The efficacy of vaccine adjuvants such as Toll-like receptor agonists (TLRa) can be improved through formulation and delivery approaches. Here, we attached small molecule TLR-7/8a to polymer scaffolds (polymer–TLR-7/8a) and evaluated how different physicochemical properties of the TLR-7/8a and polymer carrier influenced the location, magnitude and duration of innate immune activation in vivo. Particle formation by polymer–TLR-7/8a was the most important factor for restricting adjuvant distribution and prolonging activity in draining lymph nodes. The improved pharmacokinetic profile by particulate polymer–TLR-7/8a was also associated with reduced morbidity and enhanced vaccine immunogenicity for inducing antibodies and T cell immunity. We extended these findings to the development of a modular approach in which protein antigens are site-specifically linked to temperature-responsive polymer–TLR-7/8a adjuvants that self-assemble into immunogenic particles at physiologic temperatures in vivo. Our findings provide a chemical and structural basis for optimizing adjuvant design to elicit broad-based antibody and T cell responses with protein antigens.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Correlates of protection induced by vaccination. Clin. Vaccine Immunol. 17, 1055–1065 (2010).

  2. 2.

    et al. Live attenuated malaria vaccine designed to protect through hepatic CD8+ T cell immunity. Science 334, 475–480 (2011).

  3. 3.

    & Tuberculosis vaccines—rethinking the current paradigm. Trends Immunol. 35, 387–395 (2014).

  4. 4.

    , , , & Prospects of combinatorial synthetic peptide vaccine-based immunotherapy against cancer. Semin. Immunol. 25, 182–190 (2013).

  5. 5.

    The perfect mix: recent progress in adjuvant research. Nat. Rev. Microbiol. 5, 505–517 (2007).

  6. 6.

    & Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5, 987–995 (2004).

  7. 7.

    , , & Status and future prospects of lipid-based particulate delivery systems as vaccine adjuvants and their combination with immunostimulators. Expert Opin. Drug Deliv. 6, 657–672 (2009).

  8. 8.

    & Designing and building the next generation of improved vaccine adjuvants. J. Control. Release 190C, 563–579 (2014).

  9. 9.

    et al. TLR4 ligand formulation causes distinct effects on antigen-specific cell-mediated and humoral immune responses. Vaccine 31, 5848–5855 (2013).

  10. 10.

    et al. HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1 and CD8+ T cell responses in nonhuman primates. Proc. Natl. Acad. Sci. USA 102, 15190–15194 (2005).

  11. 11.

    et al. Toward self-adjuvanting subunit vaccines: model peptide and protein antigens incorporating covalently bound toll-like receptor-7 agonistic imidazoquinolines. Bioorg. Med. Chem. Lett. 21, 3232–3236 (2011).

  12. 12.

    et al. Immunotherapeutic activity of a conjugate of a Toll-like receptor 7 ligand. Proc. Natl. Acad. Sci. USA 104, 3990–3995 (2007).

  13. 13.

    et al. Programming the magnitude and persistence of antibody responses with innate immunity. Nature 470, 543–547 (2011).

  14. 14.

    , & In situ engineering of the lymph node microenvironment via intranodal injection of adjuvant-releasing polymer particles. Proc. Natl. Acad. Sci. USA 108, 15745–15750 (2011).

  15. 15.

    et al. Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release. Vaccine 32, 2882–2895 (2014).

  16. 16.

    et al. Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction. Proc. Natl. Acad. Sci. USA 109, 1080–1085 (2012).

  17. 17.

    , , & Vaccine adjuvant activity of 3M-052: an imidazoquinoline designed for local activity without systemic cytokine induction. Vaccine 29, 5434–5442 (2011).

  18. 18.

    et al. Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 519–522 (2014).

  19. 19.

    et al. Rational design of small molecules as vaccine adjuvants. Sci. Transl. Med. 6, 263ra160 (2014).

  20. 20.

    & Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat. Rev. Immunol. 10, 787–796 (2010).

  21. 21.

    et al. Nanoparticles target distinct dendritic cell populations according to their size. Eur. J. Immunol. 38, 1404–1413 (2008).

  22. 22.

    , , , & In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles. J. Control. Release 112, 26–34 (2006).

  23. 23.

    et al. Peripherally administered nanoparticles target monocytic myeloid cells, secondary lymphoid organs and tumors in mice. PLoS ONE 8, e61646 (2013).

  24. 24.

    et al. Synthetic TLR Agonists reveal functional differences between human TLR7 and TLR8. J. Immunol. 174, 1259–1268 (2005).

  25. 25.

    , , & TLR7 enables cross-presentation by multiple dendritic cell subsets through a type I IFN-dependent pathway. Blood 118, 3028–3038 (2011).

  26. 26.

    , & Vaccine adjuvants: putting innate immunity to work. Immunity 33, 492–503 (2010).

  27. 27.

    et al. Synthesis and structure-activity-relationships of 1H-imidazo[4,5-c]quinolines that induce interferon production. J. Med. Chem. 48, 3481–3491 (2005).

  28. 28.

    , & Beyond a decade of 5% imiquimod topical therapy. J. Drugs Dermatol. 8, 467–474 (2009).

  29. 29.

    et al. A phase I clinical trial of imiquimod, an oral interferon inducer, administered daily. Br. J. Cancer 74, 1482–1486 (1996).

  30. 30.

    et al. Oral resiquimod in chronic HCV infection: safety and efficacy in 2 placebo-controlled, double-blind phase IIa studies. J. Hepatol. 47, 174–182 (2007).

  31. 31.

    et al. Hepatic drug targeting: phase I evaluation of polymer-bound doxorubicin. J. Clin. Oncol. 20, 1668–1676 (2002).

  32. 32.

    , & Beyond oncology—application of HPMA copolymers in non-cancerous diseases. Adv. Drug Deliv. Rev. 62, 258–271 (2010).

  33. 33.

    , , & Effect of molecular weight (Mbarw) of N-(2-hydroxypropyl)methacrylamide copolymers on body distribution and rate of excretion after subcutaneous, intraperitoneal, and intravenous administration to rats. J. Biomed. Mater. Res. 21, 1341–1358 (1987).

  34. 34.

    et al. Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome. Proc. Natl. Acad. Sci. USA 106, 870–875 (2009).

  35. 35.

    et al. Small molecule Toll-like receptor 7 agonists localize to the MHC class II loading compartment of human plasmacytoid dendritic cells. Blood 117, 5683–5691 (2011).

  36. 36.

    , , , & Stimulation of innate immune cells by light-activated TLR7/8 agonists. J. Am. Chem. Soc. 136, 10823–10825 (2014).

  37. 37.

    , , & Characterization of chemically defined poly-N-isopropylacrylamide based copolymeric adjuvants. Vaccine 31, 3519–3527 (2013).

  38. 38.

    et al. Novel vaccine adjuvant LPS-Hydrogel for truncated basic fibroblast growth factor to induce antitumor immunity. Carbohydr. Polym. 89, 1101–1109 (2012).

  39. 39.

    et al. Hydrogel-delivered GM-CSF overcomes nonresponsiveness to hepatitis B vaccine through the recruitment and activation of dendritic cells. J. Immunol. 185, 5468–5475 (2010).

  40. 40.

    & Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature 440, 808–812 (2006).

  41. 41.

    et al. TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation. Cell 158, 506–521 (2014).

  42. 42.

    , , & Self-assembling peptide-polymer hydrogels designed from the coiled coil region of fibrin. Biomacromolecules 9, 2438–2446 (2008).

  43. 43.

    & The coiled coil motif in polymer drug delivery systems. Biotechnol. Adv. 31, 90–96 (2013).

  44. 44.

    et al. Protective T cell immunity in mice following protein-TLR7/8 agonist-conjugate immunization requires aggregation, type I IFN, and multiple DC subsets. J. Clin. Invest. 121, 1782–1796 (2011).

  45. 45.

    et al. Conjugation of a TLR7 agonist and antigen enhances protection in the S. pneumoniae murine infection model. Eur. J. Pharm. Biopharm. 87, 310–317 (2014).

  46. 46.

    et al. Dendritic cell activation and T cell priming with adjuvant- and antigen-loaded oxidation-sensitive polymersomes. Biomaterials 33, 6211–6219 (2012).

  47. 47.

    , & Membrane anchored immunostimulatory oligonucleotides for in vivo cell modification and localized immunotherapy. Angew. Chem. Intl. Edn. Engl. 50, 7052–7055 (2011).

  48. 48.

    & Phosphorothioate oligodeoxynucleotides: antisense or anti-protein? Antisense Res. Dev. 5, 241 (1995).

  49. 49.

    et al. DEC-205 is a cell surface receptor for CpG oligonucleotides. Proc. Natl. Acad. Sci. USA 109, 16270–16275 (2012).

  50. 50.

    et al. Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose. Proc. Natl. Acad. Sci. USA 110, 19902–19907 (2013).

  51. 51.

    , , , & Structure-activity relationships in human toll-like receptor 7-active imidazoquinoline analogs. J. Med. Chem. 53, 4450–4465 (2010).

  52. 52.

    et al. New bioerodable thermoresponsive polymers for possible radiotherapeutic applications. J. Control. Release 119, 25–33 (2007).

  53. 53.

    & Synthesis and properties of new N-(2-hydroxypropyl)-methacrylamide copolymers containing thiazolidine-2-thione reactive groups. React. Funct. Polym. 66, 1525–1538 (2006).

  54. 54.

    et al. Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-111f) formulated in MPL adjuvant. Vaccine 20, 3292–3303 (2002).

  55. 55.

    , , , & Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes. Immunity 37, 364–376 (2012).

  56. 56.

    et al. Comparative analysis of the magnitude, quality, phenotype, and protective capacity of simian immunodeficiency virus gag-specific CD8+ T cells following human-, simian-, and chimpanzee-derived recombinant adenoviral vector immunization. J. Immunol. 190, 2720–2735 (2013).

  57. 57.

    et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat. Med. 13, 843–850 (2007).

  58. 58.

    , & T-cell quality in memory and protection: implications for vaccine design. Nat. Rev. Immunol. 8, 247–258 (2008).

Download references


The authors wish to acknowledge M. Dillon, K. Wuddie and C. Chiedi at the Vaccine Research Center and B. Klaunberg and V. Diaz at the Mouse Imaging Facility (MIF) for their valuable support and assistance with the animal studies. We would also like to thank K. Ulbrich, R. Swenson and G. Griffiths for their support and helpful insights. This work was supported in part by the BIOPOL project (Grant of the Ministry of Education, Youth and Sports of the Czech Republic, no. EE2.3.30.0029); by the Czech Science Foundation (15-15181S); by Charles University (UNCE 204025/2012); by a Cancer Research UK grant (C552/A17720); and by the Office of AIDS Research and the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health.

Author information

Author notes

    • Geoffrey M Lynn
    •  & Richard Laga

    These authors contributed equally to this work.

    • Leonard W Seymour
    •  & Robert A Seder

    These authors jointly supervised this work.


  1. Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.

    • Geoffrey M Lynn
    • , Patricia A Darrah
    • , Andrew S Ishizuka
    • , Alexandra J Balaci
    • , Ayako Yamamoto
    • , Connor R Buechler
    • , Kylie M Quinn
    • , Kathrin Kastenmüller
    • , Joseph R Francica
    •  & Robert A Seder
  2. Department of Oncology, University of Oxford, Oxford, UK.

    • Geoffrey M Lynn
    • , Richard Laga
    • , Ryan Cawood
    • , Thomas Hills
    • , Kerry D Fisher
    •  & Leonard W Seymour
  3. Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

    • Richard Laga
    • , Michal Pechar
    • , Robert Pola
    •  & Tomas Etrych
  4. Imaging Probe Development Center, National Heart, Lung, and Blood Institute, NIH, Rockville, Maryland, USA.

    • Andrés E Dulcey
    •  & Olga Vasalatiy
  5. Lymphocyte Biology Section, Laboratory of Systems Biology, NIAID, NIH, Bethesda, Maryland, USA.

    • Michael Y Gerner
  6. Biological Imaging Section, Research Technologies Branch, NIAID, NIH, Bethesda, Maryland, USA.

    • Margery G Smelkinson
  7. Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

    • Ondrej Vanek
  8. Department of Chemistry, University of California, Irvine, Irvine, California, USA.

    • Lalisa Stutts
    • , Janine K Tom
    • , Keun Ah Ryu
    •  & Aaron P Esser-Kahn


  1. Search for Geoffrey M Lynn in:

  2. Search for Richard Laga in:

  3. Search for Patricia A Darrah in:

  4. Search for Andrew S Ishizuka in:

  5. Search for Alexandra J Balaci in:

  6. Search for Andrés E Dulcey in:

  7. Search for Michal Pechar in:

  8. Search for Robert Pola in:

  9. Search for Michael Y Gerner in:

  10. Search for Ayako Yamamoto in:

  11. Search for Connor R Buechler in:

  12. Search for Kylie M Quinn in:

  13. Search for Margery G Smelkinson in:

  14. Search for Ondrej Vanek in:

  15. Search for Ryan Cawood in:

  16. Search for Thomas Hills in:

  17. Search for Olga Vasalatiy in:

  18. Search for Kathrin Kastenmüller in:

  19. Search for Joseph R Francica in:

  20. Search for Lalisa Stutts in:

  21. Search for Janine K Tom in:

  22. Search for Keun Ah Ryu in:

  23. Search for Aaron P Esser-Kahn in:

  24. Search for Tomas Etrych in:

  25. Search for Kerry D Fisher in:

  26. Search for Leonard W Seymour in:

  27. Search for Robert A Seder in:


G.M.L., R.L., K.D.F., L.W.S. and R.A.S were involved in experimental planning, interpreting data and writing the manuscript. G.M.L., R.L., A.E.D., O. Vasalatiy, J.K.T., L.S., K.A.R. and A.P.E.-K. planned and carried out the synthesis, purification and characterization of small molecules. R.L., R.P., M.P., T.E., O. Vanek and G.M.L. planned and completed the synthesis, purification and characterization of the polymer precursors and polymer conjugates. P.A.D, A.S.I., A.J.B., A.Y., K.M.Q., C.R.B., K.K. and J.R.F. planned and conducted many of the biological studies. M.Y.G. and M.G.S. carried out the confocal microscopy studies on lymph node sections and polymer particles, respectively. T.H. and R.C. developed the plasmids to express the HIV Gag-coil fusion protein. R.L., M.P., R.P. and T.E. devised the coil-coil strategy. A.P.E.-K., T.E., K.D.F., L.W.S. and R.A.S. are principal investigators who advised the studies.

Competing interests

G.M.L., R.L., K.D.F., L.W.S. and R.A.S. are listed as inventors on patents describing polymer-based vaccines. K.D.F. and L.W.S. are scientific founders and equity holders in PsiOxus Therapeutics, Ltd. (Oxford, UK). G.M.L. and J.R.F. are scientific founders and equity holders in Avidea Technologies, Inc., which is developing polymer-based technologies for immunotherapeutic applications (Baltimore, Maryland, USA).

Corresponding author

Correspondence to Robert A Seder.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15 and Supplementary Methods

About this article

Publication history






Further reading