Recombinant fiber-1 protein of fowl adenovirus serotype 4 induces high levels of neutralizing antibodies in immunized chickens

Archives of Virology (2023) 168:84 https://doi.org/10.1007/s00705-023-05709-6 ORIGINAL ARTICLE Recombinant fiber‑1 protein of fowl adenovirus serotype 4 induces high levels of neutralizing antibodies in immunized chickens Satoko Watanabe1 · Yu Yamamoto1 · Aoi Kurokawa1 · Hiroshi Iseki1 · Taichiro Tanikawa1 · Masaji Mase1,2,3 Received: 21 July 2022 / Accepted: 22 December 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2023 Abstract Virulent fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium syndrome (HPS) with high mortality in chickens, leading to significant economic losses to the poultry industry. The development of an effective vaccine is essential for successful disease control. Here, we produced recombinant fiber-1 protein of FAdV-4, isolated from a Japanese HPS outbreak strain, JP/LVP-1/96, using a baculovirus expression system and evaluated its immunogenicity and protective efficacy. Recombinant fiber-1 protein induced high levels of neutralizing antibodies in immunized chickens, which were maintained for a minimum of 10 weeks. After being challenged with the virulent FAdV-4 strain JP/LVP-1/96, the immunized chickens did not exhibit clinical signs of infection or histopathological changes, there was a significant reduction in the viral load in various organs and total serum proteins, and albumin levels did not decline. These results suggest that the recombinant fiber-1 protein produced in this study can serve as a subunit vaccine to control HPS in chickens. Introduction Hydropericardium syndrome (HPS), also known as hepatitis hydropericardium syndrome (HHS), is a disease affecting chickens that was first reported in Pakistan approximately 35 years ago [1]. HPS caused by virulent fowl adenovirus serotype 4 (FAdV-4) is characterized by the accumulation of amber-colored fluid in the pericardial sac and an enlarged discolored liver with foci of hemorrhage and/or necrosis [2]. HPS outbreaks in poultry have been reported mainly in Asian and Latin American countries, causing significant economic losses to the poultry industry [3–6]. In Japan, HPS outbreaks in chickens were reported on Shikoku Island from 1996 to 2001, and FAdV-4 was isolated from all of the cases [7]. Phylogenetic analysis based on the part of the hexon gene that includes the L1 region revealed that all of these Handling Editor: Morgana Barboza . * Masaji Mase 1 National Institute of Animal Health, NARO, 3‑1‑5 Kannondai, Tsukuba, Ibaraki 305‑0856, Japan 2 United Graduate School of Veterinary Sciences, Gifu University, 1‑1 Yanagido, Gifu, Gifu 501‑1193, Japan 3 Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan FAdV-4 isolates were identical and were distinct from the cluster including FAdV-4 strains from chickens with HPS in other countries [8, 9]. The FAdV-4 capsid consists of four major structural proteins: hexon, penton base, fiber-1, and fiber-2 [10]. Functional analysis of these proteins is essential for understanding the infectivity and virulence of FAdV-4. Fiber-1 directly mediates FAdV-4 adsorption to host cells and triggers FAdV-4 infection [11]. Wang et al. also reported that the adsorption of FAdV-4 through fiber-1 is essential for infection [12]. Fiber-2 is an important virulence factor of FAdV-4 [11], and fiber-2 and hexon play crucial roles in the pathogenicity of FAdV-4 strains [13]. Amino acid substitutions at positions 219 and 188 in the fiber-2 and hexon protein, respectively, are commonly found in virulent FAdV-4 strains, including Japanese HPS-associated isolates [9]. Importantly, these capsid proteins induce humoral and/or cellular immune responses in chickens and have been shown to be protective against HPS [14–17], suggesting their potential application as subunit vaccines. Here, we report the production of recombinant FAdV-4 fiber-1 protein derived from a Japanese HPS isolate, using a baculovirus expression system, and we demonstrate its ability to induce high levels of neutralizing antibody titers in immunized chickens. The efficacy of this vaccine was verified by challenging immunized chickens with a pathogenic Japanese FAdV4 strain. 13 Vol.:(0123456789) 84 Page 2 of 8 Materials and methods Virus JP/LVP-1/96 (FAdV-4, GenBank accession number LC628937) was used in this study [9]. This strain was isolated from HPS-affected chickens in Japan [7, 8]. After plaque purification, the virus was grown in primary chicken kidney cell (CKC) cultures, and the culture supernatants were harvested, centrifuged, aliquoted, and stored at -80°C for further experiments. Cloning, expression, and purification of recombinant FAdV‑4 fiber‑1 protein The fiber-1 gene of JP/LVP-1/96 with a His-tag sequence at its C-terminus was amplified by polymerase chain reaction (PCR) and cloned into the baculovirus transfer vector pAcYM1 [18]. The recombinant transfer vector and the LacZ gene-recombinant Autographa californica nuclear polyhedrosis virus DNA, linearized with Eco81I, were cotransfected into Spodoptera frugiperda-derived Sf21AE cells. After plaque purification, the fiber-1-gene-containing recombinant baculovirus was isolated. To obtain the recombinant protein, Sf21AE cells were infected with recombinant baculovirus at a multiplicity of infection of 1.0, harvested after 3 days, and suspended in PBS containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). They were then sonicated using an LP-300N homogenizer (Titec, Tokyo, Japan), followed by centrifugation at 11,000 rpm for 20 min. The His-tagged recombinant protein was purified from the supernatant using Talon spin columns (Takarabio, Shiga, Japan) according to the manufacturer's protocol. Bound protein was eluted using 150 mM imidazole in 50 mM phosphate buffer (pH 7.0). The eluted fractions were collected and dialyzed against PBS for further analysis. To determine the purity of the recombinant proteins, samples were separated by SDS–PAGE under reducing conditions [19]. After electrophoresis, the gel was stained with Coomassie brilliant blue (CBB) G-250 or the proteins were transferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Walthan, USA). After blocking, the membrane was probed with anti-His antibody (MBL, Tokyo, Japan) and horseradish-peroxidase-conjugated goat anti-mouse immunoglobulin G (KPL Inc., Gaithersburg, MD, USA). Reactive bands were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA). The concentration of the protein obtained was determined using a DC protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard. 13 S. Watanabe et al. Immunization of chickens and determination of serum neutralizing antibody titers All experimental procedures and animal care were performed in compliance with the guidelines of the National Institute of Animal Health for the humane use of l (...truncated)