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Virus Res
Virus Res
Virus Research
0168-1702
1872-7492
Elsevier

S0168-1702(24)00152-7
10.1016/j.virusres.2024.199459
199459
Article
Evaluation of novel synthetic peptides of avian hepatitis E virus ORF2 as vaccine candidate in chickens
Chen Yiyang a1
Tang Yujia a1
Zhang Shiyu a
Tian Yinuo a
Xu Shenhao a
Zhang Chengwei a
Lin Huanqing b
Zhao Qin a
Zhou En-Min a2
Liu Baoyuan liubaoyuan0412@nwafu.edu.cn
a⁎
a Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
b Kongtong Animal Disease Prevention and Control Center, Pingliang, Gansu, China
⁎ Corresponding author at: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China. liubaoyuan0412@nwafu.edu.cn
1 These two authors equally contributed to this work.

2 Dr. En-Min Zhou passed away three years ago.

05 9 2024
11 2024
05 9 2024
349 19945931 5 2024
18 8 2024
30 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Highlights

• Avian HEV has led to serious economic losses in the poultry industry.

• The peptide RLLDRLSRTFPS showed effective protection against avian HEV infection in chickens.

• The study provided a foundation for future vaccine and antiviral drug development.

Avian hepatitis E virus (HEV) has resulted in significant economic losses in the poultry industry. There is currently no commercial vaccination available to prevent avian HEV infection. Previously, a novel epitope (601TFPS604) was discovered in the ORF2 protein of avian HEV. In this study, peptides were synthesized and assessed for their ability to provide immunoprotecting against avian HEV infection in poultry. Twenty-five Hy-Line Variety Brown laying hens were randomly divided into five groups; groups 1 to 3 respectively immunized with RLLDRLSRTFPS, PETRRLLDRLSR (irrelevant peptide control), or truncated avian HEV ORF2 protein (aa 339–606), while group 4 (negative control) was mock-immunized with PBS and group 5 (normal control) was not immunized or challenged. After the challenge, all hens in groups 2 and 4 showed seroconversion, fecal virus shedding, viremia, alanine aminotransferase (ALT) level increasing, liver lesions and HEV antigen in the liver. There were no pathogenic effects in other groups. Collectively, all of these findings showed that hens were completely protected against avian HEV infection when they were immunized with the peptide containing TFPS of the avian HEV ORF2 protein.

Keywords

Avian hepatitis E virus
Peptide
Protection
Vaccine
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pmc1 Introduction

Avian hepatitis E virus (HEV) usually results in 10 % - 40 % reduction in egg production and 1 % - 4 % rise in mortality in laying hens and broiler chickens aged 30–72 weeks, and it may occasionally lead to enlarged livers and spleens (Clarke et al., 1990; Handlinger and Williams, 1988; Morrow et al., 2008). Additionally, the virus has been detected in asymptomatic chickens (Sun et al., 2004a). This pathogen is often overlooked, leading to significant economic losses in the poultry industry. At present, neither specific vaccines nor therapeutic drugs are available for avian HEV prevention and treatment. Avian HEV is categorized into four primary genotypes with a single serotype and the sequence divergence is mainly on ORF1, but the pathogenicity of avian HEV varies among different genotypes (Liu et al., 2020). Genotype 1 avian HEV was often detected in big liver and spleen disease (BLS) chickens in Australia and egg-depleted chickens in Korea (Bilic et al., 2009; Kwon et al., 2012; Payne et al., 1999). Genotype 2 was commonly found in hepatitis-splenomegaly syndrome (HSS) chickens in the United States and Korea, with some occurrences in BLS chicken populations in Central Europe (Haqshenas et al., 2001; Moon et al., 2016). Genotype 3 was predominantly found in HSS chickens in Europe and China (Marek et al., 2010; Morrow et al., 2008; Zhang et al., 2019; Zhao et al., 2010), while genotype 4 was frequently detected in bile samples from commercial egg-laying hens in Taiwan, China, and diseased poultry in Hungary (Banyai et al., 2012; Hsu and Tsai, 2014). In recent years, two new genotypes of avian HEV have been found in chicken and silkie fowl in China (Liu et al., 2020; Su et al., 2020).

Avian HEV, similar to HEVs found in other animal species, is mostly composed of three open-reading frames: ORF1, ORF2, and ORF3 (Huang et al., 2004). The ORF2 encodes the viral capsid protein, which includes important immunodominant and neutralizing epitopes of the virus particles and is the focus of protective humoral immune responses (Syed et al., 2017). To date, the identified antigenic structural regions from I to VI in avian HEV are as follows: amino acids 389–410, 461–492, 556–566, 583–600, 339–389, and 23–85 (Dong et al., 2011; Haqshenas et al., 2002). The truncated protein Ca268 is located at the C-terminus of avian HEV ORF2 and consists of 268 amino acids from aa 339 to aa 606. It contains five antigenic structural domains (I-V) of the capsid protein. When Ca268 was used to immunize chickens, it could elicit a strong immune response and provide effective immunological protection (Syed et al., 2017). Generally, the Ca268 protein is expressed as inclusion bodies, requiring renaturation before use. However, this process presents challenges such as protein precipitation. These complex procedures have limited its advancement beyond the laboratory stage. In our previous study, a novel competitive ELISA was developed for the rapid detection of antibodies against the avian HEV, using a nanobody-horseradish peroxidase fusion protein as a probe (Chen et al., 2023). Furthermore, the nanobody (Nb49) identified the epitope at position 593–604 of the avian HEV capsid protein and aa 601–604 (TFPS, conserved in all avian HEVs) was important for binding. Therefore, we speculated that the peptide TFPS might be essential for the composition of immunodominant antigens and protection. In this study, the peptide containing TFPS was produced and evaluated for its efficacy in inhibiting avian HEV infection in vivo. These results provide a basis for the future development of vaccines and antiviral drugs against avian HEV.

2 Materials and methods

2.1 Synthesis peptides, Ca268, and ORF3 protein and virus

Corresponding peptide 593RLLDRLSRTFPS604 (Pep-1) was characterized and synthesized for protective capacity (Fig. 1). Additionally, peptide 589PETRRLLDRLSR600 (Pep-2) was evaluated as irrelevant epitope control. Each peptide was linked to keyhole limpet hemocyanin (KLH) for vaccination or bovine serum albumin (BSA) for ELISA detection. The peptides applied in this research were produced by GenScript in Nanjing, China and were diluted as the manufacturer's guidelines. The avian HEV truncated ORF2 protein Ca268 (aa 339–606) and the ORF3 protein were individually produced in Escherichia coli BL21 (DE3) cells and purified using established techniques (Guo et al., 2007; Zhao et al., 2013b).Fig. 1 Schematic diagram showing Ca268, 6 antigen regions (I-VI) and synthetic peptide fragments.

Fig 1

An avian HEV infectious stock originated by injecting four 8-week-old SPF chickens intravenously with 200 μL of a clinical bile sample containing avian HEV obtained from a 35-week-old chicken (CaHEV, GenBank accession no. GU954430) and contained 104 genomic equivalents (GE)/mL (Zhao et al., 2010).

2.2 Immunization of chickens with synthetic peptides

Hy-Line Variety Brown laying hens (150-day-old) were bought from Shaanxi Zhengda Company (Xianyang, China). No chickens in the farm had previously been infected with avian HEV or demonstrated decrease in egg production. All 25 chickens were healthy and exhibited no clinical symptoms of the disease. Each hen was negative for anti-HEV antibodies in the blood and HEV RNA in the feces using indirect ELISA and nested RT-PCR, as described in previous studies (Chen et al., 2023; Dong et al., 2011).

After one week of acclimation, hens (157-day-old) from groups 1–3 were inoculated intramuscularly in both sides of the breast with Pep-1, Pep-2, or Ca268, respectively. Hens in group 4 were immunized with PBS as a negative control. As normal controls, chickens in group 5 were not immunized or challenged. 200 μg of antigen was combined with Freund's complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) for the first immunization and subsequently mixed with Freund's incomplete adjuvant following a two-week interval.

2.3 Virus challenge and sample collection

All hens (185-day-old) in groups 1–4 were intravenously injected with 1 mL of CaHEV (104GE/mL) after two weeks following booster immunization. Serum and fecal samples were collected before the challenge and weekly thereafter for 5 weeks. Fecal virus shedding and viremia were assessed in each injected chicken using a nested RT-PCR test. Serum samples were analyzed for anti-avian HEV antibodies using indirect ELISA with avian HEV ORF3 (CaHEV-ORF3) as the coated antigen. Additionally, serum levels of alanine aminotransferase (ALT) were quantified. Liver tissues of hens (220-day-old) were obtained during necropsy for routine histological analysis and immunohistochemistry (IHC). All animal experiments were conducted in compliance with the guidelines for Experimental Animal Welfare and Ethical Treatment by the Ministry of Science and Technology of China. The procedures were approved by the Committee on Ethical Use of Animals at Northwest A&F University (AE124985).

2.4 Indirect ELISA

Anti-avian HEV antibody levels were specific for peptides, Ca268, or ORF3 protein and were evaluated in chicken serum samples using indirect ELISA before and after vaccination and challenge, as previously described (Zhao et al., 2013a, 2013b). In short, 200 ng/well of various peptides (with BSA-conjugated), Ca268, or ORF3 protein was coated on ELISA plates. Following the blocking and washing procedures, plates were supplemented with serum samples (100 μL/well) and incubated at 25 °C for 1 hour. Subsequently, goat anti-chicken IgG conjugated with horseradish peroxidase (HRP) from Jackson ImmunoResearch in West Grove, PA, USA was diluted at a ratio of 1:4000 (100 μL/well) and added to the wells. The plates were then incubated for 1 hour at 25 °C. Following several washes, 3,3′,5,5′-tetramethylbenzidine (TMB) was supplemented to the wells, and the plates were kept in the dark for 15 min at 25 °C. The reaction was stopped by supplementing 3 M H2SO4 (50 μL/well), and the optical density (OD) values were measured at 450 nm using an automatic microplate reader (Bio-Rad, USA). Each of the serum samples was evaluated in two duplicate wells.

2.5 Detecting avian HEV RNA by RT-PCR

Avian HEV RNA was analyzed in serum and fecal samples with the procedures by Dong et al. (Dong et al., 2011). RNA was extracted from 10 % fecal supernatant or 200 μL of serum using TRIzol reagent according to the manufacturer's guidelines. The PrimeScript™ One-Step RT-PCR Kit (TaKaRa, Japan) was applied for reverse transcription and the first PCR. The second PCR was then performed following the manufacturer's protocol using TransTaq High Fidelity DNA polymerase (TransGen Biotech Co., Beijing, China). The PCR results were then confirmed using electrophoresis on a 1 % agarose gel.

2.6 Measurement of serum alanine aminotransferase (ALT) levels

Weekly serum samples were analyzed for ALT concentrations using a Hitachi 912 analyzer, following the manufacturer's guidelines (Roche, Indianapolis, IN, USA). Hepatitis was confirmed when their post-challenge ALT concentrations were at least two-fold higher than their pre-challenge ALT levels (Ma et al., 2010).

2.7 Gross hepatic lesions, histopathological and immunohistochemical changes in liver tissues

Gross pathological lesions in the liver were assessed during necropsy. In addition, liver tissues were processed for routine histological examination after being fixed in 10 % neutral buffered formalin. IHC analyses were also conducted. The monoclonal antibody 1H5 (mouse anti-avian HEV capsid protein, 1 mg/ml, 1:1000 dilution) was used.

2.8 Statistical analysis

Student's t-test was performed in order to compare the ALT values of various groups. A level of significance of P < 0.05 was considered.

3 Results

3.1 Immune responses in chickens immunized with synthetic peptides or Ca268 protein

A cell counting kit-8 (CCK-8) assay was performed to test the toxicity of peptides in HepG2 cells. The viability of HepG2 cells was close to that of the untreated control cells at concentrations lower than 20 μM. Chicken serum samples were obtained before and at different weeks after vaccination to evaluate antibody levels specific to peptides (with BSA-conjugated for coating antigen) or Ca268 protein using an indirect ELISA. Following the second vaccination at 4 weeks post-immunization (4 wpi), the OD450mm values of serum samples from immunized hens in groups 1–3 increased to about 1.5, indicating a strong antigen-antibody interaction (Fig. 2A-C). There were no obvious changes in the serum OD450mm values of groups 4–5 (Fig. 2D-E).Fig. 2 Seroconversion in chickens immunized with Pep-1 (A), Pep-2 (B), Ca268 protein (C), PBS (D) and normal control (E). Serum samples were collected from chickens before immunization and 2 weeks after each immunization (1st and 2nd).

Fig 2

3.2 Seroconversion, fecal virus shedding, viremia, and ALT levels in the experimentally

According to the cut-off value (0.47) of the indirect ELISA, all chickens in groups 2 and 4 were seroconverted at 3 week post-challenge (wpc) and remained positive for anti-avian HEV antibodies until the end of the experiment (5 wpc) (Table 1, Fig. 3B and Fig. 3D). Conversely, seronegative chickens were observed in groups 1, 3, and 5 throughout the experiment (Table 1, Fig. 3A, C, and E).Table 1 Seroconversion, viremia and fecal shedding of all chickens during the course of the study.

Table 1Group	Immunization	No. of seroconversion/viremia/fecal shedding tested at indicated wpi	
0	1	2	3	4	5	
1	Pep-1	0/0/0	0/0/0	0/0/0	0/0/0	0/0/0	0/0/0	
2	Pep-2	0/0/0	0/5/5	0/4/5	5/3/4	5/0/4	5/0/5	
3	Ca268	0/0/0	0/0/0	0/0/0	0/0/0	0/0/0	0/0/0	
4	PBS (NC)	0/0/0	0/5/5	0/4/5	5/4/5	5/0/4	5/0/5	
5	PBS (Normal)	0/0/0	0/0/0	0/0/0	0/0/0	0/0/0	0/0/0	

Fig. 3 Fecal viral shedding/viremia, ALT levels, and antibody levels in chickens experimentally challenged with CaHEV. Chickens were immunized with Pep-1 (A), Pep-2 (B), Ca268 protein (C), or PBS (D); Chickens challenged with PBS (E) as normal control. “+” and “-” represent positive and negative for fecal virus shedding and viremia, respectively. Statistical significance (*P < 0.05).

Fig 3

Fecal and serum samples from all chickens were negative for avian HEV RNA prior to infection, and hens in groups 1, 3, and 5 remained negative throughout the study (Table 1, Fig. 3A, C, and E). Viremia and fecal virus shedding were observed in all hens in groups 2 and 4 at 1 wpc. Until the end of the study (5 wpc), HEV RNA was consistently detected in fecal samples from all chickens. However, it was detectable for 2–3 weeks in serum samples. (Table 1, Fig. 3B, and D).

There were no obvious changes in the serum ALT concentrations of groups 1, 3, and 5 (Fig. 3A, C, and E). In contrast, the average serum ALT levels in groups 2 and 4 increased (96–110 U/L) at 1 wpc and then returned to normal levels (Fig. 3B and Fig. 3D). In addition, at 1 wpc, the serum ALT levels of all hens in groups 2 and 4 increased significantly more than those in the negative control group (P < 0.05), and this difference was not significant at other wpc.

3.3 Gross hepatic lesions, histopathological and immunohistochemical changes in liver tissues

No gross hepatic lesions (swelling and bleeding) were exhibited in the livers of all necropsied hens across any groups (groups 1–5). However, hepatic lesions were seen in all infected chickens in groups 2 and 4 under the microscope, revealing severe lymphocytic periphlebitis (Fig. 4B and D). In the other groups (groups 1, 3 and 5), none of the chickens showed apparent microscopic liver lesions (Fig. 4A, C, and E). With IHC staining, no specific brown staining was observed in the livers of all hens in groups 1, 3 and 5 (Fig. 5A, C, and E), and HEV antigens were detected in the groups 2 and 4 (Fig. 5B and D).Fig. 4 Microscopic lesions of the livers from the necropsied Chickens. Liver sections showed no apparent pathological signs of HEV infection in chickens in groups 1, 3 and 5; Severe lymphocytic periphlebitis (arrow) in chickens in groups 2 and 4. Chickens were immunized with Pep-1 (A), Pep-2 (B), Ca268 protein (C), or PBS (D); Chickens challenged with PBS (E) as normal control. Tissues were stained with hematoxylin and eosin.

Fig 4

Fig. 5 HEV antigens detection in the liver from the necropsied hens. Liver sections from hens showing no specifc brown staining in groups 1, 3 and 5; HEV antigens in the livers (arrow) of all hens in groups 2 and 4. Hens were immunized with Pep-1 (A), Pep-2 (B), Ca268 protein (C), or PBS (D); Chickens challenged with PBS (E) as normal control.

Fig 5

4 Discussion

Avian HEV strains had been detected in various avian species, even rabbits (Liu et al., 2020, 2018; Reuter et al., 2016a, b; Sun et al., 2004b; Yang et al., 2018; Zhang et al., 2017), and caused significant economic losses in the poultry industry (Morrow et al., 2008). Previously, ORF2 capsid protein or Ca268 protein could confer complete immunoprotection against CaHEV infection in chicken, and the ORF3 protein only provided partial immunoprotection (Syed et al., 2017). Additionally, an important epitope (TFPS) had been identified in the avian HEV capsid protein, which might play a critical role in the dominant antigen composition and immune protection (Chen et al., 2023). Therefore, in this study, chickens were immunized with a synthetic peptide based on this epitope and then evaluated for their immune protection against avian HEV infection. The findings indicated that peptide 593RLLDRLSRTFPS604 or Ca268 protein conferred complete protection.

The Welling method was used to predict 5 antigen regions (I-V) in the Ca268 protein in the previous study (Dong et al., 2011; Haqshenas et al., 2002). Studies have revealed that region IV (aa 583–600) did not act as a dominant antigen or provide protection against avian HEV infection. However, the Ca268 protein (aa 339–606) showed effective immunological protection (Guo et al., 2007; Zhao et al., 2012). In this study, the peptide 593RLLDRLSRTFPS604 (aa 601–604, located outside region IV) provided complete protection comparable to Ca268 protein against avian HEV infection, while the peptide 589PETRRLLDRLSR600 had no immunoprotective effects. These findings indicate that the peptide TFPS is crucial for antigenic region IV and has a significant protective role. In our previous study, Nb49 was found to bind strongly to the peptide 593RLLDRLSRTFPS604 after identification with different truncated proteins and peptides, particularly the peptide TFPS (Chen et al., 2023). The peptide was conserved in all avian HEV sequences of various genotypes. This suggested that the peptide TFPS may offer spectral protection against all avian HEV genotypes in chickens, and the Nb49 also had antiviral neutralizing activity. Generally, T cells response is included to demonstrate the immunogenic properties of a novel peptide. The study had shown that type І interferon was down-regulated after HEV infection (Nan et al., 2014), and it played an important role in antiviral infection. Besides, the activations of cytotoxic T cells, helper lymphocytes and other cytokines need to be considered. Other experiments will be executed to verify our hypothesis.

In conclusion, chickens vaccinated with peptide 593RLLDRLSRTFPS604 or Ca268 protein did not exhibit seroconversion, fecal virus shedding, viremia, elevated ALT levels, liver lesions or HEV antigen in liver. The study revealed that the peptide TFPS effectively protected hens against avian HEV infection, laying the foundation for future vaccination and antiviral drugs research.

Author statement

No AI technologies were used in the analysis or preparation of this manuscript.

CRediT authorship contribution statement

Yiyang Chen: Writing – original draft, Methodology, Investigation, Funding acquisition, Data curation. Yujia Tang: Validation, Methodology, Investigation, Data curation. Shiyu Zhang: Visualization, Software, Formal analysis. Yinuo Tian: Software, Formal analysis. Shenhao Xu: Validation, Software. Chengwei Zhang: Software, Formal analysis. Huanqing Lin: Visualization, Software, Funding acquisition. Qin Zhao: Visualization, Software. En-Min Zhou: Visualization, Software. Baoyuan Liu: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no competing interests.

Data availability

Data will be made available on request.

Acknowledgments

The study was supported by grants from the 10.13039/501100001809 National Natural Science Foundation of China (No. 32302863 ), the 10.13039/501100002858 China Postdoctoral Science Foundation (No. 2021M692659 ) and Kongtong Science and Technology Project of Pingliang (PL-KT-2023A-005).
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