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Production of Classical Swine Fever Virus Envelope Glycoprotein E2 as Recombinant Polyhedra in Baculovirus-Infected Silkworm Larvae

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Abstract

Although, classical swine fever virus (CSFV) envelope glycoprotein E2 subunit vaccine has been developed using the baculovirus expression system, the expression of viral antigens in baculovirus-infected insect cells is often ineffective. Therefore, an alternative strategy to the traditional baculovirus expression system is needed that is more productive and effective. Here, we report a novel strategy for the large-scale production of a CSFV E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg/ml of hemolymph and 0.53 mg/larva at 6-days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedra elicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that this strategy can be used for the large-scale production of CSFV E2 antigen.

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References

  1. Lindenbach, B. D., & Rice, C. N. (2001). Flaviviridae: the viruses and their replication. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, & B. Rzman (Eds.), Fields Virology (4th ed., pp. 991–1041). Philadelphia: Lippincott Williams & Wilkins.

    Google Scholar 

  2. Rümenapf, T., Unger, G., Strauss, J. H., & Thiel, H. J. (1993). Processing of the envelope glycoproteins of pestiviruses. Journal of Virology, 67, 3288–3294.

    Google Scholar 

  3. Van Zijl, M., Wensvoort, G., De Klijver, E. P., Hulst, M. M., Van der Gulden, H., Gielkens, A. L. J., et al. (1991). Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera. Journal of Virology, 65, 2761–2765.

    Google Scholar 

  4. Hulst, M. M., Westra, D. F., Wensvoort, G., & Moormann, R. J. M. (1993). Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera. Journal of Virology, 67, 5435–5442.

    CAS  Google Scholar 

  5. König, M., Lengsfeld, T., Pauly, T., Stark, R., & Thiel, H. J. (1995). Classical swine fever virus: independent induction of protective immunity by two structural glycoproteins. Journal of Virology, 69, 6479–6486.

    Google Scholar 

  6. Van Rijn, P. A., Bossers, A., Wensvoort, G., & Moormann, R. J. M. (1996). Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pig from lethal CSFV challenge. Journal of General Virology, 77, 2737–2745.

    Article  Google Scholar 

  7. Wensvoort, G. (1989). Topographical and functional mapping of epitopes on hog cholera virus with monoclonal antibodies. Journal of General Virology, 70, 2865–2876.

    Article  Google Scholar 

  8. Van Rijn, P. A., van Gennip, R. G. P., de Meijer, E. J., & Moormann, R. J. M. (1992). A preliminary map of epitopes on envelope glycoprotein E1 of HCV strain Brescia. Veterinary Microbiology, 33, 221–230.

    Article  Google Scholar 

  9. Van Rijn, P. A., van Gennip, H. G. P., de Meijer, E. J., & Moormann, R. J. M. (1993). Epitope mapping of envelope glycoprotein E1 of hog cholera virus strain Brescia. Journal of General Virology, 74, 2053–2060.

    Article  Google Scholar 

  10. Van Rijn, P. A., Miedema, G. W., Wensvoort, G., van Gennip, H. G. P., de Meijer, E. J., & Moormann, R. J. M. (1994). Antigenic structure of envelope glycoprotein E1 of hog cholera virus. Journal of Virology, 68, 3934–3942.

    Google Scholar 

  11. Lin, M., Lin, F., Mallory, M., & Clavijo, A. (2000). Deletions of structural glycoprotein E2 of classical swine fever virus strain Alfort/187 resolve a linear epitope of monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum. Journal of Virology, 74, 11619–11625.

    Article  CAS  Google Scholar 

  12. Beer, M., Reimann, I., Hoffmann, B., & Depner, K. (2007). Novel marker vaccines against classical swine fever. Vaccine, 25, 5665–5670.

    Article  CAS  Google Scholar 

  13. Toledo, J. R., Sánchez, O., Montesino, R., Farnós, O., Rodríguez, M. P., Alfonso, P., et al. (2008). Highly protective E2-CSFV vaccine candidate produced in the mammary gland of adenoviral transduced goats. Journal of Biotechnology, 133, 370–376.

    Article  CAS  Google Scholar 

  14. Barrera, M., Sánchez, O., Farnós, O., Rodríguez, M. P., Domínguez, P., Tait, H., et al. (2010). Early onset and long lasting protection in pigs provided by a classical swine fever E2-vaccine candidate produced in the milk of goats. Veterinary Immunology and Immunopathology, 133, 25–32.

    Article  CAS  Google Scholar 

  15. Shao, H. B., He, D. M., Qian, K. X., Shen, G. F., & Su, Z. L. (2008). The expression of classical swine fever virus structural protein E2 gene in tobacco chloroplasts for applying chloroplasts as bioreactors. C. R. Biologies, 331, 179–184.

    Article  CAS  Google Scholar 

  16. Lin, G. J., Liu, T. Y., Tseng, Y. Y., Chen, Z. W., You, C. C., Hsuan, S. L., et al. (2009). Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response. Veterinary Microbiology, 139, 369–374.

    Article  CAS  Google Scholar 

  17. Rümenapf, T., Stark, R., Meyers, G., & Thiel, H. J. (1991). Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity. Journal of Virology, 65, 589–597.

    Google Scholar 

  18. Hammond, J. M., McCoy, R. J., Jansen, E. S., Morrissy, C. J., Hodgson, A. L., & Johnson, M. A. (2000). Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55 (E2) gene protects pigs against classical swine fever. Vaccine, 18, 1040–1050.

    Article  CAS  Google Scholar 

  19. Sun, Y., Liu, D. F., Wang, Y. F., Liang, B. B., Cheng, D., Li, N., et al. (2010). Generation and efficacy evaluation of a recombinant adenovirus expressing the E2 protein of classical swine fever virus. Research in Veterinary Science, 88, 77–82.

    Article  CAS  Google Scholar 

  20. Bouma, A., de Smit, A. J., de Kluijier, E. P., Terpstra, C., & Moormann, R. J. M. (1999). Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus. Veterinary Microbiology, 66, 101–114.

    Article  CAS  Google Scholar 

  21. Xu, X. G., & Liu, H. J. (2008). Baculovirus surface display of E2 envelope glycoprotein of classical swine fever virus and immunogenicity of the displayed proteins in a mouse model. Vaccine, 26, 5455–5460.

    Article  CAS  Google Scholar 

  22. Li, M., Wang, Y. F., Wang, Y., Gao, H., Li, N., Sun, Y., et al. (2009). Immune responses induced by a BacMam virus expressing the E2 protein of classical swine fever virus in mice. Immunology Letters, 125, 145–150.

    Article  CAS  Google Scholar 

  23. Dewulf, J., Laevens, H., Koenen, F., Vanderhallen, H., Mintiens, K., Deluyker, H., et al. (2001). An experimental infection with classical swine fever in E2 subunit marker-vaccine vaccinated and in non-vaccinated pigs. Vaccine, 19, 475–482.

    Article  Google Scholar 

  24. Uttenthal, A., le Potier, M. F., Romero, L., de Mia, G. M., & Floegel-Niesmann, G. (2001). Classical swine fever (CSF) marker vaccine. Trial 1. Challenge studies in Weaner pigs. Veterinary Microbiology, 83, 85–106.

    Article  CAS  Google Scholar 

  25. O’Reilly, D. R., Miller, L. K., & Luckow, V. A. (1992). Baculovirus Expression Vectors: A Laboratory Manual. New York: W.H. Freeman & Co.

    Google Scholar 

  26. Maeda, S. (1989). Expression of foreign genes in insects using baculovirus vectors. Annual Review of Entomology, 34, 351–372.

    Article  CAS  Google Scholar 

  27. Kato, T., Kajikawa, M., Maenaka, K., & Park, E. Y. (2010). Silkworm expression system as a platform technology in life science. Applied Microbiology and Biotechnology, 85, 459–470.

    Article  CAS  Google Scholar 

  28. Gui, Z. Z., Lee, K. S., Kim, B. Y., Choi, Y. S., Wei, Y. D., Choo, Y. M., et al. (2006). Functional role of aspartic proteinase cathepsin D in insect metamorphosis. BMC Developmental Biology, 6, 49.

    Article  Google Scholar 

  29. Je, Y. H., Chang, J. H., Kim, M. H., Roh, J. Y., Jin, B. R., & O’Reilly, D. R. (2001). The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-positive and occlusion-negative expression vectors. Biotechnology Letters, 23, 1809–1817.

    Article  CAS  Google Scholar 

  30. Lee, K. S., Kim, B. Y., Je, Y. H., Woo, S. D., Sohn, H. D., & Jin, B. R. (2007). A technique for producing recombinant baculovirus directly in silkworm larvae. Biotechnology Letters, 29, 175–180.

    Article  CAS  Google Scholar 

  31. Je, Y. H., Jin, B. R., Park, H. W., Roh, J. Y., Chang, J. H., Seo, S. J., et al. (2003). Baculovirus expression vector that incorporate the foreign protein into viral occlusion bodies. Biotechniques, 34, 81–87.

    CAS  Google Scholar 

  32. Jarvis, D. L., Bohlmeyer, D. A., & Garcia, A, Jr. (1991). Requirements for nuclear localization and supramolecular assembly of a baculovirus polyhedrin protein. Virology, 185, 795–810.

    Article  CAS  Google Scholar 

  33. Terzić, S., Jemeršić, L., Lojkić, M., Madić, J., Grom, J., Toplak, I., et al. (2003). Comparison of antibody values in sera of pigs vaccinated with a subunit or an attenuated vaccine against classical swine fever. Veterinary Research Communications, 27, 329–339.

    Article  Google Scholar 

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Acknowledgments

This study was supported by the Rural Development Administration (BioGreen 21 Project 20070401034002), Republic of Korea

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Authors and Affiliations

  1. College of Natural Resources and Life Science, Dong-A University, Busan, 604-714, Republic of Korea

    Kwang Sik Lee, Mi Ri Sohn, Bo Yeon Kim, Young Moo Choo & Byung Rae Jin

  2. College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju, Republic of Korea

    Soo Dong Woo & Hyun Na Koo

  3. Choong-Ang Vaccine Laboratory, Daejeon, Republic of Korea

    Sung Sik Yoo

  4. Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea

    Yeon Ho Je, Jae Young Choi & Jong Yul Roh

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  1. Kwang Sik Lee
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Correspondence to Byung Rae Jin.

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Kwang Sik Lee and Mi Ri Sohn contributed equally to this study.

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Lee, K.S., Sohn, M.R., Kim, B.Y. et al. Production of Classical Swine Fever Virus Envelope Glycoprotein E2 as Recombinant Polyhedra in Baculovirus-Infected Silkworm Larvae. Mol Biotechnol 50, 211–220 (2012). https://doi.org/10.1007/s12033-011-9431-5

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