- Academic Editor
Background: Outbreaks of highly pathogenic avian influenza viruses
cause huge economic losses to the poultry industry worldwide. Vaccines that can
protect chickens from infections caused by various variants of highly pathogenic
H5Nx avian influenza viruses are needed owing to the continuous emergence of new
variants. We previously showed that vaccines containing the H5 cleavage-site
peptide from clade 2.3.4.4. H5N6 avian influenza virus protects chickens from
infection with homologous clade 2.3.4.4. H5N6 avian influenza virus, but not from
infection with the heterologous clade 1 H5N1 avian influenza virus. Therefore, we
developed bivalent peptide vaccines containing H5 cleavage sites of viruses from
both clades to protect chickens from both H5N1 and H5N6 avian influenza viruses.
Methods: Chickens were vaccinated with two doses of a combined peptide
vaccine containing cleavage-site peptides from clade 1 and clade 2.3.4.4. highly
pathogenic H5N1 and H5N6 avian influenza viruses and then challenged with both
viruses. The infected chickens were monitored for survival and their tracheae and
cloacae were sampled to check for viral shedding based on the median tissue
culture infectious dose of 50 (log
Avian influenza viruses infect wild birds and domestic poultry. Based on their pathogenicity, avian influenza viruses are classified as low or highly pathogenic (HP) avian influenza viruses. Avian influenza viruses contain a single-stranded negative-sense RNA genome and belong to the Orthomyxoviridae family [1]. Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA) are present on the surface of avian influenza virus particles. These viruses can be classified into different subtypes based on their HA and NA proteins; birds have been found to be infected with viruses containing 15 types of HA and 9 types of NA [2]. HP avian influenza viruses contain polybasic amino acids in the HA cleavage site, which enables the replication of these viruses in cells of poultry birds. Among the subtypes of avian influenza viruses, few H5 and H7 viruses contain polybasic amino acids at the HA cleavage site [2].
Outbreaks of HP H5Nx avian influenza viruses have caused major economic losses to the poultry industry worldwide [3, 4, 5, 6, 7, 8, 9, 10]. Additionally, HP H5Nx avian influenza viruses infect mammals, such as minks, red foxes, polecats, otters, and badgers [3, 4]. Various methods have been used to develop vaccines against HP H5Nx avian influenza viruses. The major types of vaccines include inactivated recombinants, virus-like particles (VLPs), and DNA vaccines [11, 12, 13]. We previously showed that vaccines containing the H5 cleavage-site peptide from clade 2.3.4.4. H5N6 avian influenza virus protects chickens from infection with homologous clade 2.3.4.4. H5N6 avian influenza virus, but not from infection with the heterologous clade 1 H5N1 avian influenza virus [14]. Therefore, we developed bivalent peptide vaccines containing clades 1 and 2.3.4.4. HP H5 cleavage sites to protect chickens from HP H5N1 and H5N6 avian influenza viruses of both clades.
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
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.
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
The bivalent peptide-immunized chickens were intranasally infected with 1 mL (1
MDCK cells were cultured in MEM containing 10% FBS and 1
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
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).
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.
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
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
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
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
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).
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.
HP H5Nx avian influenza viruses cause major economic losses to the poultry industry worldwide. HP H5Nx viruses continue to evolve and create new variants. Therefore, the development of vaccines to protect poultry from various variants of HP H5Nx avian influenza virus is necessary. We used a dual-antigen vaccine containing clade 1 and clade 2.3.4.4. H5 cleavage-site peptides that contain polybasic amino acids to protect chickens against HP H5Nx avian influenza viruses of both clades.
Antibody response was similarly induced in chickens immunized with the bivalent peptide antigens containing HA-cleavage site RRRK and RRRKK. The results suggest that KLH-labeled H5 cleavage site peptides of clade 1 and clade 2.3.4.4. are immunogenic in chickens.
Chickens immunized with dual antigens containing H5 cleavage-site peptides of both clades 1 and 2.3.4.4. were completely protected against H5N1 and H5N6 avian influenza viruses. Chickens immunized with the bivalent peptide antigens did not show any clinical signs such as hemorrhage in comb, wattle, and legs, which appeared in the non-immunized infected chickens. We previously found that chickens immunized with clade 2.3.4.4. The previous vaccine studies did not provide complete protection for chickens against highly pathogenic avian influenza viruses, in contrast to our current study. H5 cleavage-site peptides are protected from lethal H5N6 infections of the H5N6 (clade 2.3.4.4.) avian influenza virus, but not protected from lethal infection of the H5N1 (clade 1) avian influenza virus [14]. The clade 1 H5 cleavage site contains RRRKK as polybasic amino acids, whereas the clade 2.3.4.4. H5 cleavage site contains RRRK as polybasic amino acids. The efficacy of immunization using antigens based on various ectodomains of influenza matrix protein 2 (M2e) expressed in Escherichia coli in chickens has been tested against HP H5N1 (A/chicken/Guangdong/04) avian influenza virus. Vaccinated chickens are partially protected from infection with HP H5N1 avian influenza virus [16]. The HA stalk regions (HA2 domain) were previously used to create a universal vaccine against avian influenza viruses. Sf9 insect cells have been used to prepare triple H5N1/NA-HA-M1 influenza VLPs by coexpressing NA, HA, and matrix proteins. Purified VLPs have been used to immunize broiler hens to produce universal antibodies. The generated universal antibodies recognize mammalian-expressed HA-stalk recombinant proteins from homologous H5N1 and heterologous H7N9 avian influenza viruses [17]. A chimeric vaccine (HA2-AtCYN) that contains subunit 2 of HA2 and Arabidopsis thaliana cyanase protein (AtCYN) induces HA2-specific IgA production in tears and serum IgG production, and it partially protected chickens against low-pathogenic H5N2 infections.
Antigens of bivalent H5-cleavage site peptides protected the immunized chickens from the lethal infections of HP H5N1 or H5N6 avian influenza viruses without showing clinical signs such as hemorrhage in comb, wattle, and legs. For future studies, the protective efficacy of the bivalent vaccines against the diverse strains of HP H5Nx avian influenza viruses in chickens needs to be investigated to examine the possibility of the vaccine as a universal vaccine.
The data are available from the corresponding author upon reasonable request.
DC & XHL performed animal experiments. SHS conceived the works and wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
Animal experiments were performed according to the protocol (CNU-01191) approved by the Internal Animal Use Committee of Chungnam National University (CNU). The relevant guidelines and regulations of Chungnam National University, Republic of Korea were applied to all animal experiments.
Not applicable.
This work was supported by a grant by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (2019R1A2C2002166812).
The authors declare no conflict of interest. Given Sang Heui Seo as Guest Editor, had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Dr. Giuseppe Murdaca.
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