Molecular Biotechnology

, Volume 56, Issue 10, pp 903–910 | Cite as

Optimization of the Immunogenicity of a DNA Vaccine Encoding a Bacterial Outer Membrane Lipoprotein

  • Arun Buaklin
  • Tanapat Palaga
  • Drew Hannaman
  • Ruthairat Kerdkaew
  • Kanitha PatarakulEmail author
  • Alain JacquetEmail author


Bacterial outer membrane lipoproteins represent potent immunogens for the design of recombinant subunit vaccines. However, recombinant lipoprotein production and purification could be a challenge notably in terms of expression yield, protein solubility, and post-translational acylation. Together with the cost effectiveness, facilitated production, and purification as well as good stability, DNA-based vaccines encoding lipoproteins could become an alternative strategy for antibacterial vaccinations. Although the immunogenicity and the efficacy of DNA-based vaccines can be demonstrated in small rodents, such vaccine candidates could request concrete optimization as they are weak immunogens in primates and humans and particularly when administered by conventional injection. Therefore, the goal of the present study was to optimize the immunogenicity of a DNA vaccine encoding an outer membrane lipoprotein. LipL32, the major outer membrane protein from pathogenic Leptospira, was selected as a model antigen. We evaluated the influence of antigen secretion, the in vivo DNA delivery by electroporation, the adjuvant co-administration, as well as the heterologous prime-boost regimen on the induction of anti-LipL32 specific immune responses. Our results clearly showed that, following transfections, a DNA construct based on the authentic full-length LipL32 gene (containing leader sequence and the N-terminus cysteine residue involved in the protein anchoring) drives antigen secretion with the same efficiency as a plasmid-encoding anchor-less LipL32 and for which the bacterial leader sequence was replaced with a viral signal peptide. The in vivo DNA delivery by electroporation drastically enhanced the production of strong Th1 responses characterized by specific IgG2a antibodies and the IFNγ secretion in a restimulation assay, regardless of the DNA constructs used. In comparison with the heterologous prime-boost regimen, the homologous prime-boost vaccinations with DNA co-administrated with polyinosinic-polycytidylic acid (poly I:C) generated the highest specific IgG and IgG2a titers as well as the greatest IFNγ production. Taken together, these data suggest that optimization of outer membrane lipoprotein secretion is not critical for the induction of antigen-specific responses through DNA vaccination. Moreover, the potent antibody response induced by DNA plasmid encoding lipoprotein formulated with poly I:C and delivered through electroporation provides the rationale for the design of new prophylactic vaccines against pathogenic bacteria.


Bacterial lipoprotein DNA vaccine Electroporation Poly I:C Prime-boost LipL32 



This work was supported by the National Research Council of Thailand and the Higher Education Research Promotion and the National Research University Project of Thailand, Office of the Higher Education Commission (HR1164A2). Dr. Arun Buaklin was supported by a Post-doctoral fellowship from Chulalongkorn University Graduate School.


  1. 1.
    Williams, J. A. (2013). Vector design for improved DNA vaccine efficacy, safety and production. Vaccines, 1, 225–249.CrossRefGoogle Scholar
  2. 2.
    Kutzler, M. A., & Weiner, D. B. (2008). DNA vaccines: ready for prime time? Nature Reviews Genetics, 9, 776–788.CrossRefGoogle Scholar
  3. 3.
    Ingolotti, M., Kawalekar, O., Shedlock, D. J., Muthumani, K., & Weiner, D. B. (2010). DNA vaccines for targeting bacterial infections. Expert Review Vaccines., 9, 747–763.CrossRefGoogle Scholar
  4. 4.
    Kovacs-Simon, A., Titball, R. W., & Michell, S. L. (2011). Lipoproteins of bacterial pathogens. Infection and Immunity, 79, 548–561.CrossRefGoogle Scholar
  5. 5.
    Schenk, M., Belisle, J. T., & Modlin, R. L. (2009). TLR2 looks at lipoproteins. Immunity, 31, 847–849.CrossRefGoogle Scholar
  6. 6.
    Haake, D. A., Chao, G., Zuerner, R. L., Barnett, J. K., Barnett, D., Mazel, M., et al. (2000). The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infection and Immunity, 68, 2276–2285.CrossRefGoogle Scholar
  7. 7.
    Yang, C. W., Hung, C. C., Wu, M. S., Tian, Y. C., Chang, C. T., Pan, M. J., et al. (2006). Toll-like receptor 2 mediates early inflammation by leptospiral outer membrane proteins in proximal tubule cells. Kidney International, 69, 815–822.CrossRefGoogle Scholar
  8. 8.
    Hsu, S. H., Lo, Y. Y., Tung, J. Y., Ko, Y. C., Sun, Y. J., Hung, C. C., et al. (2010). Leptospiral outer membrane lipoprotein LipL32 binding on toll-like receptor 2 of renal cells as determined with an atomic force microscope. Biochemistry, 49, 5408–5417.CrossRefGoogle Scholar
  9. 9.
    Massaer, M., Mazzu, P., Haumont, M., Magi, M., Daminet, V., Bollen, A., et al. (2001). High-level expression in mammalian cells of recombinant house dust mite allergen ProDer p 1 with optimized codon usage. International Archives of Allergy and Immunology, 125, 32–43.CrossRefGoogle Scholar
  10. 10.
    Baldwin, S. L., D’Souza, C. D., Orme, I. M., Liu, M. A., Huygen, K., Denis, O., et al. (1999). Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A. Tubercle and Lung Disease, 79, 251–259.CrossRefGoogle Scholar
  11. 11.
    Wang, S., Farfan-Arribas, D. J., Shen, S., Chou, T. H., Hirsch, A., He, F., et al. (2005). Relative contributions of codon usage, promoter efficiency and leader sequence to the antigen expression and immunogenicity of HIV-1 Env DNA vaccine. Vaccine, 24, 4531–4540.CrossRefGoogle Scholar
  12. 12.
    Jacquet, A., Magi, M., Haumont, M., Jurado, M., Garcia, L., & Bollen, A. (2003). Absence of immunoglobulin E synthesis and airway eosinophilia by vaccination with plasmid DNA encoding ProDer p 1. Clinical and Experimental Allergy, 33, 218–225.CrossRefGoogle Scholar
  13. 13.
    Flingai, S., Czerwonko, M., Goodman, J., Kudchodkar, S. B., Muthumani, K., & Weiner, D. B. (2013). Synthetic DNA vaccines: improved vaccine potency by electroporation and co-delivered genetic adjuvants. Frontiers in Immunology, 4, 354.CrossRefGoogle Scholar
  14. 14.
    Grossmann, C., Tenbusch, M., Nchinda, G., Temchura, V., Nabi, G., Stone, G. W., et al. (2009). Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands. BMC Immunology, 10, 43–48.CrossRefGoogle Scholar
  15. 15.
    Tewari, K., Flynn, B. J., Boscardin, S. B., Kastenmueller, K., Salazar, A. M., Anderson, C. A., et al. (2010). Poly(I:C) is an effective adjuvant for antibody and multi-functional CD4 + T cell responses to Plasmodium falciparum circumsporozoite protein (CSP) and αDEC-CSP in non human primates. Vaccine, 28, 7256–7266.CrossRefGoogle Scholar
  16. 16.
    Ranasinghe, C., & Ramshaw, I. A. (2009). Genetic heterologous prime-boost vaccination strategies for improved systemic and mucosal immunity. Expert Review of Vaccines, 8, 1171–1181.CrossRefGoogle Scholar
  17. 17.
    Lugade, A. A., Bianchi-Smiraglia, A., Pradhan, V., Elkin, G., Murphy, T. F., & Thanavala, Y. (2011). Lipid motif of a bacterial antigen mediates immune responses via TLR2 signaling. PLoS ONE, 6, e19781.CrossRefGoogle Scholar
  18. 18.
    Hansson, L., Noppa, L., Nilsson, A. K., Stromqvist, M., & Bergstrom, S. (1995). Expression of truncated and full-length forms of the Lyme disease Borrelia outer surface protein a in Escherichia coli. Protein Expression and Purification, 6, 15–24.CrossRefGoogle Scholar
  19. 19.
    Shang, E. S., Summers, T. A., & Haake, D. A. (1996). Molecular cloning and sequence analysis of the gene encoding LipL41, a surface exposed lipoprotein of pathogenic Leptospira species. Infection and Immunity, 64, 2322–2330.Google Scholar
  20. 20.
    Hall, J., Hazlewood, G. P., Surani, M. A., Hirst, B. H., & Gilbert, H. J. (1990). Eukaryotic and prokaryotic signal peptides direct secretion of a bacterial endoglucanase by mammalian cells. Journal of Biological Chemistry, 265, 19996–19999.Google Scholar
  21. 21.
    Coban, C., Kobiyama, K., Aoshi, T., Takeshita, F., Horii, T., Akira, S., et al. (2011). Novel strategies to improve DNA vaccine immunogenicity. Current Gene Therapy, 11, 479–484.CrossRefGoogle Scholar
  22. 22.
    Rosazza, C., Buntz, A., Rieß, T., Wöll, D., Zumbusch, A., & Rols, M.-P. (2013). Intracellular tracking of single-plasmid DNA particles after delivery by electroporation. Molecular Therapy, 21, 2217–2226.CrossRefGoogle Scholar
  23. 23.
    Blasius, A. L., & Beutler, B. (2010). Intracellular Toll-like receptors. Immunity, 32, 305–315.CrossRefGoogle Scholar
  24. 24.
    Babiuk, S., Mookherjee, N., Pontarollo, R., Griebel, P., van Drunen Littel-van den Hurk, S., Hecker, R., et al. (2004). TLR9−/− and TLR9+/+mice display similar immune responses to a DNA vaccine. Immunology, 113, 114–120.CrossRefGoogle Scholar
  25. 25.
    Vabret, N., & Blander, J. M. (2013). Sensing microbial RNA in the cytosol. Front: Immunol. doi: 10.3389/fimmu.2013.00468.Google Scholar
  26. 26.
    Coutinho, M. L., Choy, H. A., Kelley, M. M., Matsunaga, J., Babbitt, J. T., Lewis, M. S., et al. (2011). A LigA three-domain region protects hamsters from lethal infection by Leptospira interrogans. PLOS Neglected Tropical Diseases, 5, e1422.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Arun Buaklin
    • 1
  • Tanapat Palaga
    • 2
  • Drew Hannaman
    • 3
  • Ruthairat Kerdkaew
    • 4
  • Kanitha Patarakul
    • 1
    Email author
  • Alain Jacquet
    • 5
    Email author
  1. 1.Department of Microbiology, Faculty of MedicineChulalongkorn UniversityBangkokThailand
  2. 2.Department of Microbiology, Faculty of ScienceChulalongkorn UniversityBangkokThailand
  3. 3.Ichor Medical SystemsSan DiegoUSA
  4. 4.Department of Internal Medicine, Faculty of MedicineChulalongkorn UniversityBangkokThailand
  5. 5.Department of Medicine, Faculty of MedicineChulalongkorn UniversityBangkokThailand

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