Improving the potency of DNA vaccine against Chicken Anemia Virus (CAV) by fusing VP1 protein of CAV to Marek's Disease Virus (MDV) Type-1 VP22 protein
© Moeini et al; licensee BioMed Central Ltd. 2011
Received: 22 September 2010
Accepted: 14 March 2011
Published: 14 March 2011
Studies have shown that the VP22 gene of Marek's Disease Virus type-1 (MDV-1) has the property of movement between cells from the original cell of expression into the neighboring cells. The ability to facilitate the spreading of the linked proteins was used to improve the potency of the constructed DNA vaccines against chicken anemia virus (CAV).
The VP1 and VP2 genes of CAV isolate SMSC-1 were amplified and inserted into eukaryotic co-expression vector, pBudCE4.1 to construct pBudVP2-VP1. We also constructed pBudVP2-VP1/VP22 encoding CAV VP2 and the VP22 of MDV-1 linked to the CAV VP1. In vitro expression of the genes was confirmed by using RT-PCR, Western blot and indirect immunofluorescence. The vaccines were then tested in 2-week-old SPF chickens which were inoculated with the DNA plasmid constructs by the intramuscular route. After in vivo expression studies, immune responses of the immunized chickens were evaluated pre- and post-immunization.
Chickens vaccinated with pBudVP2-VP1/VP22 exhibited a significant increase in antibody titers to CAV and also proliferation induction of splenocytes in comparison to the chickens vaccinated with pBudVP2-VP1. Furthermore, the pBudVP2-VP1/VP22-vaccinated group showed higher level of the Th1 cytokines IL-2 and IFN-γ.
This study showed that MDV-1 VP22 gene is capable of enhancing the potency of DNA vaccine against CAV when fused with the CAV VP1 gene.
Chicken anemia virus (CAV) is a small non-enveloped virus of genus Gyrovirus from Circoviridae family, which causes anemia in young susceptible chickens and subclinical infections in older chickens [1–3]. Commercially available vaccines against CAV infection which are based on non-attenuated virulent CAV propagated in chicken embryos  or attenuated live vaccine  cannot be used in chickens in lay and within 21 days of slaughter. Furthermore, live attenuated vaccine may cause clinical disease if not attenuated sufficiently and sometimes spreading of the modified viruses to young chickens may cause the disease. In a recent development, plasmid DNA-based vaccines have emerged as one of the more promising applications of non-viral gene therapy. One such subunit vaccine against infectious chicken anemia was developed by using recombinant baculovirus as a vector for the expression of the CAV proteins . They found that co-synthesis of VP1 and VP2 is required for the induction of neutralizing antibodies.
A limitation in the use of DNA vaccine is its inability to spread in vivo. Thus, a strategy to facilitate the spread of the antigen may significantly enhance the potency of the vaccine. This can be demonstrated by the fusion of the VP22 gene of Marek's disease virus type-1 (MDV-1) to the target gene encoding the antigenic protein. It has been shown that the VP22 protein of Marek's disease virus type-1 (MDV-1) possesses the ability to improve DNA vaccine potency by facilitating intercellular spreading of the linked protein . The MDV-1 VP22 is a phosphorylated protein with a DNA-binding activity located at the N terminus of the protein that displays cell trafficking properties . In fact, VP22 is a tegument protein involved in intercellular transport and movement between cells from the original cell of expression into the neighboring cells [9, 10].
We therefore investigated the use of MDV-1 VP22 linked to CAV VP1 gene in a recombinant DNA plasmid, namely pBudVP2-VP1/VP22 which allows the CAV VP1 fused to MDV-1 VP22 to be simultaneously expressed with the CAV VP2.
Material and methods
Expression vector and viral genes
The pBudCE4.1 co-expression vector (Invitrogen, USA) was used to construct the DNA vaccines. The vector contains the human cytomegalovirus (CMV) immediate-early promoter and the human elongation factor 1α-subunit (EF-1α) promoter for high-level, constitutive, independent expression of two recombinant proteins. The VP22 gene of MDV-1 strain CVI988/Rispens (Accession No: AY311498) and recombinant pCRVP1-VP2 cloning vector containing VP1 and VP2 genes of CAV isolate SMSC-1 (Accession No. AF285882) were obtained from the Faculty of Veterinary Medicine, University Putra Malaysia (UPM).
Construction of DNA vaccines
Primers for (A): PCR amplification and RT-PCR analysis of the viral genes; (B): RT-PCR analysis of chicken cytokines
Sequence (5' to 3')
Expected product size (base pair)
In vitro transcription and translation of the constructs
In vitro expression of the recombinant plasmids was carried out in chicken MDCC-MSB1 cell line. The MSB1 cells were transfected with the plasmids using Lipofectamine™2000 reagent (Invitrogen, USA) according to the manufacturer's recommendation with some modifications. After 48 h of transfection, expression of the genes was evaluated by RT-PCR, Western blot and indirect immunofluorescence.
Total RNA was extracted from the transfected MSB1 cells using Trizol and then treated with DNase I to remove DNA contamination. To confirm DNA removal from the samples, they were tested by PCR using specific primers for the inserted genes. The RNA from normal MSB1 cells was used as negative control. RT-PCR reactions were performed in 25 μl volume in a Gradient Thermal Cycler (BioRad, USA). Following a reverse transcription step at 45°C for 45 min, the samples were denatured at 94°C for 3 min and amplification was carried out in 35 cycles 94°C for 40 s, 55°C for 1 min and 68°C for 2 min; and a final elongation step at 68°C for 10 min. RT-PCR products were then analyzed by electrophoresis on a 1% agarose gel.
In vitro translation of the genes was studied by Western blotting using chicken anti-CAV serum and mouse anti-His monoclonal antibody (Promega, USA) as primary antibodies. Expression of the genes was also evaluated by immunofluorescence test according to Richter and Wick  using chicken anti-CAV serum as primary antibody and fluorescein isothiocyanate (FITC)-conjugated anti-chicken IgY (Promega, USA) as secondary.
Evaluation of DNA vaccines in SPF chickens
Specific pathogen free (SPF) chicken eggs taken from Malaysian Vaccines & Pharmaceuticals Sdn Bhd, Malaysia were hatched and maintained under specific-pathogen free condition with free access to feed and water. Two-week-old SPF chickens (n = 10) were vaccinated with 150 μg of pBudVP2-VP1, pBudVP2-VP1/VP22, parental plasmid or 1x PBS via intramuscularly injection. Chickens were boosted two times with the same regimen as the first injection, at 2 weeks intervals. Blood was withdrawn from the wing vein of the chickens before and 10 days after the last injection and collected sera were kept in -20°C for further analysis. All procedures were conducted with the protocols approved by Animal Care and Use Committee of the Faculty of Veterinary Medicine, University Putra Malaysia (UPM).
In vivo expression analysis
Transcriptional expression of VP1, VP2 and MDV-1 VP22 genes was determined in the vaccinated groups by RT-PCR using the specific primers. Ten days after the last vaccination, the chickens were sacrificed and their skeletal muscle (at the site of injection) was harvested; homogenized and their total RNA was extracted. After treatment with DNase I, DNA removal was confirmed by PCR and then DNA-free samples were subjected to RT-PCR as described above.
Western blotting was also used to show in vivo expression of the proteins, where a section of the harvested muscles at the site of injection was homogenized and then treated with RIPA (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 5 μg/ml Aprotinin, 5 μg/ml Leupeptin, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS) containing 1 mM protease inhibitor PMSF (phenylmethylsulfonyl fluoride). The extracted proteins were mixed with 2× sample buffer (1.25 ml Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.02% (w/v) bromophenol blue, 10% 2-mercaptoethanol) followed by 5-10 min boiling at 95°C. The protein mixtures were subjected to Western blot analysis using primary chicken anti-CAV serum and secondary goat anti-chicken IgY conjugated to alkaline phosphatase.
Antibody titer to CAV
Serum antibody titers against CAV were determined, pre- and post-vaccination, using IDEXX ELISA kit (IDEXX, Portland, ME, USA). The kit uses an anti-CAV monoclonal antibody in a blocking format ELISA for the detection of antibodies to CAV in chicken serum. Briefly, Samples were diluted at 1:100 dilutions and then added into microtiter wells coated with CAV antigen followed by 60 min incubation at RT. After 3 times washing with washing buffer, anti-CAV monoclonal antibody conjugated with horseradish peroxidase was added and then incubated at RT for 30 min. Following the incubation period, the unreacted anti-CAV conjugate was removed by 3 times washing and TMB substrate solution (100 μl) was then added into the wells followed by 15 min incubation at RT. The reaction was stopped by adding 100 μl of the stop solution and the absorbance value was measured at 650 nm. All sample ODs were normalized to the negative control (S/N = sample OD650/negative control OD650). The antibody titers were calculated based as the following calculation, log10 titer = (S/N-2.72)/-0.64. ELISA titers higher than 1000 were considered as positive.
Neutralizing antibody titer
To investigate whether the produced antibodies have virus neutralization activity, the collected sera were analyzed for neutralization antibody titer against CAV infection in chicken MSB1 cells. The assay was carried out by the measurement of cell proliferation and viability using WST-1 reagent (Roche, Germany) as described by Moeini et al.  and Lehtoranta et al. .
Cytokines production assay
The serum level of Th1 cytokines, interleukin 2 (IL-2) and interferon γ (IFN-γ) was determined by ELISA kits (Cusabio Biotech, USA). The procedure was following the procedure recommended by the manufacturer. For the reason that no commercial ELISA kits were available for the detection of chicken IL-12, IL-4 and IL-6 which have important roles in the differentiation of CD4+ T cells into Th1 or Th2 cells, transcriptional expression of these cytokines were studied in the spleen of the immunized chickens by RT-PCR using the specific primers listed in Table 1.
Splenocyte proliferation assay
The proliferation response of the chicken splenocytes to the CAV VP1 protein was studied after immunization. The VP1 protein of CAV was prepared and purified from a recombinant E. coli BL21 (DE3) expressing the VP1 and VP2 genes of CAV, synchronously (data not shown). At 10 days of immunization, single cell suspensions were generated from the harvested spleens in PBS-EDTA solution (1X PBS, pH 7.4, 2 mg/ml EDTA) supplemented with 2% penicillin/streptomycin. Red blood cells were removed by washing the cell suspensions with red blood cell lysis solution containing 0.84% NH4CL, 0.1% NaHCO3 and 1.8 ml of 5% EDTA. The splenocytes were pelleted at 1000 rpm for 10 min and then resuspended in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Viable splenocytes were added to 96-well plates in 0.1 ml at 2 × 104 cells/well and incubated in triplicate with CAV antigen (purified VP1 protein), mitogen phytohemagglutinin (PHA, Sigma) as positive control, or medium alone as negative control followed by 3 days incubation at 37°C in an atmosphere of 5% CO2. The proliferation response was evaluated by BrdU cell proliferation assay kit (Exalpha Biologicals, USA). Data was reported as stimulation indices (SI), which was the mean of experimental wells/mean of antigen free wells (negative control).
The data was analyzed by t-test and statistical significance was set at P < 0.05. The results were expressed as means ± standard error of mean. All the analysis was carried out using GraphPad Prism 5 and Windows Microsoft Excel 2007.
Construction of plasmids
In vitro characterization of the constructs
In vitro translation of the DNA vaccines was confirmed by Western blot analysis of the transfected MSB1 cells (Figure 2C). The expression of VP1 (~56 KDa) and VP1/VP22 (~82 KDa) proteins were detected in the cells transfected with pBudVP2-VP1 or pBudVP2-VP1/VP22, respectively using both anti-His and anti-CAV primary antibodies. The VP2 protein (~27 KDa) was detected in the both transfected cells using anti-His monoclonal antibody (Figure 2C, lanes 4 and 5).
In vivo evaluation of the DNA vaccines
Serum antibody titer and VN titer to CAV in the vaccinated chickens
Neutralization activities of the serum antibodies in the vaccinated groups.
Virus neutralization test
No. of samples
No. of positive
VN antibody titer
Cell-mediated responses after DNA vaccination
According to the previous studies [6, 14, 15] and the finding of our group , co-synthesis of the VP1 and VP2 proteins is required to produce the essential neutralizing form of VP1 resulting in the efficient induction of antibody response to CAV, whereas separate expression of VP1 does not. In fact, VP2 may play a role as a scaffold protein during virion assembly, but is removed in the next step [14, 15]. Therefore, pBudVP2-VP1 plasmid co-expressing the VP1 and VP2 genes of CAV was developed as a DNA vaccine against CAV. To explore whether the linkage of MDV-1 VP22, as a tegument protein with a property of intercellular transport, to CAV VP1 protein lead to enhance the potency of the DNA vaccine, the pBudVP2-VP1/VP22 plasmid encoding the fusion protein VP1/VP22 and VP2 protein was also constructed.
In vitro analysis of the constructs revealed the successful transcriptional and translational expression of the genes in MSB1 cells. In agreement with the immunoblotting results, immunofluorescence staining of the transfected cells showed the reaction of the VP1 and the fusion protein, VP1/VP22 with anti-CAV polyclonal antibody resulting in bright cytoplasmic fluorescence. These observations indicated that the VP1 protein has proper secondary folding to induce immune system to CAV. The plasmid constructs were then tested in SPF chickens. In vivo expression analysis matched up well with in vitro findings, confirmed the transcriptional and translation expression of the genes in the immunized chickens.
To investigate the ability of the linkage MDV-1 VP22 to CAV VP1 to enhance the potency of the DNA vaccine, immune responses were evaluated in the chickens vaccinated with the plasmid encoding VP1/VP22 and the results were compared with those from the chickens vaccinated with pBudVP2-VP1. Antibody titer to CAV was significantly higher (P < 0.05) in the group vaccinated with pBudVP2-VP1/VP22 in comparison with the group vaccinated with pBudVP1-VP2. Virus neutralization (VN) test indicated positive neutralization activity of the antibodies in the both vaccinated groups with moderate protective titer ranging from 1:256 to 1:512. However, the number of samples with VN titer of 1:512 was obviously higher in the pBudVP2-VP1/VP22-vaccinated group. Similarly such presence of neutralizing antibodies against CAV has been shown to provide protection against CAV in young chickens [16–21]. Nevertheless, in order to confirm this issue, it is suggested that breeder flocks producing day-old chicks with maternal antibodies be vaccinated for virus challenge.
Cytokine assay by ELISA showed high level of IL-2 and IFN-γ in the both vaccinated groups post-immunization, although their levels were higher in the pBudVP2-VP1/VP22 group. Furthermore, the expression of IL-12 which triggers the differentiation of Th1  was detected by RT-PCR in the spleen of the both groups, whereas IL-4 and IL-6 which are involved in differentiation of Th2 [22, 23] were not detected in any samples. These observations indicate a pattern of cytokine production that most closely promotes the Th1 T-helper cell responses which are known to be involved in cellular-mediated immunity [22, 24].
To further investigation whether the linkage of MDV-1 VP22 lead to enhance antigen-specific immune responses, we compared antigen-stimulated proliferative response in the splenocytes of the vaccinated groups. Compared to the pBudVP2-VP1-vaccinated group, the pBudVP2-VP1/VP22-vaccinated group exhibited a significant increase in the proliferation induction of splenocytes indicating higher VP1-specific immune responses in the presence of the MDV-1 VP22 protein.
The results of the present study showed that the fusion of the MDV-1 VP22 to the target gene, CAV VP1, could significantly increase CAV-specific immune responses. This view is supported by Hung et al. , who showed the capacity of the VP22 protein of Marek's disease virus type-1 (MDV-1) to improve DNA vaccine potency by facilitating intercellular spreading of the linked protein.
Hassan Moeini is supported by the Graduate Research Fellowship, University Putra Malaysia. We are thankful to Ms. Razieh Monjezi for her assistance in animal challenge.
- Von Bülow V: Avian infectious anemia and related syndromes caused by chicken anemia virus. Critical Reviews in Poultry Biology 1991, 3: 1-17.Google Scholar
- McNulty MS: Chicken anemia agent: a review. Avian Pathol 1991, 20: 187-203. 10.1080/03079459108418756View ArticlePubMedGoogle Scholar
- Schat KA: Chicken anemia virus. Curr Top Microbiol Immunol 2009, 331: 151-183. full_textPubMedGoogle Scholar
- Vielitz E, Landgraf H: Anaemia - dermatitis of broilers: field observations on its occurrence, transmission and prevention. Avian Pathol 1988, 17: 113-120. 10.1080/03079458808436432View ArticlePubMedGoogle Scholar
- Steenhuisen W, Jagt HJM, Schrier CC: The use of a live attenuated CAV vaccine in breeder flocks in the Netherlands. Proceedings of the International Symposium on Infectious Bursal Disease and Infectious Chicken Anaemia; Rauischholzhausen, Germany: University of Giesen 1994, 482-497.Google Scholar
- Koch G, van Roozelaar DJ, Verschueren CAJ, van der Eb AJ, Noteborn MHM: Immunogenic and protective properties of chicken anemia virus protein expressed by baculovirus. Vaccine 1995, 13: 763-770. 10.1016/0264-410X(94)00034-KView ArticlePubMedGoogle Scholar
- Hung CF, He L, Juang J, Lin TJ, Ling M, Wu TC: Improving DNA vaccine potency by linking Marek's Disease Virus Type-1 VP22 to an antigen. J Virol 2002, 76: 2676-2682. 10.1128/JVI.76.6.2676-2682.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Dorange F, El Mehdaoui S, Pichon C, Coursaget P, Vautherot J-F: Marek's disease virus (MDV) homologues of herpes simplex virus type 1 UL49 (VP22) and UL48 (VP16) genes: high-level expression and characterization of MDV-1 VP22 and VP16. J Gen Virol 2000, 81: 2219-2230.View ArticlePubMedGoogle Scholar
- Elliott G, O'Hare P: Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell 1997, 88: 223-233. 10.1016/S0092-8674(00)81843-7View ArticlePubMedGoogle Scholar
- Wills KN, Atencio IA, Avanzini JB, Neuteboom S, Phelan A, Philopena J, Sutjipto S, Vaillancourt MT, Wen SF, Ralston RO, Johnson DE: Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus. J Virol 2001, 75: 8733-8741. 10.1128/JVI.75.18.8733-8741.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Richter E, Wick G: Fluoro-immuno-cytoadherence (FICA): A new method for the identification and enumeration of antigen-binding cells. Z immunitatsforsch Immunobiol 1977, 152: 351-362.PubMedGoogle Scholar
- Moeini H, Omar A, Rahim R, Yusoff K: Development of a DNA vaccine against chicken anemia virus by using a bicistronic vector expressing VP1 and VP2 proteins of CAV. Comp Immunol Microbiol Infect Dis, in press.Google Scholar
- Lehtoranta L, Villberg A, Santanen R, Ziegler T: A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses. J Virol Methods 2009, 159: 271-276. 10.1016/j.jviromet.2009.04.015View ArticlePubMedGoogle Scholar
- Lacorte C, Lohuis H, Goldbach R, Prins M: Assessing the expression of chicken anemia virus proteins in plants. Virus Res 2007, 129: 80-86. 10.1016/j.virusres.2007.06.020View ArticlePubMedGoogle Scholar
- Noteborn MH, Verschueren CA, Koch G, Van der Eb AJ: Simultaneous expression of recombinant baculovirus-encoded chicken anemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope. J Gen Virol 1998, 79: 3073-3077.View ArticlePubMedGoogle Scholar
- Otaki Y, Saito K, Tajima M, Momura Y: Persistence of maternal antibody to chicken anemia agent and its effect on the susceptibility of young chicken. Avian Pathology 1992, 21: 147-151. 10.1080/03079459208418828View ArticlePubMedGoogle Scholar
- Malo A, Weingarten M: Determination of the minimum protective neutralizing antibody titer to CAV in adult chickens. Intervet VSD Newslett 1995, 11: 1-5.Google Scholar
- Markowski-Grimsrud CJ, Schat KA: Infection with chicken anaemia virus impairs the generation of pathogen-specific cytotoxic T lymphocytes. Immunol 2003, 109: 283-294. 10.1046/j.1365-2567.2003.01643.xView ArticleGoogle Scholar
- Rosenberger JK, Cloud SS: The isolation and characterization of chicken anemia agent (CAA) from broilers in the United States. Avian Dis 1989, 33: 707-713. 10.2307/1591148View ArticlePubMedGoogle Scholar
- Saif YM: Diseases of poultry. 11th edition. Ames, IA USA: Iowa State University Press; 2003.Google Scholar
- Yuasa N, Noguchi T, Furuta K, Yoshida I: Maternal antibody and its effect on the susceptibility of chicks to chicken anemia agent. Avian Dis 1980, 24: 197-201. 10.2307/1589779View ArticleGoogle Scholar
- Goldsby RA, Kindt TJ, Osborne BA, Kuby J: Immunology. 5th edition. New York, NY: W.H. Freeman; 2003.Google Scholar
- Rincón M, Anguita J, Nakamura T, Fikrig E, Flavell RA: Interleukin (IL)-6 directs the differentiation of IL-4-producing CD4 + T cells. J Exp Med 1997, 185: 461-469.PubMed CentralView ArticlePubMedGoogle Scholar
- Wigley P, Kaiser P: Avian cytokines in health and disease. Brazilian Journal of Poultry Science 2003, 5: 1-14.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.