Immune Responses in Pigs Induced by Recombinant Canine Adenovirus 2 Expressing M Protein of Porcine Reproductive and Respiratory Syndrome Virus Zhou Jing-Xiang1 Wang Xin-Tong4 Xue Jiang-Dong5 Yu Tao1 Liu Ye3 Zhang Jia-Bao2,* Hu Rong-Liang3,† 1 College of Animal Science and Technology, JiLin Agriculture University, Changchun, P.R. China; 2 Centre of experimental Animal, JiLin University, Changchun, P.R. China; 3 Laboratory of Epidemiology, Veterinary Research Institute, Academy of Military Medical Science, Changchun, P.R. China; 4 Undergraduate with Entrance in 2008 into Bethune Medical School of Jilin University 5 College of Animal Science and Technology, Inner Mongolia University for Nationalities, Tongliao, P.R. China KEY WORDS: : Porcine reproductive virus CAV-2-M was obtained by transfect- and respiratory syndrome virus, M ing the recombinant CAV-2-M genome into protein, Canine adenovirus vector, Pigs, MDCK cells together with Lipofectamine™ Recombinant vaccine, Immunization 2000. Immunization trials in piglets with ABSTRACT the recombinant CAV-2-M showed that In order to develop a new type vaccine CAV-2-M could stimulate a specific immune for porcine reproductive and respiratory response to PRRSV. Immune response to syndrome (PRRS) prevention using canine the MP and PRRS virus was confirmed by adenovirus 2 (CAV-2) as vector, the expres- ELISA, western blot analysis, neutralization sion cassette of M protein (MP) derived test and lymphocyte proliferation assays. from plasmid pMD18T-M was cloned into These results indicated that CAV-2 may the CAV-2 genome in which E3 region had serve as a vector for development of PRRSV been partly deleted, and the recombinant vaccine in pigs and the CAV-2-M might be a 332 Vol. 9, No. 4, 2011 • Intern J Appl Res Vet Med. candidate vaccine to be tested for preventing be modified to serve as the vector for genetic PRRSV infection. vaccine development against canine and InTRoduCTIon porcine diseases. Porcine reproductive and respiratory In order to explore the possibility of this syndrome (PRRS), characterized by severe recombinant virus as a vaccine against reproductive problems in sows and respira- PRRSV, we developed a live recombinant tory distress in piglets and growing pigs, is virus vaccine (CAV-2-M) expressing M pro- a kind of communicable disease caused by tein (MP) of PRRSV using CAV-2 as vector, PRRS virus (PRRSV) infection 1-3 and is investigated MP expression, and examined now one of the most important diseases in its immunological characteristics in piglets. the swine breeding industry 4,5. MaterialS And METhodS Currently, commercially conventional Vaccines (inactivated and attenuated) vaccines can- The commercial PRRSV KV vaccine was not provide satisfactory immunoprotection serotype Ch-1R strain (Harbin Weike Bio- against PRRSV 6-8 because of the poor technology Development Company, Harbin, immune effect 9,10 or the intrinsic risk of re- China), batch No. 0702001, approval no. version to virulence under natural condition (2007) 080011063. 11,12 or residual pathogenicity . Thus, more Virus, plasmid and cells effective, safer vaccines are urgently needed. PRRSV-JL (a wild PRRSV strain) was PRRSV is an enveloped, positive- isolated from clinical specimens in our strand RNA virus belonging to the family laboratory. Marc-145 and MDCK cells were of Arteriviridae and the genus of Okavirus supplied by Epidemiological Laboratory, 13 . The genome of PRRSV is approximately Chinese Academy of Military Medical Sci- 15 kb in length and contains 7 open reading ences and cultured in DMEM (Gibco, Inc., 14 frames (ORFs) . According to the sequence Rockville, Maryland, USA) supplemented data, the ORF1 encodes functional proteins with 10% new calf serum (NCS, Hyclone, involved in virus replication. The ORFs 2-7 USA), 1% penicillin (100 U/ml), and are postulated to encode structural proteins, streptomycin (100 mg/ml). Plasmid pPolyII- in which M protein (MP) is one of the CAV-2 containing the whole genome of major structural proteins. MP as a candidate canine adenovirus 2 (CAV-2) was con- antigen has been mentioned in many reports structed by Zhang et al.22. It is an infectious regarding genetic vaccine development of plasmid that can produce canine adenovirus 6,15-18 PRRS . particles after being transfected into MDCK Recombinant adenoviruses are one of cells 22. Plasmid pMD18T-M containing the the preferred viral vectors used in vaccine expressing cassette of M protein (MP) was production. Canine adenovirus 2 (CAV-2) constructed in Epidemiological Labora- has biological advantages, similar to human tory, Chinese Academy of Military Medical adenovirus, including (1) genetic stability, Sciences23. The restriction enzymes, reverse a wide range host, good replication in pigs transcriptases and T4 DNA ligases were [19-21]; (2) a good recombinant virus vec- purchased from NEB, Beijing, China. tor. The E3 region, unnecessary for replica- Plasmid construction tion, is partially deleted to provide space The 4.8 kb KpnΙ fragment containing the E3 for the exogenous gene insertion. There region from pPolyII-CAV-2 was first cloned will not be pathogenicity after infection, or into pVAX1 (Invitrogen, Carlsbad, Califor- potential harm to the external environment nia, USA), forming pVAX-E3. The EcoRV/ [18,19]; (3) the low-level replication in hu- BamHI double-digested fragment containing man cell lines without packaging new virus MP cDNA from plasmid pMD18T-M was particles or recombination with wild type cloned into pEGFP-C1 (Clontech Laborato- human adenovirus. Therefore, CAV-2 may Intern J Appl Res Vet Med • Vol. 9, No. 4, 2011. 333 ries, Inc., San Jose, California, USA) at the ing 10 µg/ml RNase A, and placed at 37 oC restriction site of NheΙ and BamHΙ, forming for 20 min. Then they were digested with pEM-C1. The AseΙ/MLuΙ fragment of pEM- restriction enzymes and separated on 1% C1 containing the MP cDNA expression agarose gel electrophoresis. The recombi- cassette was filled in and cloned into the nant virus genome was digested with differ- SspΙ/SspΙ site and Klenow/dNTPs blunted ent restriction enzymes, meanwhile pPolyII- pVAX-E3, forming pVAX-∆E3-M. The frag- CAV-2 served as the negative control. ment of pVAX-∆E3-M double digested with Titration of the recombinant virus SalΙ and NruΙ, containing the deleted E3 The TCID50 of the recombinant canine ad- into which the MP expression cassette was enovirus was assayed on 96-well cell culture inserted, was cloned into pPolyII-CAV-2 by plate (Nalge Nunc International, Denmark) replacing the fragment between the SalΙ and according to the protocol described else- NruΙ sites, forming pPolyII-CAV-2-∆E3-M. where25. This recombinant genome was prepared for MP expression from the recombinant transfection. The cloning steps and plasmid virus preparation were performed as described elsewhere24, the constructing route was The recombinant virus infected cell lysates presented in Fig.1. were separated on 10% SDS-PAGE, the proteins were blotted onto a nitrocellulose Production of the recombinant virus membrane (Pall Corporation) probed with Recombinant pPolyII-CAV-2-M was piglet positive serum against PRRSV and transfected into MDCK cells using Lipo- detected using horseradish peroxidase- fectamine 2000TM (Invitrogen) according to labeled goat anti-pig IgG antibody (Sigma) the protocol described. Briefly, 4μg puri- and chromophore DAB as described else- fied recombinant genome and 25 μl Lipo- where26. fectamine 2000TM were dispersed in 300 Immunization of pigs with the μl DMEM respectively, mixed up at room temperature for 20 min, supplemented with recombinant virus 1.4 ml DMEM and then overlaid onto the Eighteen piglets, which have not been 80% confluent MDCK cells. After 12 hours infected with PRRSV (demonstrated by RT- of incubation, the medium was replaced by PCR and indirect ELISA), were purchased a complete DMEM containing 10% NCS. from the Changchun Animal Breeding Transfected cells were cultured at 37 oC and Center for Medicine, Changchun, China, propagated for days until cytopathic effect and randomly divided into three groups, six (CPE) appeared. piglets for each group. Groups 1 and 2 were Identification of recombinant virus via intramuscularly (in the neck) injected only restriction digestion once with 1.5 ml recombinant CAV-2-M (107.8/0.1 ml TCID50) and PRRSV KV Transfected cells were rinsed 2 times by vaccine, respectively; group 3 was injected PBS and suspended in 800 µl of fresh lysis with CAV-2 (negative control) and PBS solution (0.6% SDS, 10 mmol/l EDTA, 100 (blank control) respectively, three piglets for µg/ml Proteinase K) at 37 oC for 1h; supple- each injection. Blood was collected before mented with 200 µl of 5 mol/l NaCl, placed inoculation and at an interval of 2 weeks af- on ice for 1h, and then precipitated by ter immunization (p.i.). Sera were separated centrifugation at 4 oC with 12,000 rpm for for detection of specific antibody against 20 min. The supernatant was extracted once PRRSV. The lymphocytes were separated by using equivalent phenol and chloroform from the spleens of mice for detection of respectively and precipitated in anhydrous specific cell-mediated immune responses. alcohol. The sediments were rinsed by 70% ethanol, dried at room temperature for 5 Serum antibody against PRRSV via min, dissolved in aqueous solution contain- ELISA and western blot analysis 334 Vol. 9, No. 4, 2011 • Intern J Appl Res Vet Med. Figure 1. Routes of construct CAV-2 expressing M protein Lymphocyte proliferation of PRRSV. assay Lymphocyte proliferation assay was performed using peripheral blood mononuclear cells (PBMCs) of immunized piglets. Swine PBMCs were isolated and plated in 96-well flat-bottom plate at 100 μl/ well (106 cells/well). Immedi- ately, 100 μl/well of medium with respective 5 µg/ml of CAV-2 and 2 μg/ml of attenu- ated PRRSV-JL proteins (an extract of PRRSV-JL-infected Marc-145 cells concentrated by ultracentrifugation at 80,000×g for 2 hr) or simple medium (control) was added and mixed.