Construction And Expression Of Adenovirus-based CVB3 Vaccines RAd/VP1 And RAd/VP22-L-VP1 Directed Against The VP1 Protein

Objective: Coxsackievirus Group B Type 3(CVB3) is a major pathogen of human viral myocarditis, which is the leading cause of neonate sudden death as well. There is no effective method available to prevent or treat this infection until now.The VP1 protein of CVB3, which can induce both humoral and cellular immune responses, is the major capsid protein of CVB3. Our laboratory have shown that the plasmid-based CVB3 VP1 DNA vaccine, could induce a immune response in mice but could not protect the mice from a lethal challenge of CVB3, for the lower titers of antibody induced. Therefore, enhancing the antigenicity and protective effects of VP1 gene vaccine is a problem need to be sovled.Development of a transfer vector with high efficiency of gene delivery and targetting specificity is a key point to enhance the immune effection of DNA vaccine. Replication-defective adenovirus, which has shown having many advantages over other vector systems, such as infecting more types of cells with no requirement for cell division, higher efficiency of gene transfer, robust antigen expression and higher protective immunity, has made it a promising system for human gene therapy in vivo.Althouth the adenoviral vector can transfect the somatic cells efficiently, if the protein encoded by adenoviral vector in nonantigen-presentation cells can not spread to professional antigen-presenting cells (APCs) rapidly, the immune effect still can not be improved. Therefore it is another key point to develop a competent delivery system for intercellular transport in the development of DNA vaccine. Encoded by the UL49 gene of herpes simplex virus type 1 (HSV-1), VP22 is an alkaline protein of 301 amino acid residues.With the protein transduction domain (PTD), VP22 can mediate a transmembrane transduction of VP22-linked protein or DNA from the cells in which it is synthesised endogenously to adjacent cells. Taken the advantage of this property, VP22 can be used to improve the immunity of DNA vaccine by co-delivering the antigen fused with VP22 to APCs. In this study, we constructed adenovirus-based CVB3 vaccines rAd/VP1 and rAd/VP22-L-VP1, and detected the expression of VP1 and VP22-L-VP1 in 293 cells, holding promise for protecting human beings against CVB3 infection.Methods: 1 Amplification and identification of genes of interest The DNA fragment of VP22-Linker (VP22-L) was amplified by PCR from the plasmid pAP85H encoding VP22 protein. Similarly, the DNA fragments of VP1 and Linker-VP1 (L-VP1) were amplified by PCR from the plasmid pcDNA3/VP1. Gene fragments were inserted into pGEM-T vector respectively. The competent E.coli DH5αwas transformed and cultured in ampicillin-containing 2×YT broth and the recombinants were identified by endonuclease fragments analyses and gene sequencing.2 Construction of transfer plasmids The plasmids constructed above were digested by endonucleases and the goal fragments were purified by agarose gel electrophoresie.The fragments gotten were subcloned to the transfer vector pAdTrack-CMV cut by the same endonucleases to construct the recombinant adenoviral transfer plasmids AdTrack-CMV/VP1 and AdTrack-CMV/VP22-L-VP1. Transformed DH5αwere selected by Kanamycin, the recombined plasmids were verified by endonuclease analyses.3 Generation of recombinant adenoviral plasmids The recombinant plasmids were then linearized with Pme I and co-transformed into E. coli strain Bj5183 respectively together with pAdEasy-1, the viral DNA plasmid. Recombinants were selected with kanamycin and screened by restriction enzyme analysis as well as PCR. Once achieved and verified, the recombinant adenoviral plasmids were transformed to competent E. coli DH5αfor gigantic DNA preparation and the DNA were then purified by PEG.4 Screening and amplification of recombinant adenoviral particles Linearized by Pac I digestion, the recombinant adenoviral plasmids were transferred into HEK 293 cells (human embryo kidney 293 derived cell line) using LipofectamineTM2000 as a transfection reagent according to the manufacturer's guidelines, to produce viral particles, screened by the expression of green fluorescent protein(GFP). The recombinant viruses were further amplified by passaging the viruses in 293 cells for 2 to 4 times.5 The Cells were lysed by freezing and thawing 48h post infection and Western blotting analysis was used to detect the protein expressed.6 Viral particle titration Cell-free virus stocks were diluted serially in serum-free medium at ten times, and were titrated by blue forming units methods according to AdEasy vector system application manual.Result: 1 Clone vectors pGEM-T/VP22-L, pGEM-T/VP1 and pGEM-T/L-VP1 were constructed successfully; endonuclease analyses and sequencing showed that the inserted genes were the same as reported.2 Recombinant adenoviral transfer plasmids AdTrack-CMV/VP1 and AdTrack-CMV/VP22-L-VP1 were constructed successfully.3 Recombinant adenoviral plasmids pAd, pAd/VP1 and pAd/VP22-L-VP1 were generated successfully. Restriction digest with Pac I yielded a large fragment of approximately 30 kb, and a smaller fragment of either 3.0 kb or 4.5 kb. The lengths of the fragments were the same as expected through verification of PCR.4 Indicated by the expression of report gene GFP in the cells 48h post infection, recombinant adenoviruses rAd, rAd/VP1 and rAd/VP22-L-VP1 were assembled successfully. The infected cells were lysed by freezing and thawing, and supernatant was used to reinfect fresh HEK 293 cells. The infected cells showed the characteristic comet-like fluorescent plaques.5 VP1 and VP22-L-VP1 encoded by the recombinant adenoviruses were expressed in the infected cells, which were confirmed by Western blotting.6 The titers of recombinant adenoviruses rAd from passage 1 to passage 4 were as follows, 1.2×10⁵ pfu/ml, 4.07×106pfu/ml, 1.47×10⁷pfu/ml and 9.6×10⁷ pfu/ml;rAd/VP1, 2.67×10⁵pfu/ml,1.82×10⁶pfu/ml,3.67×10⁶pfu/ml and 1.6×10⁷pfu/ml; rAd/VP22-L-VP1, 2.53×10⁵pfu/ml, 1.53×106pfu/ml, 1.40×10⁷pfu/ml and 6.77×10⁷pfu/ml.Conclusion: 1 Recombinant adenoviral plasmids rAd, rAd/VP1 and rAd/VP22-L-VP1 were generated successfully. And the expression of VP1 or VP22-L-VP1 was verified by Western blotting.2 With the further amplification of the recombinant viruses, the viral particle titrations were augmented at ten times by each passage.3 We constructed adenovirus-based CVB3 vaccines rAd/VP1 and rAd/VP22-L-VP1 successfully, by use of AdEasy^TM technology, providing a promising approach to prevent CVB3-caused myocardities.