with dual peptides of H5 cleavage sites (clade 1 and clade 2.3.4.4.) (5.0 µg per each peptide) and 4 weeks later, were boosted with the same dose. Sera were collected one week after boosting. Antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) using bovine serum albumin (BSA)-labeled each peptide.

Chickens (n = 13 per group) vaccinated with two doses of bivalent peptide antigens were intranasally infected with A/Hong Kong/213/2003 (H5N1) (clade 1) or A/waterfowl/Korea/S57/2016 (H5N6) (clade 2.3.4.4.), and mortality was monitored (Fig. 3). All chickens immunized with bivalent peptides survived, as did the uninfected control chickens. All PBS-inoculated chickens died at 4 days post-infection. On day 2 post-infection, the mortality rate increased up to 50–60% in PBS-mock immunized and challenged chickens.

Fig. 3.

Survival rate of the challenged chickens immunized with bivalent peptide antigens. The vaccinated chickens (Fig. 1.) were intranasally (i.n.) infected with 1 mL (10${}^5$TCID${}_{50}$/mL) of A/Waterfowl/Korea/S57/2016 (H5N6) (clade 2.3.4.4.), or A/Waterfowl/Korea/S57/2016 (H5N6). The challenged chickens were monitored for survival rate for 10 days.

The challenged chickens immunized with bivalent peptides did not show any clinical signs during the 10-day observation period, while the PBS-mock immunized and challenged chickens showed severe clinical signs such as hemorrhage in combs, wattles, and legs, and drooped heads and wings.

The tracheae and cloacae of challenged chickens were swabbed for 10 d, and viral titers in MDCK cells were measured based on log${}_{10}$TCID${}_{50}$/mL (Fig. 4A,B). No viruses were detected in the tracheae (Fig. 4A) and cloacae (Fig. 4B) of the challenged and bivalent peptide-immunized chickens. The mean viral titers in the tracheae of PBS-inoculated chickens on day 2 after challenge with HP H5N1 (clade 1) and HP H5N6 (clade 2.3.4.4.) were 4.0 and 4.5, respectively (Fig. 4A). The mean viral titers in cloacae of PBS-inoculated chickens on day 2 after challenge with HP H5N1 (clade 1) and HP H5N6 (clade 2.3.4.4.) were 3.0 and 3.5, respectively (Fig. 4A).

Fig. 4.

Viral titers in swabbed samples and tissues in the challenged chickens. The bivalent-peptide immunized chickens were intranasally (i.n.) challenged with 1 mL (10${}^5$ TCID${}_{50}$/mL) of H5N1 (A/Hong Kong/213/2003, clade 1) or H5N6 HP (A/Waterfowl/Korea/S57/2016, clade 2.3.4.4). The surviving chickens were swabbed in the tracheas (A) and cloacae (B) with PBS (pH 7.4) for 10 days after the challenge. Three chickens per group were euthanized two days after the challenge to collect the tissues of lungs and brains (C) for viral titers. Viral titers in the samples were measured in MDCK cells by log${}_{10}$TCID${}_{50}$/mL. The limit of detection was 1 TCID${}_{50}$/mL. **p 0.001.

To evaluate viral infections in the tissues of challenged chickens, the chickens (n = 3 per group) were euthanized at 2 days post-infection for the collection of tissues (lungs and brains). Viral titers in tissues were measured in MDCK cells based on log${}_{10}$TCID${}_{50}$/0.1 g of tissue. No viruses were detected in the lungs and brains of bivalent peptide-immunized and challenged chickens (Fig. 4C). The mean viral titers in the lungs and brains of PBS-inoculated chickens infected with HP H5N1 (clade 1) viruses were 3.75 and 4.0 TCID${}_{50}$/0.1 g, respectively (Fig. 4C). Mean viral titers in the lungs and brains of PBS-inoculated chickens infected with HP H5N6 (clade 2.3.4.4.) viruses were 4.0 and 4.5 TCID${}_{50}$/0.1 g, respectively (Fig. 4C).

Portions of lung tissues used for estimating viral titers were stained with H&E to evaluate pathological damage. The lung tissues (Fig. 5C,F) of bivalent peptide-immunized and challenged chickens did not show any signs of pneumonia and were similar to uninfected control chickens (Fig. 5A,D), whereas the lung tissues of PBS-inoculated and challenged chickens showed severe pneumonia with inflammatory cell infiltration (Fig. 5B,E).

Fig. 5.

Histopathology of lung tissues of the challenged immunized chickens. The lung tissues of challenged immunized chickens (n = 3 per group) with H5N1 or H5N6 avian influenza viruses were stained with hematoxylin and eosin. The stained tissues were observed under a microscope. (A,D) PBS-mock challenged naïve lung tissues of immunized chickens. (B) Lung tissue of PBS-mock immunized and challenged chickens with H5N1 avian influenza virus. (C) Lung tissue of bivalent-peptide immunized and challenged chickens with H5N1 avian influenza virus. (E) Lung tissue of PBS-mock immunized and challenged chickens with H5N6 avian influenza virus. (F) Lung tissue of bivalent-peptide immunized and challenged chickens with H5N6 avian influenza virus.