DNA-Encoded Flagellin Activates Toll-Like Receptor 5 (TLR5), Nod-like Receptor Family CARD Domain-Containing Protein 4 (NRLC4), and Acts as an Epidermal, Systemic, and Mucosal-Adjuvant
Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes.
References
[1]
Jones, S.; Evans, K.; McElwaine-Johnn, H.; Sharpe, M.; Oxford, J.; Lambkin-Williams, R.; Mant, T.; Nolan, A.; Zambon, M.; Ellis, J.; et al. DNA vaccination protects against an influenza challenge in a double-blind randomised placebo-controlled phase 1b clinical trial. Vaccine 2009, 27, 2506–2512, doi:10.1016/j.vaccine.2009.02.061.
[2]
Girard, M.P.; Bansal, G.P. HIV/AIDS vaccines: A need for new concepts? Int. Rev. Immunol. 2008, 27, 447–471, doi:10.1080/08830180802432160.
[3]
Jechlinger, W. Optimization and delivery of plasmid DNA for vaccination. Expert Rev. Vaccines 2006, 5, 803–825, doi:10.1586/14760584.5.6.803.
[4]
Miao, E.A.; Andersen-Nissen, E.; Warren, S.E.; Aderem, A. TLR5 and Ipaf: Dual sensors of bacterial flagellin in the innate immune system. Semin. Immunopathol. 2007, 29, 275–288, doi:10.1007/s00281-007-0078-z.
[5]
Applequist, S.E.; Rollman, E.; Wareing, M.D.; Liden, M.; Rozell, B.; Hinkula, J.; Ljunggren, H.G. Activation of innate immunity, inflammation, and potentiation of DNA vaccination through mammalian expression of the TLR5 agonist flagellin. J. Immunol. 2005, 175, 3882–3891.
[6]
Song, L.; Nakaar, V.; Kavita, U.; Price, A.; Huleatt, J.; Tang, J.; Jacobs, A.; Liu, G.; Huang, Y.; Desai, P.; et al. Efficacious recombinant influenza vaccines produced by high yield bacterial expression: A solution to global pandemic and seasonal needs. PLoS One 2008, 3, e2257, doi:10.1371/journal.pone.0002257.
[7]
Mizel, S.B.; Graff, A.H.; Sriranganathan, N.; Ervin, S.; Lees, C.J.; Lively, M.O.; Hantgan, R.R.; Thomas, M.J.; Wood, J.; Bell, B. Flagellin-F1-V fusion protein is an effective plague vaccine in mice and two species of nonhuman primates. Clin. Vaccine Immunol. 2009, 16, 21–28, doi:10.1128/CVI.00333-08.
[8]
Mizel, S.B.; Bates, J.T. Flagellin as an adjuvant: Cellular mechanisms and potential. J. Immunol. 2010, 185, 5677–5682, doi:10.4049/jimmunol.1002156.
[9]
Lycke, N. Recent progress in mucosal vaccine development: Potential and limitations. Nat. Rev. Immunol. 2012, 12, 592–605, doi:10.1038/nri3251.
[10]
Chen, W.; Patel, G.B.; Yan, H.; Zhang, J. Recent advances in the development of novel mucosal adjuvants and antigen delivery systems. Hum. Vaccine 2010, 6, 706–714, doi:10.4161/hv.6.9.11561.
[11]
Hinkula, J.; Bratt, G.; Gilljam, G.; Nordlund, S.; Broliden, P.A.; Holmberg, V.; Olausson-Hansson, E.; Albert, J.; Sandstrom, E.; Wahren, B. Immunological and virological interactions in patients receiving passive immunotherapy with HIV-1 neutralizing monoclonal antibodies. J. Acq. Immune Defic. Syndr. 1994, 7, 940–951.
[12]
Sutlu, T.; Nystrom, S.; Gilljam, M.; Stellan, B.; Applequist, S.E.; Alici, E. Inhibition of intracellular anti-viral defense mechanisms augments lentiviral transduction of human natural killer cells: Implications for gene therapy. Hum. Gene Ther. 2012, 23, 1090–1100, doi:10.1089/hum.2012.080.
[13]
Hinkula, J.; Devito, C.; Zuber, B.; Benthin, R.; Ferreira, D.; Wahren, B.; Schroder, U. A novel DNA adjuvant, N3, enhances mucosal and systemic immune responses induced by HIV-1 DNA and peptide immunizations. Vaccine 2006, 24, 4494–4497, doi:10.1016/j.vaccine.2005.08.015.
[14]
Petersson, P.; Hedenskog, M.; Alves, D.; Brytting, M.; Schroder, U.; Linde, A.; Lundkvist, A. The Eurocine L3 adjuvants with subunit influenza antigens induce protective immunity in mice after intranasal vaccination. Vaccine 2010, 28, 6491–6497, doi:10.1016/j.vaccine.2010.07.001.
[15]
Meyers, G.; Korber, B.; Foley, K.T.; Jeang, J.W.; Mellers, J.W.; Wain-Hobson, S. A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences; Los Alamos, Los Alamos National Laboratory: Washington, DC, USA, 1992.
[16]
Brave, A.; Hallengard, D.; Schroder, U.; Blomberg, P.; Wahren, B.; Hinkula, J. Intranasal immunization of young mice with a multigene HIV-1 vaccine in combination with the N3 adjuvant induces mucosal and systemic immune responses. Vaccine 2008, 26, 5075–5078, doi:10.1016/j.vaccine.2008.03.066.
[17]
Hinkula, J.; Hagbom, M.; Wahren, B.; Schroder, U. Safety and immunogenicity, after nasal application of HIV-1 DNA gagp37 plasmid vaccine in young mice. Vaccine 2008, 26, 5101–5106, doi:10.1016/j.vaccine.2008.03.098.
Devito, C.; Levi, M.; Broliden, K.; Hinkula, J. Mapping of B-cell epitopes in rabbits immunised with various gag antigens for the production of HIV-1 gag capture ELISA reagents. J. Immunol. Methods 2000, 238, 69–80, doi:10.1016/S0022-1759(00)00141-1.
[20]
Lightfield, K.L.; Persson, J.; Brubaker, S.W.; Witte, C.E.; von Moltke, J.; Dunipace, E.A.; Henry, T.; Sun, Y.H.; Cado, D.; Dietrich, W.F.; et al. Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin. Nat. Immunol. 2008, 9, 1171–1178, doi:10.1038/ni.1646.
Takeshita, F.; Ishii, K.J. Intracellular DNA sensors in immunity. Curr. Opin. Immunol. 2008, 20, 383–388.
[23]
Schroder, K.; Muruve, D.A.; Tschopp, J. Innate immunity: Cytoplasmic DNA sensing by the AIM2 inflammasome. Curr. Biol. 2009, 19, R262–R265, doi:10.1016/j.cub.2009.02.011.
[24]
Uematsu, S.; Fujimoto, K.; Jang, M.H.; Yang, B.G.; Jung, Y.J.; Nishiyama, M.; Sato, S.; Tsujimura, T.; Yamamoto, M.; Yokota, Y.; et al. Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5. Nat. Immunol. 2008, 9, 769–776, doi:10.1038/ni.1622.
[25]
Atif, S.M.; Uematsu, S.; Akira, S.; McSorley, S.J. CD103-CD11b+ dendritic cells regulate the sensitivity of CD4 T-cell responses to bacterial flagellin. Mucosal. Immunol. 2013, doi:10.1038/mi.2013.25.
[26]
Wiley, S.R.; Raman, V.S.; Desbien, A.; Bailor, H.R.; Bhardwaj, R.; Shakri, A.R.; Reed, S.G.; Chitnis, C.E.; Carter, D. Targeting TLRs expands the antibody repertoire in response to a malaria vaccine. Sci. Transl. Med. 2011, doi:10.1126/scitranslmed.3002135.
[27]
Vajdy, M.; Srivastava, I.; Polo, J.; Donnelly, J.; O’Hagan, D.; Singh, M. Mucosal adjuvants and delivery systems for protein-, DNA- and RNA-based vaccines. Immunol. Cell. Biol. 2004, 82, 617–627, doi:10.1111/j.1440-1711.2004.01288.x.
Han, T.K.; Dao, M.L. Enhancement of salivary IgA response to a DNA vaccine against Streptococcus mutans wall-associated protein A in mice by plasmid-based adjuvants. J. Med. Microbiol. 2007, 56, 675–680, doi:10.1099/jmm.0.47020-0.
[30]
Kataoka, K.; McGhee, J.R.; Kobayashi, R.; Fujihashi, K.; Shizukuishi, S. Nasal Flt3 ligand cDNA elicits CD11c+CD8+ dendritic cells for enhanced mucosal immunity. J. Immunol. 2004, 172, 3612–3619.
[31]
Melkebeek, V.; Sonck, E.; Verdonck, F.; Goddeeris, B.M.; Cox, E. Optimized FaeG expression and a thermolabile enterotoxin DNA adjuvant enhance priming of an intestinal immune response by an FaeG DNA vaccine in pigs. Clin. Vaccine Immunol. 2007, 14, 28–35, doi:10.1128/CVI.00268-06.
[32]
Van Gils, M.J.; Sanders, R.W. Broadly neutralizing antibodies against HIV-1: Templates for a vaccine. Virology 2013, 435, 46–56, doi:10.1016/j.virol.2012.10.004.
[33]
Girard, M.P.; Osmanov, S.K.; Kieny, M.P. A review of vaccine research and development: The human immunodeficiency virus (HIV). Vaccine 2006, 24, 4062–4081, doi:10.1016/j.vaccine.2006.02.031.
[34]
McGuire, A.T.; Hoot, S.; Dreyer, A.M.; Lippy, A.; Stuart, A.; Cohen, K.W.; Jardina, J.; Menis, S.; Scheid, J.F.; West, A.P.; et al. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies. J. Exp. Med. 2013, 210, 656–663.
[35]
Narayan, K.M.; Agrawal, N.; Du, S.X.; Murakana, J.E.; Bauer, K.; Leaman, D.P.; Phung, P.; Limoli, K.; Chen, H.; Boenig, R.I.; et al. Prime-boost immunization of rabbits with HIV-1 gp120 elicits potent neutralizing activity against a primary viral isolate. PLoS One 2013, 8, e52732.
[36]
Naito, S.; Maeyama, J.-I.; Mizukami, T.; Takahashi, M.; Hamaguchi, I.; Yamaguchi, K. Transcutaneous immunization by merely prolonging the duration of antigen presence in the skin of mice induces a potent antigen-specific immune response even in the absence of an adjuvant. Vaccine 2007, 25, 8762–8770, doi:10.1016/j.vaccine.2007.10.031.
[37]
Scharton-Kersten, T.; Glenn, G.M.; Vassell, R.; Yu, J.; Walwender, D.; Alving, C.R. Principles of transcutaneous immunization using cholera toxin as an adjuvant. Vaccine 1999, 17, S37–S43, doi:10.1016/S0264-410X(99)00233-9.
[38]
Kasturi, S.P.; Skountzou, I.; Albrecht, R.A.; Koutsonanos, D.; Hua, T.; Nakaya, H.I.; Ravindran, R.; Stewart, S.; Alam, M.; Kwissa, M.; et al. Programming the magnitude and persistence of antibody responses with innate immunity. Nature 2011, 470, 543–547, doi:10.1038/nature09737.
[39]
Napoliatani, G.; Rinaldi, A.; Bertoni, F.; Sallusto, F.; Lanzavecchia, A. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat. Immunol. 2005, 6, 769–776, doi:10.1038/ni1223.
[40]
Shen, S.-S.; Yang, Y.-W. Antigen delivery for cross-priming via emulsion vaccine adjuvants. Vaccine 2012, 30, 1560–1571, doi:10.1016/j.vaccine.2011.12.120.
[41]
Marichal, T.; Ohata, K.; Bedoret, D.; Mesnil, C.; Sabatel, C.; Kobiyama, K.; Lekeux, B.; Coban, C.; Ishii, K.J.; Bureau, F.; et al. DNA released from dying host cells mediate aluminium adjuvant activity. Nat. Med. 2011, 17, 996–1003, doi:10.1038/nm.2403.
[42]
Yang, Y.-W.; Wei, A.-C.; Shen, S.-S. The immunogenicity-enhancing effect of emulsion vaccine adjuvant independent of the dispersion type and antigen release rate—A revisit of the role of the hydrophile-lipophile balance (HLB) valus. Vaccine 2005, 23, 2665–2675, doi:10.1016/j.vaccine.2004.09.007.
[43]
O’Hagan, D.T.; Ott, G.S.; van Nest, G.; Rappuoli, R.; del Guidice, G. The history of M59 adjuvant: A phoenix that rose from the ashes. Exp. Rev. Vaccines 2013, 12, 13–30, doi:10.1586/erv.12.140.
[44]
Awasti, A.; Kuchroo, V.K. Th17 cells: From precursors to players in inflammation and infection. Int. Immunol. 2009, 20, 489–498.
[45]
Makidon, P.E.; Belyakov, I.M.; Blanco, L.P.; Janczak, K.W.; Landers, J.; Bielinska, A.U.; Groom, J.V., 2nd; Naker, J.R., Jr. Nanoemulsion mucosal adjuvant uniquely activates cytokine production by nasal epithelium and induces dendritic cell trafficing. Eur. J. Immunol. 2012, 42, 2073–2086, doi:10.1002/eji.201142346.
[46]
Albert, M.L.; Sauter, B.; Bhardwaj, N. Dendritic cells aquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 1998, 392, 86–89, doi:10.1038/32183.
[47]
Valensi, J.P.; Carlson, J.R.; van Nest, G.A. Systemic cytokine profiles in BALB/c mice immunized with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants. J. Immunol. 1994, 153, 4029–4039.
[48]
Van Duin, D.; Medzhitov, R.; Shaw, A.C. Triggering TLR signaling in vaccination. Trends Immunol. 2006, 27, 49–55, doi:10.1016/j.it.2005.11.005.
[49]
Honko, A.N.; Mizel, S.B. Mucosal administration of flagellin induces innate immunity in the mouse lung. Infect. Immun. 2004, 72, 6676–6679, doi:10.1128/IAI.72.11.6676-6679.2004.
[50]
Pritts, T.; Hungness, E.; Wang, Q.; Robb, B.; Hershko, D.; Hasselgren, P.O. Mucosal and enterocyte IL-6 production during sepsis and endotoxemia-role of transcription factors and regulation of stress response. Am. J. Surg. 2002, 183, 372–383, doi:10.1016/S0002-9610(02)00812-7.
[51]
Kinnebrew, M.A.; Buffie, C.G.; Diehl, G.E.; Zenewicz, L.A.; Leiner, I.; Hohl, T.M.; Flavelli, R.A.; Littman, D.R.; Pamer, E.G. Interleukin 23 production by intestinal CD103(+)CD11b(+) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defence. Immunity 2012, 36, 276–287, doi:10.1016/j.immuni.2011.12.011.
[52]
Van Maele, L.; Carnoy, C.; Cayet, D.; Songhet, P.; Dumoutier, L.; Ferrero, I.; Janot, L.; Erard, F.; Bertout, J.; Leger, H.; et al. TLR5 signaling stimulates the innate production of IL-17 and IL-22 by CD3(neg)CD127+ immune cells in spleen and mucosa. J. Immunol. 2010, 185, 1177–1185, doi:10.4049/jimmunol.1000115.
[53]
Krieg, A.M. CpG still rocks update on an accidental drug. Nucleic Acid Ther. 2012, 22, 77–89.
