The AAPS Journal

, 20:73 | Cite as

Vaccine Adjuvant Incorporation Strategy Dictates Peptide Amphiphile Micelle Immunostimulatory Capacity

  • Rui Zhang
  • Jake S. Kramer
  • Josiah D. Smith
  • Brittany N. Allen
  • Caitlin N. Leeper
  • Xiaolei Li
  • Logan D. Morton
  • Fabio Gallazzi
  • Bret D. UleryEmail author
Research Article Theme: Pioneering Pharmaceutical Science by Emerging Investigators
Part of the following topical collections:
  1. Theme: Pioneering Pharmaceutical Science by Emerging Investigators


Current vaccine research has shifted from traditional vaccines (i.e., whole-killed or live-attenuated) to subunit vaccines (i.e., protein, peptide, or DNA) as the latter is much safer due to delivering only the bioactive components necessary to produce a desirable immune response. Unfortunately, subunit vaccines are very weak immunogens requiring delivery vehicles and the addition of immunostimulatory molecules termed adjuvants to convey protective immunity. An interesting type of delivery vehicle is peptide amphiphile micelles (PAMs), unique biomaterials where the vaccine is part of the nanomaterial itself. Due to the modularity of PAMs, they can be readily modified to deliver both vaccine antigens and adjuvants within a singular construct. Through the co-delivery of a model antigenic epitope (Ovalbumin319–340—OVABT) and a known molecular adjuvant (e.g., 2,3-dipalmitoyl-S-glyceryl cysteine—Pam2C), greater insight into the mechanisms by which PAMs can exert immunostimulatory effects was gained. It was found that specific combinations of antigen and adjuvant can significantly alter vaccine immunogenicity both in vitro and in vivo. These results inform fundamental design rules that can be leveraged to fabricate optimal PAM-based vaccine formulations for future disease-specific applications.

Graphical Abstract


Adjuvant Co-localization Peptide amphiphile micelles Subunit vaccines 



We thank Professor Thomas Phillips, Professor Jeffrey Adamovicz, Alexis Dadelahi, and Dr. Curtis Pritzl for their useful input on this work. We also thank Biolegend technical support team for their assistance on flow cytometry and cytokine multiplex assays.

Funding Information

This work is supported by the University of Missouri start-up funding, the University of Missouri research council board, and the PhRMA Foundation.

Supplementary material

12248_2018_233_MOESM1_ESM.docx (681 kb)
ESM 1 (DOCX 681 kb)


  1. 1.
    Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID, Organization WH. Smallpox and its eradication. 1988.Google Scholar
  2. 2.
    Fine PE, Carneiro IA. Transmissibility and persistence of oral polio vaccine viruses: implications for the global poliomyelitis eradication initiative. Am J Epidemiol. 1999;150(10):1001–21.CrossRefPubMedGoogle Scholar
  3. 3.
    Mast E, Mahoney F, Kane M, Margolis H. Hepatitis B vaccine. Vaccines, 4th ed Philadelphia: WB Saunders Company 2004:299–338.Google Scholar
  4. 4.
    Babiuk LA. Broadening the approaches to developing more effective vaccines. Vaccine. 1999;17(13):1587–95.CrossRefPubMedGoogle Scholar
  5. 5.
    Brown F. Peptide vaccines: fantasy or reality? World J Microbiol Biotechnol. 1992;8:52–3.CrossRefPubMedGoogle Scholar
  6. 6.
    Levine MM, Sztein MB. Vaccine development strategies for improving immunization: the role of modern immunology. Nat Immunol. 2004;5(5):460–4.CrossRefPubMedGoogle Scholar
  7. 7.
    Zhang R, Ulery BD. Synthetic vaccine characterization and design. J Bionanosci. 2018;12(1):1–11.CrossRefGoogle Scholar
  8. 8.
    Chang TZ, Stadmiller SS, Staskevicius E, Champion JA. Effects of ovalbumin protein nanoparticle vaccine size and coating on dendritic cell processing. Biomater Sci. 2017;5(2):223–33.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Deng L, Mohan T, Chang TZ, Gonzalez GX, Wang Y, Kwon Y-M, et al. Double-layered protein nanoparticles induce broad protection against divergent influenza a viruses. Nat Commun. 2018;9(1):359.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Acharya AP, Clare-Salzler MJ, Keselowsky BG. A high-throughput microparticle microarray platform for dendritic cell-targeting vaccines. Biomaterials. 2009;30(25):4168–77.CrossRefPubMedGoogle Scholar
  11. 11.
    Wang L, Chang TZ, He Y, Kim JR, Wang S, Mohan T, et al. Coated protein nanoclusters from influenza H7N9 HA are highly immunogenic and induce robust protective immunity. Nanomedicine. 2017;13(1):253–62.CrossRefPubMedGoogle Scholar
  12. 12.
    Ross K, Adams J, Loyd H, Ahmed S, Sambol A, Broderick S, et al. Combination nanovaccine demonstrates synergistic enhancement in efficacy against influenza. ACS Biomater Sci Eng. 2016;2(3):368–74.CrossRefGoogle Scholar
  13. 13.
    Ross KA, Loyd H, Wu W, Huntimer L, Ahmed S, Sambol A, et al. Polyanhydride-based H5 hemagglutinin influenza nanovaccines elicit protective virus neutralizing titers and cell-mediated immunity. Synthetic nanoparticle-based vaccines against respiratory pathogens 2013:149.Google Scholar
  14. 14.
    An M, Liu H. Dissolving microneedle arrays for transdermal delivery of amphiphilic vaccines. Small. 2017;13(26)Google Scholar
  15. 15.
    Hanson MC, Abraham W, Crespo MP, Chen SH, Liu H, Szeto GL, et al. Liposomal vaccines incorporating molecular adjuvants and intrastructural T-cell help promote the immunogenicity of HIV membrane-proximal external region peptides. Vaccine. 2015;33(7):861–8.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Ulery BD, Kumar D, Ramer-Tait AE, Metzger DW, Wannemuehler MJ, Narasimhan B. Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases. PLoS One. 2011;6(3):e17642.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Moon JJ, Suh H, Bershteyn A, Stephan MT, Liu H, Huang B, et al. Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses. Nat Mater. 2011;10(3):243–51.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Zhang P, Chiu Y-C, Tostanoski LH, Jewell CM. Polyelectrolyte multilayers assembled entirely from immune signals on gold nanoparticle templates promote antigen-specific T cell response. ACS Nano. 2015;9(6):6465–77.CrossRefPubMedGoogle Scholar
  19. 19.
    Tsoras AN, Champion JA. Cross-linked peptide nanoclusters for delivery of oncofetal antigen as a cancer vaccine. Bioconjug Chem. 2018;29:776–85.CrossRefPubMedGoogle Scholar
  20. 20.
    Barrett JC, Ulery BD, Trent A, Liang S, David NA, Tirrell MV. Modular peptide Amphiphile micelles improving an antibody-mediated immune response to group A Streptococcus. ACS Biomater Sci Eng. 2016;Google Scholar
  21. 21.
    Trent A, Ulery BD, Black MJ, Barrett JC, Liang S, Kostenko Y, et al. Peptide amphiphile micelles self-adjuvant group A streptococcal vaccination. AAPS J. 2015;17(2):380–8.CrossRefPubMedGoogle Scholar
  22. 22.
    Zhang R, Smith JD, Kramer Jake S, Allen BN, Martin S, Ulery BD. Peptide amphiphile micelle vaccine size and charge influences immunogenicity. ACS Biomater Sci Eng 2018;Submitted.Google Scholar
  23. 23.
    Zhang R, Morton LD, Smith JD, Gallazzi F, White TA, Ulery BD. Instructive design of tri-block peptide amphiphiles for structurally complex micelle formation. ACS Biomater Sci Eng 2018;Accepted.Google Scholar
  24. 24.
    Barrett JC, Ulery BD, Trent A, Liang S, David NA, Tirrell M. Modular peptide amphiphile micelles improve an antibody-mediated immune response to group A Streptococcus. ACS Biomater Sci Eng. 2016;3(2):144–52.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Sun T, Han H, Hudalla GA, Wen Y, Pompano RR, Collier JH. Thermal stability of self-assembled peptide vaccine materials. Acta Biomater. 2016;30:62–71.CrossRefPubMedGoogle Scholar
  26. 26.
    Chen J, Pompano RR, Santiago FW, Maillat L, Sciammas R, Sun T, et al. The use of self-adjuvanting nanofiber vaccines to elicit high-affinity B cell responses to peptide antigens without inflammation. Biomaterials. 2013;34(34):8776–85.CrossRefPubMedGoogle Scholar
  27. 27.
    Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity. 1999;11(4):443–51.CrossRefPubMedGoogle Scholar
  28. 28.
    Takeuchi O, Takeda K, Hoshino K, Adachi O, Ogawa T, Akira S. Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades. Int Immunol. 2000;12(1):113–7.CrossRefPubMedGoogle Scholar
  29. 29.
    Basto AP, Leitão A. Targeting TLR2 for vaccine development. J Immunol Res. 2014;2014:1–22.CrossRefGoogle Scholar
  30. 30.
    Chiu Y-C, Gammon JM, Andorko JI, Tostanoski LH, Jewell CM. Modular vaccine design using carrier-free capsules assembled from polyionic immune signals. ACS Biomater Sci Eng. 2015;1(12):1200–5.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Kuai R, Ochyl LJ, Bahjat KS, Schwendeman A, Moon JJ. Designer vaccine nanodiscs for personalized cancer immunotherapy. Nat Mater. 2016;Google Scholar
  32. 32.
    de Jong S, Chikh G, Sekirov L, Raney S, Semple S, Klimuk S, et al. Encapsulation in liposomal nanoparticles enhances the immunostimulatory, adjuvant and anti-tumor activity of subcutaneously administered CpG ODN. Cancer Immunol Immunother. 2007;56(8):1251–64.CrossRefPubMedGoogle Scholar
  33. 33.
    Joshi VB, Geary SM, Salem AK. Biodegradable particles as vaccine delivery systems: size matters. AAPS J. 2013;15(1):85–94.CrossRefPubMedGoogle Scholar
  34. 34.
    Keselowsky BG, Xia CQ, Clare-Salzler M. Multifunctional dendritic cell-targeting polymeric microparticles: engineering new vaccines for type 1 diabetes. Human Vaccines. 2011;7(1):37–44.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Sevimli S, Knight FC, Gilchuk P, Joyce S, Wilson JT. Fatty acid-mimetic micelles for dual delivery of antigens and Imidazoquinoline adjuvants. ACS Biomater Sci Eng. 2016;3(2):179–94.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Hudalla GA, Modica JA, Tian YF, Rudra JS, Chong AS, Sun T, et al. A self-adjuvanting supramolecular vaccine carrying a folded protein antigen. Adv Healthc Mater. 2013;2(8):1114–9.CrossRefPubMedGoogle Scholar
  37. 37.
    An M, Li M, Xi J, Liu H. Silica nanoparticle as a lymph node targeting platform for vaccine delivery. ACS Appl Mater Interfaces. 2017;9(28):23466–75.CrossRefPubMedGoogle Scholar
  38. 38.
    Zom GG, Khan S, Britten CM, Sommandas V, Camps MG, Loof NM, et al. Efficient induction of antitumor immunity by synthetic toll-like receptor ligand–peptide conjugates. Cancer Immunol Res. 2014;2(8):756–64.CrossRefPubMedGoogle Scholar
  39. 39.
    Denton AE, Wesselingh R, Gras S, Guillonneau C, Olson MR, Mintern JD, et al. Affinity thresholds for naive CD8+ CTL activation by peptides and engineered influenza A viruses. J Immunol. 2011;187(11):5733–44.CrossRefPubMedGoogle Scholar
  40. 40.
    Moyle PM, Dai W, Zhang Y, Batzloff MR, Good MF, Toth I. Site-specific incorporation of three toll-like receptor 2 targeting adjuvants into semisynthetic, molecularly defined nanoparticles: application to group a streptococcal vaccines. Bioconjug Chem. 2014;25(5):965–78.CrossRefPubMedGoogle Scholar
  41. 41.
    Shime H, Maruyama A, Yoshida S, Takeda Y, Matsumoto M, Seya T. Toll-like receptor 2 ligand and interferon-γ suppress anti-tumor T cell responses by enhancing the immunosuppressive activity of monocytic myeloid-derived suppressor cells. Oncoimmunology. 2018;7(1):e1373231.CrossRefGoogle Scholar
  42. 42.
    Dietrich N, Lienenklaus S, Weiss S, Gekara NO. Murine toll-like receptor 2 activation induces type I interferon responses from endolysosomal compartments. PLoS One. 2010;5(4):e10250.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Dowling JK, Dellacasagrande J. Toll-like receptors: ligands, cell-based models, and readouts for receptor action. Toll-Like Receptors: Springer. 2016:3–27.Google Scholar
  44. 44.
    Kulsantiwong P, Pudla M, Srisaowakarn C, Boondit J, Utaisincharoen P. Pam2CSK4 and Pam3CSK4 induce iNOS expression via TBK1 and MyD88 molecules in mouse macrophage cell line RAW264. 7. Inflamm Res. 2017;66(10):843–53.CrossRefPubMedGoogle Scholar
  45. 45.
    Natarajan M, Lin K-M, Hsueh RC, Sternweis PC, Ranganathan R. A global analysis of cross-talk in a mammalian cellular signalling network. Nat Cell Biol. 2006;8(6):571–80.CrossRefPubMedGoogle Scholar
  46. 46.
    Black M, Trent A, Kostenko Y, Lee JS, Olive C, Tirrell M. Self-assembled peptide amphiphile micelles containing a cytotoxic T-cell epitope promote a protective immune response in vivo. Adv Mater. 2012;24(28):3845–9.CrossRefPubMedGoogle Scholar
  47. 47.
    Fagan V, Hussein WM, Su M, Giddam AK, Batzloff MR, Good MF, et al. Synthesis, characterization and immunological evaluation of self-adjuvanting group A Streptococcal vaccine candidates bearing various lipidic adjuvanting moieties. Chembiochem. 2017;18(6):545–53.CrossRefPubMedGoogle Scholar
  48. 48.
    Kang JY, Nan X, Jin MS, Youn S-J, Ryu YH, Mah S, et al. Recognition of lipopeptide patterns by toll-like receptor 2-toll-like receptor 6 heterodimer. Immunity. 2009;31(6):873–84.CrossRefPubMedGoogle Scholar
  49. 49.
    Agnihotri G, Crall BM, Lewis TC, Day TP, Balakrishna R, Warshakoon HJ, et al. Structure–activity relationships in toll-like receptor 2-agonists leading to simplified monoacyl lipopeptides. J Med Chem. 2011;54(23):8148–60.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Wu W, Li R, Malladi SS, Warshakoon HJ, Kimbrell MR, Amolins MW, et al. Structure− activity relationships in toll-like receptor-2 agonistic diacylthioglycerol lipopeptides. J Med Chem. 2010;53(8):3198–213.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Azuma M, Sawahata R, Akao Y, Ebihara T, Yamazaki S, Matsumoto M, et al. The peptide sequence of diacyl lipopeptides determines dendritic cell TLR2-mediated NK activation. PLoS One. 2010;5(9):e12550.CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Fujimoto Y, Hashimoto M, Furuyashiki M, Katsumoto M, Seya T, Suda Y, et al. Lipopeptides from Staphylococcus aureus as Tlr2 ligands: prediction with mrna expression, chemical synthesis, and immunostimulatory activities. Chembiochem. 2009;10(14):2311–5.CrossRefPubMedGoogle Scholar
  53. 53.
    Kasturi SP, Skountzou I, Albrecht RA, Koutsonanos D, Hua T, Nakaya HI, et al. Programming the magnitude and persistence of antibody responses with innate immunity. Nature. 2011;470(7335):543–7.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Mohsen MO, Gomes AC, Cabral-Miranda G, Krueger CC, Leoratti FM, Stein JV, et al. Delivering adjuvants and antigens in separate nanoparticles eliminates the need of physical linkage for effective vaccination. J Control Release. 2017;251:92–100.CrossRefPubMedGoogle Scholar
  55. 55.
    Nomura F, Akashi S, Sakao Y, Sato S, Kawai T, Matsumoto M, et al. Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression. J Immunol. 2000;164(7):3476–9.CrossRefPubMedGoogle Scholar
  56. 56.
    Akashi S, Saitoh S-i, Wakabayashi Y, Kikuchi T, Takamura N, Nagai Y, et al. Lipopolysaccharide interaction with cell surface toll-like receptor 4-MD-2. J Exp Med. 2003;198(7):1035–42.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Triantafilou M, Gamper FG, Haston RM, Mouratis MA, Morath S, Hartung T, et al. Membrane sorting of toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting. J Biol Chem. 2006;281(41):31002–11.CrossRefPubMedGoogle Scholar
  58. 58.
    Kužnik A, Benčina M, Švajger U, Jeras M, Rozman B, Jerala R. Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines. J Immunol. 2011;186(8):4794–804.CrossRefPubMedGoogle Scholar
  59. 59.
    Takeshita F, Gursel I, Ishii KJ, Suzuki K, Gursel M, Klinman DM, editors. Signal transduction pathways mediated by the interaction of CpG DNA with toll-like receptor 9. Semin Immunol; 2004: Elsevier.Google Scholar
  60. 60.
    Napolitani 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(8):769–76.CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Neefjes J, Jongsma ML, Paul P, Bakke O. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol. 2011;11(12):823–36.CrossRefPubMedGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  1. 1.Department of Chemical EngineeringUniversity of MissouriColumbiaUSA
  2. 2.Department of BiochemistryUniversity of MissouriColumbiaUSA
  3. 3.Department of BioengineeringUniversity of MissouriColumbiaUSA
  4. 4.Molecular interactions CoreUniversity of MissouriColumbiaUSA

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