In vitro evaluation of chitosan-DNA plasmid complex encoding Jembrana disease virus Env-TM protein as a vaccine candidate

Open access


Introduction: The development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex.

Material and Methods: Cloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection.

Results: With a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent.

Conclusion: pEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.

1. Budiarso I.T., Hardjosworo S.: Jembrana disease in Bali cattle. Aust Vet J 1976, 52, 97–97.

2. Burkala E.J., Narayani I., Hartaningsih N., Kertayadnya G., Berryman D.I., Wilcox G.E.: Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses. J Virol Methods 1998, 74, 39–46.

3. Chadwick B.J., Desport M., Brownlie J., Wilcox G.E., Dharma D.M.N.: Detection of Jembrana disease virus in spleen, lymph nodes, bone marrow and other tissues by in situ hybridization of paraffin-embedded sections. J Gen Virol 1998, 79, 101–106.

4. Chen H., Wilcox G., Kertayadnya G., Wood C.: Characterization of the Jembrana disease virus tat gene and the cis- and trans-regulatory elements in its long terminal repeats. J Virol 1999, 73, 658–666.

5. Craigo J.K., Montelaro R.C.: Lessons in AIDS vaccine development learned from studies of equine infectious anemia virus infection and immunity. Viruses 2013, 5, 2963–2976.

6. Ditcham W.G.F.: The development of recombinant vaccines against Jembrana disease, Murdoch University 2007,

7. Ditcham W.G.F., Lewis J.R., Dobson R.J., Hartaningsih N., Wilcox G.E., Desport M.: Vaccination reduces the viral load and the risk of transmission of Jembrana disease virus in Bali cattle. Virology 2009, 386, 317–324.

8. Gregory A.E., Titball R., Williamson D.: Vaccine delivery using nanoparticles. Front Cell Infect Microbiol 2013, 3, doi: 10.3389/fcimb.2013.00013.

9. Haas J., Park E.C., Seed B.: Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol 1996, 6, 315–324.

10. Hartaningsih N., Wilcox G.E., Dharma D.M.N., Soetrismo M.: Distribution of Jembrana disease in cattle in Indonesia. Vet Microbiol 1993, 38, 23–29.

11. Hashimoto M., Yang Z., Koya Y., Sato T.: Chitosan. In: Non-viral Gene Therapy: Gene Design and Delivery, edited by Taira K., Kataoka K., Niidome T., Springer-Verlag, Tokyo, Japan, 2005, pp. 63–74.

12. Issel C.J., Horohov D.W., Lea D.F., Adams W.V., Hagius S.D., McManus J.M., Allison A.C., Montelaro R.C.: Efficacy of inactivated whole-virus and subunit vaccines in preventing infection and disease caused by equine infectious anemia virus. J Virol 1992, 66, 3398–3408.

13. Kharia A.A., Singhai A.K., Verma R.: Formulation and evaluation of polymeric nanoparticles of an antiviral drug for gastroretention. Int J Pharm Pharm Sci 2012, 4, 1557–1562.

14. Kusumawati A., Hidayat B., Sardjono B., Widada J.S.: Isolation-amplification of env-tm subunit genes of Jembrana disease virus by a single step RT-PCR and its direct cloning in pCR2.1-TOPO plasmid. Buletin Peternakan 2012, 27, 1–7, doi: 10.21059/buletinpeternak.v27i1.1459.

15. Leutenegger C.M., Hofmann-Lehmann R., Holznagel E., Cuisinier A.M., Wolfensberger C., Duquesne V., Cronier J., Allenspach K., Aubert A., Ossent P., Lutz, H.: Partial protection by vaccination with recombinant feline immunodeficiency virus surface glycoproteins. AIDS Res Hum Retroviruses 1998, 14, 275–283.

16. Martien R., Loretz B., Thaler M., Majzoob S., Bernkop-Schnürch A.: Chitosan – thioglycolic acid conjugate: an alternative carrier for oral nonviral gene delivery? J Biomed Mater Res A 2007, 82, 1–9.

17. Masotti A., Bordi F., Ortaggi G., Marino F., Palocci C.: A novel method to obtain chitosan/DNA nanospheres and a study of their release properties, Nanotechnology 2008, 19, doi:

18. Pachuk C.J., McCallus D.E., Weiner, D.B., Satishchandran C.: DNA vaccines challenges in delivery. Current Curr Opin Mol Ther 2000, 2, 188–198.

19. Pearson L.D., Poss M.L., Demartini J.C.: Animal lentivirus vaccines: problems and prospects. Vet Immunol Immunopathol 1989, 20, 183–212.

20. Siegert W., Nitsche A.: Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 2004, 313, 856–862.

21. Sinha V., Singla A., Wadhawan S., Kaushik R., Kumria R., Bansal K., Dhawan S.: Chitosan microspheres as a potential carrier for drugs. Int J Pharm 2004, 274, 1–33.

22. Soeharsono S., Wilcox G.E., Dharma D.M.N., Hartaningsih N., Kertayadnya G., Budiantono A.: Species differences in the reaction of cattle to Jembrana disease virus infection. J Comp Pathol 1995, 112, 391–402.

23. Wang G., Pan L., Zhang Y.: Approaches to improved targeting of DNA vaccines. Hum Vac Immunother 2011, 7, 1271–1281.

24. Wilcox G.E., Chadwick B.J., Kertayadnya G.: Recent advances in the understanding of Jembrana disease. Vet Microbiol 1995, 46, 249–255.

25. Wilcox G.E., Soeharsono S., Dharma D.M.N., Copland J.W.: Jembrana disease and the bovine lentiviruses. Aciar Proc 1997, 75, 10–75.

26. Williams J.A.: Vector design for improved DNA vaccine efficacy, safety, and production. Vaccines 2013, 1, 225–249.

27. Winarti L., Martien R., Sismindari: Formulation of nanoparticles from short chain chitosan as gene delivery system and transfection against T47D cell line. Majalah Farmasi Indonesia 2011, 22, 204–211.

28. Xu K., Ling Z.Y., Sun L., Xu Y., Bian C., He Y., Lu W., Chen Z., Sun B.: Broad humoral and cellular immunity elicited by a bivalent DNA vaccine encoding HA and NP genes from an H5N1 virus. Viral Immunol 2011, 24, 45–56.

29. Ye Y., Xu Y., Liang W., Leung G.P.H., Cheung K., Zheng C., Chen F., Lam J.K.W.: Permeability across Calu-3 cells. J Drug Target 2013, 2330, 1–13.

30. Zhou J., Wang L., Lu G., Zhou A., Zhu M., Li Q., Wang Z., Arken M., Wang A., He S.: Epitope analysis and protection by a ROP19 DNA vaccine against Toxoplasma gondii. Parasite 2016, 23, 17.

Journal of Veterinary Research

formerly Bulletin of the Veterinary Institute in Pulawy

Journal Information

IMPACT FACTOR J Vet Res 2017: 0.811

CiteScore 2017: 0.68

SCImago Journal Rank (SJR) 2017: 0.29
Source Normalized Impact per Paper (SNIP) 2017: 0.484


All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 215 215 128
PDF Downloads 106 106 40