Tropical Animal Health and Production

, Volume 51, Issue 8, pp 2529–2538 | Cite as

Evaluation of a fusion gene-based DNA prime-protein boost vaccination strategy against Newcastle disease virus

  • Sagar A. Khulape
  • Hemanta Kumar Maity
  • Dinesh Chandra Pathak
  • Narayan Ramamurthy
  • Saravanan Ramakrishnan
  • Madhan Mohan ChellappaEmail author
  • Sohini DeyEmail author
Regular Articles


The low potency of genetic immunization has to date impeded development of commercial vaccines against major infectious diseases. The aim of this study was to develop and evaluate a fusion gene-based DNA prime-protein boost vaccination strategy to improve the efficacy of both DNA and subunit vaccines against Newcastle disease virus (NDV). The fusion (F) protein, a viral surface glycoprotein, is responsible for the cell membrane fusion and spread, also is one of the major targets for immune response. In this study, groups of chickens were vaccinated twice intramuscularly at 14-day interval either with plasmid DNA encoding F protein gene of NDV or with recombinant F protein alone or with plasmid DNA and boosted with the recombinant F protein and compared with birds that were vaccinated with live NDV vaccine. The immune response was evaluated by indirect ELISA, lymphocyte transformation test, virus neutralization test, cytokine analysis, immunophenotyping of peripheral blood mononuclear cells, and protective efficacy study against virulent NDV challenge virus infection. Chickens in prime-boost group developed a higher level of humoral and cellular immune responses as compared with those immunized with plasmid or protein alone. The DNA prime-protein boost using F protein of NDV yielded 91.6% protection against virulent NDV challenge infection better than immunization with DNA vaccine (66.6%) or rF protein (83.3%) alone. These findings suggest that the “DNA prime-protein boost” approach using full-length F gene could enhance the immune response against NDV in the chickens.


Newcastle disease virus Fusion protein DNA prime-protein boost Protective immunity 



The authors are thankful to the Director of the Indian Veterinary Research Institute, Izatnagar, for providing necessary facilities and infrastructure.

Funding information

This work was supported by grants from the National Fund for Basic and Strategic Research in Agriculture of ICAR (NFBSFARA/BS-3010) awarded to SD and the Department of Biotechnology, Government of India BT/PR15373/AAQ/57/116/2011 and DBT-JRF/2012-13/107 awarded to MMC and SAK respectively.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Statement of animal rights

Animal experiments were approved by the Institute Animal Ethics Committee of ICAR-Indian Veterinary Research Institute, Izatnagar, and performed in accordance with animal ethics guidelines and approved protocols of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment, Forest, Climate change, Government of India.


  1. Aravind, S., Kamble, N.M., Gaikwad, S.S., Shukla, S.K., Saravanan, R., Dey, S. and Mohan, C.M., 2015. Protective effects of recombinant glycoprotein D based prime boost approach against duck enteritis virus in mice model, Microbial Pathogenesis, 88, 78-86CrossRefGoogle Scholar
  2. Casares, S., Inaba, K., Brumeanu, T.D., Steinman, R.M. and Bona, C.A., 1997. Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope, Journal of Experimental Medicine, 186, 1481-1486CrossRefGoogle Scholar
  3. Cattoli, G., Fusaro, A., Monne, I., Molia, S., Le Menach, A., Maregeya, B., Maina, A.G., Koffi, J.N., Thiam, H., Bezeid, O.E., Salviato, A., Nisi, R., Terregino, C. and Capua, I., 2010. Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa-implications for diagnosis and control, Veterinary Microbiology, 142, 168-176CrossRefGoogle Scholar
  4. Degefa, T., Dadi, L., Yami, A., Gmariam, K. and Nassir, M., 2004. Technical and economic evaluation of different methods of Newcastle disease vaccine administration, Journal of Veterinary Medicine A, Physiology, Pathology, Clinical Medicine, 51, 365–369CrossRefGoogle Scholar
  5. Dey, S., Upadhyay, C., Mohan, C.M., Kataria, J.M. and Vakharia, V., 2009. Formation of subviral particles of the capsid protein VP2 of infectious bursal disease virus and its application in serological diagnosis, Journal of Virological Methods, 157, 84-89CrossRefGoogle Scholar
  6. Dortmans, J.C., Peeters, B.P. and Koch, G., 2012. Newcastle disease virus outbreaks: vaccine mismatch or inadequate application?, Veterinary Microbiology, 160, 17-22CrossRefGoogle Scholar
  7. Gao, H., Li, K., Gao, L., Qi, X., Gao, Y., Qin, L., Wang, Y. and Wang, X., 2013. DNA prime-protein boost vaccination enhances protective immunity against infectious bursal disease virus in chickens, Veterinary Microbiology, 164, 9-17CrossRefGoogle Scholar
  8. Gurunathan, S., Klinman, D.M. and Seder, R.A., 2000. DNA vaccines: immunology, application, and optimization, Annual Review of Immunology, 18, 927-974CrossRefGoogle Scholar
  9. Heckert, R.A., Elankumaran, S., Oshop, G.L. and Vakharia, V.N., 2002. A novel transcutaneous plasmid-dimethylsulfoxide delivery technique for avian nucleic acid immunization, Veterinary Immunology and Immunopathology, 89, 67-81CrossRefGoogle Scholar
  10. Kamiya, N., Niikura, M., Ono, M., Kai, Ch., Matsuura, Y., and Mikami, T., 1994. Protective effect of individual glycoproteins of Newcastle disease virus expressed in insect cells: the fusion protein derived from avirulent strain had lower protective efficacy, Virus Research, 32, 373-379CrossRefGoogle Scholar
  11. Khulape, S.A., Gaikwad, S.S., Chellappa, M.M., Mishra, B.P. and Dey, S., 2014. Complete Genome Sequence of a Newcastle Disease Virus Isolated from Wild Peacock (Pavo cristatus) in India. Genome Announcements, 2, e00495-14CrossRefGoogle Scholar
  12. Kim, S.H., Wanasen, N., Paldurai, A., Xiao, S. and Collins, P.L., 2013. Newcastle Disease Virus fusion protein is the major contributor to protective immunity of genotype-matched vaccine, PLoS One, 8, e74022CrossRefGoogle Scholar
  13. Kumar, S., Nayak, B., Collins, P.L. and Samal, S.K., 2011. Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus type 3 vector in chickens, Journal of Virology, 85, 6521-6534CrossRefGoogle Scholar
  14. Lamb, R. and Parks, G., 2007. Paramyxoviridae: the viruses and their replication, In: D.M. Knipe, P.M. Howley, D.E. Griffin, R.A. Lamb, M.A. Martin, Philadelphia: Lippincott Williams & Wilkins, pp 1449–1496Google Scholar
  15. Law, M., Cardoso, R.M., Wilson, I.A. and Burton, D., 2007. Antigenic and immunogenic study of membrane-proximal external region-grafted gp120 antigens by a DNA prime-protein boost immunization strategy, Journal of Virology, 81, 4272-4285CrossRefGoogle Scholar
  16. Lee, Y.J., Sung, H.W., Cho, J.G., Lee, E.K., Yoon, H., Kim, J.H. and Song, C.S., 2008. Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutinin-neuraminidase proteins, Journal of Veterinary Science, 9, 301-308CrossRefGoogle Scholar
  17. Liang, R., van den Hurk, J.V., Zheng, C., Yu, H., Pontarollo, R.A., Babiuk, L.A. and van Drunen Littel-van den Hurk, S., 2005. Immunization with plasmid DNA encoding a truncated, secreted form of the bovine viral diarrhea virus E2 protein elicits strong humoral and cellular immune responses, Vaccine, 23, 5252-5262CrossRefGoogle Scholar
  18. Liu, H., Zhang, M., Han, H., Yuan, J. and Li, Z., 2010. Comparison of the expression of cytokine genes in the bursal tissues of the chickens following challenge with infectious bursal disease viruses of varying virulence, Virology Journal, 7, 364-371CrossRefGoogle Scholar
  19. Loke, C.F., Omar, A.R., Raha, A.R. and Yusoff, K., 2005. Improved protection from velogenic Newcastle disease virus challenge following multiple immunizations with plasmid DNA encoding for F and HN genes, Veterinary Immunology and Immunopathology, 106, 259-267CrossRefGoogle Scholar
  20. Maity, H.K., Dey, S., Mohan, C.M., Khulape, S.A., Pathak, D.C. and Vakharia, V.N., 2015. Protective efficacy of a DNA vaccine construct encoding the VP2 gene of infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens, Vaccine, 33, 1033-1039CrossRefGoogle Scholar
  21. Mattanovich, D., Branduardi, P., Dato, L., Gasser, B., Sauer, M. and Porro, D., 2012. Recombinant Protein Production in Yeasts, In: Lorence A. (eds) Recombinant Gene Expression, Methods in Molecular Biology (Methods and Protocols), 824, Humana Press, Totowa, NJGoogle Scholar
  22. Meulemans, G., Gonze, M., Carlier, M.C., Petit, P., Burny, A. and Long, L., 1986. Protective effects of HN and F glycoprotein-specific monoclonal antibodies on experimental Newcastle disease, Avian Pathology, 15, 761-768CrossRefGoogle Scholar
  23. Miller, P.J., King, D.J., Afonso, C.L. and Suarez, D.L., 2007. Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge, Vaccine, 25, 7238-7246CrossRefGoogle Scholar
  24. Miller, P.J., Estevez, C., Yu, Q., Suarez, D.L. and King, D.K., 2009. Comparison of viral shedding following vaccination with inactivated and live Newcastle disease vaccines formulated with wild-type and recombinant viruses, Avian Diseases, 53, 39-49CrossRefGoogle Scholar
  25. Miller, P.J., Decanini, E.L. and Afonso, C.L., 2010. Newcastle disease: evolution of genotypes and the related diagnostic challenges, Infection, Genetics and Evolution, 10, 26-35CrossRefGoogle Scholar
  26. Miyamoto, T., Taura, Y., Une, S., Yoshitake, M., Nakama, S. and Watanabe, S., 1995. Immunological responses to polyvalent canine vaccines in dogs, Journal of Veterinary Medical Science, 57, 347-349CrossRefGoogle Scholar
  27. Mohan, C.M., Dey, S., Rai, A. and Kataria, J.M., 2006. Recombinant haemagglutinin neuraminidase antigen-based single serum dilution ELISA for rapid serological profiling of Newcastle disease virus, Journal of Virological Methods, 138, 117-182CrossRefGoogle Scholar
  28. Morrison, T.G. and Portner, A., 1991. In H. Fraenkel Conrat and R.R. Wagner (Eds), The Paramyxovirus, Plenum Press, New York, 347–82CrossRefGoogle Scholar
  29. Nagai, Y., Klenk, H.D. and Rott, R., 1976. Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus, Virology, 72, 494-508CrossRefGoogle Scholar
  30. Reynolds, D.L. and Maraqa, A.D., 2000. Protective immunity against Newcastle disease virus: the role of cell-mediated immunity, Avian Diseases, 44, 145-154CrossRefGoogle Scholar
  31. Sakaguchi, M., Nakamura, H., Sonoda, K., Hamada, F. and Hirai, K., 1996. Protection of chickens from Newcastle disease by vaccination with a linear plasmid DNA expressing the F protein of Newcastle disease virus, Vaccine, 14, 747-752CrossRefGoogle Scholar
  32. Sawant, P.M., Verma, P.C., Subudhi, P.K., Chaturvedi, U., Singh, M., Kumar, R. and Tiwari, A.K., 2011. Immunomodulation of bivalent Newcastle disease DNA vaccine induced immune response by co-delivery of chicken IFN-gamma and IL-4 gene, Veterinary Immunology and Immunopathology, 144, 36-44CrossRefGoogle Scholar
  33. Shedlock, D.J. and Weiner, D.B., 2000. DNA vaccination: antigen presentation and the induction of immunity, Journal of Leukocyte Biology, 68, 793-806PubMedGoogle Scholar
  34. Wang, K., Holtz, K.M., Anderson, K., Chubet, R., Mahmoud, W. and Cox, M.M., 2006. Expression and purification of an influenza hemagglutinin-one step closer to a recombinant protein-based influenza vaccine, Vaccine, 24, 2176-2185CrossRefGoogle Scholar
  35. Yusoff, K. and Tan, W.S., 2001. Newcastle disease virus: Macromolecules and opportunities, Avian Pathology, 30, 439-455CrossRefGoogle Scholar
  36. Zhao, K., Li, W., Huang, T., Luo, X. and Chen, G., 2013. Preparation and efficacy of Newcastle disease virus DNA vaccine encapsulated in PLGA nanoparticles, PLoS One, 8, e82648CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Sagar A. Khulape
    • 1
  • Hemanta Kumar Maity
    • 2
  • Dinesh Chandra Pathak
    • 2
  • Narayan Ramamurthy
    • 2
  • Saravanan Ramakrishnan
    • 3
  • Madhan Mohan Chellappa
    • 2
    Email author
  • Sohini Dey
    • 2
    Email author
  1. 1.Directorate on Foot and Mouth Disease VirusNainitalIndia
  2. 2.Recombinant DNA Laboratory, Division of Veterinary BiotechnologyIndian Veterinary Research InstituteBareillyIndia
  3. 3.Immunology SectionIndian Veterinary Research InstituteBareillyIndia

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