Development of a subunit vaccine based on fiber2 and hexon against fowl adenovirus serotype 4
Introduction
The family Adenoviridae has two genera, Mastadenoviruses and Aviadenoviruses which are divided into three groups (groups I–III) (Hess, 2000), Group Ⅰ has five species (A–E) based on restriction enzyme digest patterns and twelve serotypes based on the results of serum cross-neutralization tests (FAdV-1 to 8a and b to 11) (Chen et al., 2019). Fowl aviadenovirus serotype 4 (FAdV-4) is a member of species fowl aviadenovirus C, group I, which belongs to Aviadenovirus genus of the Adenoviridae family (Li et al., 2016). FAdV-4 is the causative agent of hepatitis-hydropericardium syndrome (HHS) in poultry (Liu et al., 2016). In 1987, a new disease called Angara disease was reported in an Angara Gothic broiler flock near Karachi, Pakistan (Anjum et al., 1989). It was later reported in Russia, Central America, South America, Japan and South Korea (Balamurugan and Kataria, 2004; Schachner et al., 2018). The first experimental proof that HHS is caused by FAdV-4 was reported in 1998(Mazaheri et al., 1998). In 2015, fowl adenoviruses strain JSJ was isolated from chickens with HHS in China, and this was the first report of a FAdV-4 strain in China (Zhao et al., 2015). Since 2015, the FAdV-4 strain has caused substantial economic losses to the poultry industry in China (Li et al., 2016; Liu et al., 2016). From 2015 to 2018, FAdV-4 was the dominant serotype in China (Chen et al., 2019). Aviadenoviruses are non-enveloped particles with a double-stranded DNA genome and a diameter of 70–90 nm. The virions are icosahedral shaped consist of the major structural proteins, hexon, penton and fiber non-covalently bound to the penton base by its N terminus (Hess et al., 1995; Russell, 2009). All FAdVs have two fibers per penton base but only FAdV-1, FAdV-4 and FAdV-10 have two fiber genes (Marek et al., 2012). One fiber functions in virus attachment and one for internalization (Hess et al., 1995). The fiber protein promotes virus infection by attaching the viral capsid to the host cell surface and by interacting with cellular receptors (Shah et al., 2017). Among the major structural proteins, fiber 2 protein plays an important role in mediating FAdV-4 infection (Wang et al., 2018a). Compared to the inactivated vaccine, fiber 2 protein can provide better protection against avian adenovirus infection and induce rapid proliferation of CD4+ T-cells (Chen et al., 2018).
In this study, the soluble truncated fiber 2 protein, fused with one epitope of hexon, was expressed in an E. coli expression system. In a vaccination trial, the efficacy of different doses of the fiber2 (Gly275- Pro479 aa)-hexon (Met21-Val51 aa) protein (rFH) to induce protective immunity in 14-d-old chickens was assessed. These results could be useful for evaluating the application potential of the recombinant fiber 2 and hexon protein as a subunit vaccine against FAdV-4.
Section snippets
Virus
FAdV-4 strain was isolated from a poultry farm of the Hubei Province, and the liver of this infected chicken was homogenated in phosphate buffer (pH 7.2) at a ratio of 1:5 (W:V), with a high-throughput tissue homogenizer (BIONOON, Shanghai, China) at 50 Hz for 2 min at −30 °C. The homogenate was frozen and thawed three times, centrifuged at 4000 g for 5 min at 4 °C, then the supernatant was filtered through 0.22 μm membrane and named FAdV-4 HB1505 (Genbank accession: MZ054256).
Construction of recombinant plasmids
The viral DNA was
Expression and purification of recombinant protein
The molecular mass of the rFH protein was detected by reducing SDS-PAGE, approximately 28 kDa. Furthermore, expression analysis showed that large fractions of rFH protein present as soluble proteins in the supernatant of the solution. After purification using ammonium sulfate precipitation, Sephacryl S-300 gel filtration chromatography and DEAE Sepharose Fast Flow anion exchange chromatography, the rFH protein was obtained with a yield of 256 mg/L and a purity of 92% (Fig. 1). The purified
Discussion
HHS outbreaks are characterized by their sudden occurrence. Most cases have been observed in 3–6 week old chickens with mortality approaching 60% (Balamurugan and Kataria, 2004; Liu et al., 2016), causing large economic losses to the poultry industry of China. Many countries and regions have developed an inactivated vaccine and a live attenuated vaccine for prevention of this disease (Schachner et al., 2018). However, there are still some risks of incomplete virus inactivation or weakening, the
Conclusions
In summary, we successfully expressed the recombinant fiber2 (Gly275- Pro479 aa)-hexon (Met21-Val51 aa) protein in an E. coli expression system. The recombinant fiber2-hexon protein showed good solubility and good immunity protection. A 2.5 μg dose per chicken provided partial protection, and a single 5 μg dose per chicken provided 100% protection against FAdV-4 virus attack. These findings suggest that FAdV-4 fiber2-hexon is a good candidate for the development of an effective and safe subunit
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests.
Acknowledgments
Jixiong Hu took part in all the experiments, performed the statistical analyzes and wrote the manuscript. Jun Rong designed the study, supervised the analyzes and helped to draft the manuscript. Guopan Li, Xi Wang, Mingxuan Rong, Huan Li, Ming Xie, Zhixiang Zhang and Qingqing Chen participated in the procedure of the animal experiment. All co-authors revised the manuscript and approved the final submitted version.
This work was supported by Higher Education Comprehensive Award fund (2018-
Ethics statement
All animal experiments procedures were conducted in compliance with the Guide for Care and Use of Laboratory Animals and the Policy of Animal Care and Use Committee of Yangtze University.
Author statement
The authors confirm the availability of all data presented in the manuscript.
References (37)
Recombinant protein expression in Escherichia coli
Curr. Opin. Biotechnol.
(1999)- et al.
Immunogenicity and protective efficacy of recombinant fiber-2 protein in protecting SPF chickens against fowl adenovirus 4
Vaccine
(2018) - et al.
Epidemiological investigation of fowl adenovirus infections in poultry in China during 2015-2018
BMC Vet. Res.
(2019) - et al.
Multi-epitope proteins for improved serological detection of trypanosoma cruzi infection and chagas disease
Diagn. Microbiol. Infect. Dis.
(2016) - et al.
A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein
Vaccine
(2003) - et al.
The avian adenovirus penton: two fibres and one base
J. Mol. Biol.
(1995) - et al.
Prime-boost vaccine strategy against viral infections: mechanisms and benefits
Vaccine
(2016) - et al.
Fowl adenovirus species C serotype 4 is attributed to the emergence of hepatitis-hydropericardium syndrome in chickens in China
Infect. Genet. Evol.
(2016) - et al.
Fowl adenovirus serotype 4 SD0828 infections causes high mortality rate and cytokine levels in specific pathogen-free chickens compared to ducks
Front. Immunol.
(2018) - et al.
Two fiber genes of nearly equal lengths are a common and distinctive feature of Fowl adenovirus C members
Vet. Microbiol.
(2012)
Immunosuppressive potential of fowl adenovirus serotype 4
Poult. Sci.
Pathogenicity of a fowl adenovirus serotype 4 isolated from chickens associated with hydropericardium-hepatitis syndrome in China
Poult. Sci.
A subunit vaccine based on fiber-2 protein provides full protection against fowl adenovirus serotype 4 and induces quicker and stronger immune responses than an inactivated oil-emulsion vaccine
Infect. Genet. Evol.
Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS)
Vaccine
Development of novel subunit vaccine based on truncated fiber protein of egg drop syndrome virus and its immunogenicity in chickens
Virus Res.
Molecular relationship of the fowl adenovirus serotype 4 isolated from the contaminated live vaccine and wild strains isolated in China, 2013-2018
Poult. Sci.
Research note: molecular relationship of the fowl adenovirus serotype 4 isolated from the contaminated live vaccine and wild strains isolated in China, 2013-2018
Poult. Sci.
A novel monoclonal antibody efficiently blocks the infection of serotype 4 fowl adenovirus by targeting fiber-2
Vet. Res.
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2022, Veterinary MicrobiologyCitation Excerpt :The Fiber-2 of FAdV-4 is a hydrophilic protein without the transmembrane region and signal peptide (Chen et al., 2018). Both E. coli (Hu et al., 2021; Ruan et al., 2018a) and Baculovirus expression system (Schachner et al., 2014; Zhang et al., 2019) have been reported to be capable of successfully expressing the protective Fiber-2. However, the Fiber-2 all need to be purified and then immunized by injection in previous studies, which was complicated and expensive.