Journal of Virological Methods 211 (2015) 36–42

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Eliciting specific humoral immunity from a plasmid DNA encoding infectious bursal disease virus polyprotein gene fused with avian influenza virus hemagglutinin gene Yung-Yi C. Mosley, Ming Kun Hsieh 1 , Ching Ching Wu, Tsang Long Lin ∗ Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA

a b s t r a c t Article history: Received 20 July 2014 Received in revised form 16 October 2014 Accepted 21 October 2014 Available online 28 October 2014 Keywords: IBDV AIV Dual function DNA vaccine

DNA vaccine coding for infectious bursal disease virus (IBDV) polyprotein gene and that for avian influenza virus (AIV) hemagglutinin (HA) gene have been shown to induce immunity and provide protection against the respective disease. The present study was carried out to determine whether an IBDV polyprotein gene-based DNA fused with AIV HA gene could trigger immune response to both IBDV and AIV. After transfection, VP2 and HA were detected in the cytoplasm and at cell membrane, respectively, by immunofluorescent antibody double staining method, suggesting the fusion strategy did not affect the location of protein expression. VP4 cleavage between VP2 and HA was confirmed by Western blot, indicating the fusion strategy did not affect VP4 function in transfected cells. After vaccination in chickens, the DNA construct VP24-HA/pcDNA induced ELISA and virus neutralizing antibodies against VP2 and hemagglutination inhibition antibody against the HA subtype. The results indicated that a single plasmid construct carrying IBDV VP243 gene-based DNA fused with AIV HA gene can elicit specific antibody responses to both IBDV and AIV by DNA vaccination. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Infectious bursal disease virus (IBDV) is a member of the family Birnaviridae and genus Avibirnavirus. IBDV is an immunosuppressive viral agent due to the fact that the infection causes severe depletion of B lymphocytes in the bursa of Fabricius (Cho, 1970; Wyeth, 1975; Rosenberger and Gelb, 1978). Therefore, the infected chickens have increased susceptibility to secondary infections and decreased vaccination responses (Allan et al., 1972; Faragher et al., 1972; Winterfield et al., 1978). The genome of IBDV contains bipartite double-stranded RNA namely segment A and segment B. There are two partially overlapped open reading frames in segment A (3.3 kb). The first open reading frame encodes the nonstructural protein, VP5 (17 kDa). The second open reading frame encodes the polyprotein VP243 (109 kDa) which is processed through the

Abbreviations: IBDV, infectious bursal disease virus; AIV, avian influenza virus; HA, hemagglutinin; FITC, fluorescein isothiocyanate; TMB, 3,3 ,5,5 tetramethylbenzidine. ∗ Corresponding author at: Purdue University, ADDL, 406 South University Street, West Lafayette, IN 47907-2065, USA. Tel.: +1 765 4947927; fax: +1 765 4949181. E-mail address: [email protected] (T.L. Lin). 1 Present address: Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan. http://dx.doi.org/10.1016/j.jviromet.2014.10.011 0166-0934/© 2014 Elsevier B.V. All rights reserved.

autoproteolysis of VP4 (28 kDa) to generate pVP2, VP4 and VP3 (Azad et al., 1987; Jagadish et al., 1988). The premature form of the capsid protein, pVP2, is further cleaved at its C-terminus by VP4 and its autoproteolytic activity to form VP2 (37 kDa) (Sánchez and Rodriguez, 1999; Irigoyen et al., 2009). VP2 is the only component in the capsid and thus is the major immunogen for inducing virus neutralizing antibody to protect against IBDV infection (Becht et al., 1988; Fahey et al., 1989; Coulibaly et al., 2005). Avian influenza virus (AIV) belongs to the family Orthomyxoviridae which possesses eight segments of single-stranded minus-sense RNA genome. Hemagglutinin (HA), one of the major surface glycoprotein recognizes and attaches to sialic acid on the cell surface as the receptor for AIV (Webster et al., 1992). Thus the HA-receptor binding specificity determines the viral tropism and host range (Suzuki et al., 2000; Wilks et al., 2012). HA is also one of the major viral proteins responsible for inducing neutralizing antibody and provides protection from AIV infection (Kostolansky´ et al., 2000; Gao et al., 2006; Swayne, 2009; Vareˇcková et al., 2013). In recent years, high pathogenic H5N1 subtype has become a global concern after the direct transmission from domestic chickens to humans (Claas et al., 1998; Subbarao et al., 1998; Zhang et al., 2013). Generally, wild aquatic birds are considered as natural reservoirs for all AIVs and these viruses must require adaptation to infect and replicate more efficiently in other animal spices (Webster et al.,

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1992; Horimoto and Kawaoka, 2001). It has been suggested that the immunosuppression caused by IBDV in chickens could facilitate the adaptation process for water fowl associated AIV to domestic poultry (Ramirez-Nieto et al., 2010). Thus, it is critical for the poultry industry to prevent both viral infections in the same flock. DNA vaccines containing VP2 or VP243 gene of IBDV has been shown to provide protection of chickens against IBDV (Chang et al., 2001, 2003; Hsieh et al., 2010; Chen et al., 2011). Vaccination with DNA encoding HA gene has also shown protection against AIV challenge in previous studies (Fynan et al., 1993; Robinson et al., 1993; Kodihalli et al., 2000). The present study was carried out to determine whether an IBDV large segment gene-based DNA fused with AIV HA gene could trigger dual expression of both proteins and induce specific humoral immune responses to both IBDV and AIV by a single plasmid construct. 2. Materials & methods 2.1. Chickens Specific pathogen free chicks were hatched out after 21 days of incubation from embryos (Charles River Laboratories, Wilmington, MA, USA). After hatching, one-day-old chicks (n = 30) were transferred to Horsfall-Bauer isolators with food and water provided ad libitum. The protocol for using chickens in the present study was approved by Purdue University Animal Care and Use Committee. 2.2. Construction of DNA 2.2.1. VP243/pcDNA VP243 sequence (from variant E strain of IBDV) was PCR amplified from VP243/pCR3.1 (Hsieh et al., 2006) and subcloned with pcDNA3.1/V5-His© TOPO® TA expression kit (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s instruction. Primer sequences were VP2 forward (5 -ACGATCGCAGCGATGACAAA-3 ) and VP3 reverse (5 -TCACTCAA GGTCCTCATC-3 ). 2.2.2. HA/pcDNA AIV HA gene was subcloned to pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) with restriction enzymes BamHI and XhoI (New England Biolabs, Beverly, MA, USA) from cDNA of avian influenza virus stain TK/WI/68 H5, provided by Dr. David Suarez, Southeastern Poultry Research Laboratory, USDA, Athens, GA. Primers used were H5-BamHI-F (5 -GCTCGGATCCATGGAAAGAATAGTGATTGC-3 ) and H5-XhoI-R (5 -GTACTCTC GAGCTAGATGCAAATTCTGCAC-3 ). 2.2.3. VP24-HA/pcDNA Three steps were performed for preparing VP24-HA/pcDNA, including subcloning of VP243 gene to pNEB193 vector (New England Biolabs, Beverly, MA, USA) with KpnI and PmeI sites, inserting HA gene into VP243/pNEB193 with restriction enzymes DraIII and BsaAI (New England Biolabs) and subcloning VP24HA fusion gene into pcDNA3.1 (Invitrogen). Primers used for VP24-HA subcloning were as the following: H5-DraIII-SNF (5 -GTGCTCACTCAGTGTAATGGAAAGAATAGTGATTGC-3 ) and H5BsaAI-R (5 -GCATGCACGTAG ATGCAAATTCTGCACTG-3 ). 2.3. Protein expression from DNA in Vero cells Vero cells were transfected with the constructed plasmids by TransIT® -LT1 transfection reagent (Mirus, Madison, WI, USA) following the manufacture’s instruction. After incubation for 48 h, cells were fixed with acetone at room temperature for 10 min and subjected to immunofluorescent antibody assay as described previously (Chang et al., 2003) to detect the transient protein expression with antibody R63 (monoclonal antibody against VP2,

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American Type Culture Collection, Manassas, VA, USA) or anti-HA chicken serum (provided by Dr. Janice C. Pedersen, National Veterinary Service Laboratory, USDA, Ames, IA) as the primary antibody. Goat anti-mouse or anti-chicken IgG conjugated with fluorescein isothiocyanate (FITC) was used as the secondary antibody (KPL, Gaithersburg, MD, USA). The immunofluorescence was examined in an epifluorescence microscope (Optiphot-2, Nikon, Tokyo, Japan). 2.4. Location of expressed proteins from DNA in Vero cells Vero cells were grown on cover slips in 6-wells and transfection was followed the next day. Forty-eight hours later, cells in 6-wells were stained by anti-HA or R63 antibodies for 30 min directly without acetone fixation. After washing three times with PBS, secondary antibodies were applied for 30 min. After another three-time PBS wash, cells were fixed with acetone. Then immunofluorescent antibody staining for the second protein, either VP2 or HA was followed. For example, the first staining for HA by anti-HA chicken serum without acetone fixation and using FITC-labeled goat antichicken antibody as the secondary antibody, followed by the second staining for VP2 by R63 after acetone fixation and using Texas redlabeled goat anti-mouse antibody (KPL) as the secondary antibody. 2.5. Western blotting for expressed proteins from DNA in DF-1 cells DF-1 cells (ATCC) in 6-wells were transfected with TransIT® -LT1 (Mirus). Dilution of transfection reagent and mix with plasmids were done as described in the manufacturer’s manual. After 48 h of incubation, cell extract was prepared in CytoBusterTM (EMD, Novagen® , Gibbstown, NJ, USA) supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and subjected to SDS-PAGE and Western blotting. The blots were stained with R63 or anti-HA chicken serum first and then followed with goat anti-mouse or anti-chicken secondary antibody conjugated with horseradish peroxidase (KPL). Final incubation with 3,3 ,5,5 -tetramethylbenzidine (TMB) membrane peroxidase substrate system (KPL) was done according to the manufacturer’s instruction. 2.6. DNA vaccination Four groups of one-day-old SPF chickens were inoculated in the thigh muscle with 500 ␮g of the plasmids pcDNA vector (without any gene insert) (n = 10), VP243/pcDNA (n = 5), HA/pcDNA (n = 5), or VP24-HA/pcDNA (n = 10), respectively, as described previously (Chang et al., 2003; Chen et al., 2011). Same dose boosters were performed when chickens were 7, 14 and 28 days of age, respectively. 2.7. ELISA antibody titer to IBDV Blood was withdrawn from the wing vein of chickens (n = 30) on day 14, 21, 28, 35 and 41 after the first inoculation with DNA. Serum ELISA antibody titers were determined by using commercially available ELISA kit for IBD (IDEXX, Portland, ME, USA) following the manufacturer’s instruction. The ELISA plates were read at the wavelength of 595 nm in an ELISA reader (VMaxTM , Molecular devices, Sunnyvale, CA, USA). 2.8. Virus neutralizing antibody titer to IBDV Chicken sera were obtained as described above. The virus neutralizing titer was determined by using a protocol reported previously from our laboratory (Chen et al., 2011). Briefly, the serum was 4-fold serial diluted and transferred to the well in a 96-well

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Fig. 1. The translated VP24-HA protein. HA protein was fused with the remaining VP3 sequences at both ends with 32 amino acids at N-terminus and 106 amino acids at C-terminus (A). Vero cells were transfected with VP243/pcDNA, HA/pcDNA or VP24-HA/pcDNA respectively and subjected to immunofluorescent antibody assay. After 48 h, VP2 protein was detected in cells transfected with VP243/pcDNA or VP24-HA/pcDNA (B) and HA protein was detected in cells transfected with HA/pcDNA or VP24-HA/pcDNA (C). There was no detectable fluorescence in pcDNA-transfected cells (data not shown).

plate containing confluent chicken embryo fibroblasts. One thousand plaque forming unit of IBDV strain PBG98 (Dr. Rosenberger, University of Delaware, DE, USA) was added to each well and incubated for 5 days at 37 ◦ C. Titers were read by staining the cells

with 1% crystal violet. The virus neutralizing antibody titer was decided as the highest serum dilution in which chicken embryo fibroblasts were completely intact and the titer was presented as log4 values.

Fig. 2. Location of expressed VP2 protein from DNA in Vero cells. Vero cells were transfected with VP243/pcDNA, HA/pcDNA or VP24-HA/pcDNA respectively and subjected to immunofluorescent antibody double staining after 48 h of transfection. Staining for VP2 with acetone fixation (A) or without acetone fixation (B) revealed the cytoplasmic expression of VP2.

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Fig. 3. Location of expressed HA protein from DNA in Vero cells. Vero cells were transfected with VP243/pcDNA, HA/pcDNA or VP24-HA/pcDNA respectively and subjected to immunofluorescence antibody double staining after 48 h of transfection. Staining for HA with acetone fixation (A) or without acetone fixation (B) revealed the plasma membrane expression of HA.

2.9. Antibody to AIV HA by hemagglutinin inhibition assay The serum was 2-fold serial diluted by adding equal volume of chicken serum to H5 antigen (with final antigen concentration as 4 HA unit/well) in an U-bottomed 96-well microtiter plate. After 30 min of incubation, 0.5% chicken erythrocyte suspension was added to each well. The hemagglutination inhibition titer was read after 30 min of incubation as the highest dilution with complete hemagglutination inhibition. Hemagglutinin antigen (H5 antigen) was provided by Dr. Janice C. Pedersen, National Veterinary Service Laboratory, USDA, Ames, IA. 2.10. Statistical analysis Antibody titers were analyzed by one-way ANOVA followed with Tukey’s test for post hoc analysis using SPSS 19 (IBM SPSS, Chicago, IL, USA). Different number superscript indicates statistical difference among different groups at each time point with statistical significance set at the level of p 0.05). b c

decrease the vaccine efficacy (Fattom et al., 1999). However, the immune responses generated by multi/bi-valent DNA vaccines are not always decreased when compared to vaccination with individual antigen (Riemenschneider et al., 2003; Shen et al., 2009). The negative effect seems to be dependent on the antigen itself and independent on whether the injection sites are the same location or not when each antigen is encoded in a single plasmid (Wang et al., 2007). There are multiple methods to improve the efficacy of DNA vaccines, including applying adjuvants such as cytokines or signaling molecules; enhancing DNA delivery by cationic lipid or electroporation after muscle injection; or combining with other vaccine formats such as subunit or viral vector vaccines as the prime-boost regimen (Saade and Petrovsky, 2012). These methods have been proven to induce broader and higher immune responses for DNA vaccination (Abdulhaqq and Weiner, 2008; Wang et al., 2008; Huber et al., 2009; Gao et al., 2013) and could be utilized to enhance the efficacy of the VP24-HA DNA vaccine. DNA vaccines encoding different HAs have been applied to produce reference antisera to determine the HA subtype of newly isolated influenza viruses (Lee et al., 2003, 2006). There are several advantages to employ DNA vaccines for this purpose when compared to the traditional killed vaccines. The specificity of the antiserum is increased since it is HA-exclusive without recognizing other viral proteins; the safety and humanity during the vaccine production are also increased since there are no live viruses nor chicken embryos were involved, respectively. Therefore, VP24-HA DNA generated in the present study could be used as a platform to produce reference antisera for specific HA subtype in combination with diagnostic antisera for different IBDV strains. The production of polyclonal antibody cocktail by VP24-HA DNA vaccine could reduce the number of animals used in the antiserum production and decrease the vaccination frequency for each chicken; thus lower the stress by injection and the cost of vaccine production. In conclusion, a bivalent DNA vaccine (VP24-HA) for IBD and AI as a single plasmid construct was produced and elicited specific antibody responses to both IBDV VP2 and AIV HA proteins.

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