Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Development of a minimal saponin vaccine adjuvant based on QS-21


Adjuvants are materials added to vaccines to enhance the immunological response to an antigen. QS-21 is a natural product adjuvant under investigation in numerous vaccine clinical trials, but its use is constrained by scarcity, toxicity, instability and an enigmatic molecular mechanism of action. Herein we describe the development of a minimal QS-21 analogue that decouples adjuvant activity from toxicity and provides a powerful platform for mechanistic investigations. We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required. Biodistribution studies revealed that active adjuvants were retained preferentially at the injection site and the nearest draining lymph nodes compared with the attenuated variants. Overall, these studies have yielded critical insights into saponin structure–function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Aryl iodide saponin 6 exhibits potent adjuvant activity and low toxicity in a preclinical mouse-vaccination model.
Figure 2: Radioiodinated saponin [131I]-6 and fluorescent saponin 3 localize to lymph nodes and injection site in mice.
Figure 3: Truncated saponin 16 lacks the entire branched trisaccharide domain of QS-21 but retains potent adjuvant activity and low toxicity in a preclinical mouse-vaccination model.
Figure 4: Oleanolic acid derivative 18, which lacks both the C4-aldehyde substituent and the C16-alcohol in the triterpene domain of QS-21, exhibits poor adjuvant activity in a preclinical mouse-vaccination model.
Figure 5: Caulophyllogenin derivative 19 and echinocystic acid derivative 20, which lack the C4-aldehyde substituent but retain the C16-alcohol in the triterpene domain of QS-21, exhibit potent adjuvant activity and no toxicity in a preclinical mouse-vaccination model.
Figure 6: Adjuvant-active quillaic acid derivative 16 localizes to the injection site and lymph nodes in mice, but adjuvant-attenuated oleanolic acid derivative 18 does not.


  1. Moyle, P. M. & Toth, I. Modern subunit vaccines: development, components, and research opportunities. ChemMedChem 8, 360–376 (2013).

    CAS  Article  Google Scholar 

  2. Leroux-Roels, G. Unmet needs in modern vaccinology: adjuvants to improve the immune response. Vaccine 28, C25–C36 (2010).

    Article  Google Scholar 

  3. Reed, S. G. et al. New horizons in adjuvants for vaccine development. Trends Immunol. 30, 23–32 (2009).

    CAS  Article  Google Scholar 

  4. Kensil, C. R., Patel, U., Lennick, M. & Marciani, D. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. J. Immunol. 146, 431–437 (1991).

    CAS  PubMed  Google Scholar 

  5. Kim, S. K. et al. Comparison of the effect of different immunological adjuvants on the antibody and T-cell response to immunization with MUC1–KLH and GD3–KLH conjugate cancer vaccines. Vaccine 18, 597–603 (1999).

    CAS  Article  Google Scholar 

  6. Soltysik, S., Bedore, D. A. & Kensil, C. R. Adjuvant activity of QS-21 isomers. Ann. NY Acad. Sci. 690, 392–395 (1993).

    CAS  Article  Google Scholar 

  7. Jacobsen, N. E. et al. Structure of the saponin adjuvant QS-21 and its base-catalyzed isomerization product by 1H and natural abundance 13C NMR spectroscopy. Carbohydr. Res. 280, 1–14 (1996).

    CAS  Article  Google Scholar 

  8. Ragupathi, G., Gardner, J. R., Livingston, P. O. & Gin, D. Y. Natural and synthetic saponin adjuvant QS-21 for vaccines against cancer. Expert Rev. Vaccines 10, 463–470 (2011).

    CAS  Article  Google Scholar 

  9. Agnandji, S. T. et al. First results of Phase 3 trial of RTS,S/AS01 malaria vaccine in African children. New Engl. J. Med. 365, 1863–1875 (2011).

    Article  Google Scholar 

  10. Kennedy, J. S. et al. The safety and tolerability of an HIV-1 DNA prime-protein boost vaccine (DP6-001) in healthy adult volunteers. Vaccine 26, 4420–4424 (2008).

    CAS  Article  Google Scholar 

  11. Vandepapeliere, P. et al. Vaccine adjuvant systems containing monophosphoryl lipid A and QS21 induce strong and persistent humoral and T cell responses against hepatitis B surface antigen in healthy adult volunteers. Vaccine 26, 1375–1386 (2008).

    CAS  Article  Google Scholar 

  12. Von Eschen, K. et al. The candidate tuberculosis vaccine Mtb72F/AS02A: tolerability and immunogenicity in humans. Hum. Vaccin. 5, 475–482 (2009).

    CAS  Article  Google Scholar 

  13. Vellas, B. et al. Long-term follow-up of patients immunized with AN1792: reduced functional decline in antibody responders. Curr. Alzheimer Res. 6, 144–151 (2009).

    CAS  Article  Google Scholar 

  14. Wang, P., Kim, Y. J., Navarro-Villalobos, M., Rohde, B. D. & Gin, D. Y. Synthesis of the potent immunostimulatory adjuvant QS-21A. J. Am. Chem. Soc. 127, 3256–3257 (2005).

    CAS  Article  Google Scholar 

  15. Deng, K. et al. Synthesis of QS-21-xylose: establishment of the immunopotentiating activity of synthetic QS-21 adjuvant with a melanoma vaccine. Angew. Chem. Int. Ed. 47, 6395–6398 (2008).

    CAS  Article  Google Scholar 

  16. Adams, M. M. et al. Design and synthesis of potent Quillaja saponin vaccine adjuvants. J. Am. Chem. Soc. 132, 1939–1945 (2010).

    CAS  Article  Google Scholar 

  17. Chea, E. K. et al. Synthesis and preclinical evaluation of QS-21 variants leading to simplified vaccine adjuvants and mechanistic probes. J. Am. Chem. Soc. 134, 13448–13457 (2012).

    CAS  Article  Google Scholar 

  18. Soltysik, S. et al. Structure/function studies of QS-21 adjuvant: assessment of triterpene aldehyde and glucuronic acid roles in adjuvant function. Vaccine 13, 1403–1410 (1995).

    CAS  Article  Google Scholar 

  19. Seevers, R. H. & Counsell, R. E. Radioiodination techniques for small organic molecules. Chem. Rev. 82, 575–590 (1982).

    CAS  Article  Google Scholar 

  20. Duewell, P. et al. ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells. J. Immunol. 187, 55–63 (2011).

    CAS  Article  Google Scholar 

  21. Scott, M. T., Goss-Sampson, M. & Bomford, R. Adjuvant activity of saponin: antigen localization studies. Int. Arch. Allergy Appl. Immunol. 77, 409–412 (1985).

    CAS  Article  Google Scholar 

  22. Elliott, D. F. & Kon, G. A. R. Sapogenins. VI. Quillaic acid. J. Chem. Soc. 1130–1135 (1939).

  23. Nico, D., Santos, F. N., Borja-Cabrera, G. P., Palatnik, M. & Palatnik de Sousa, C. B. Assessment of the monoterpene, glycidic and triterpene-moieties' contributions to the adjuvant function of the CP05 saponin of Calliandra pulcherrima Benth during vaccination against experimental visceral leishmaniasis. Vaccine 25, 649–658 (2007).

    CAS  Article  Google Scholar 

  24. Castro-Diaz, N. et al. Saponins from the Spanish saffron Crocus sativus are efficient adjuvants for protein-based vaccines. Vaccine 30, 388–397 (2012).

    CAS  Article  Google Scholar 

  25. Oda, K. et al. Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants. Biol. Chem. 381, 67–74 (2000).

    CAS  Article  Google Scholar 

  26. Pink, J. R. & Kieny, M. P. Fourth meeting on novel adjuvants currently in/close to human clinical testing. Vaccine 22, 2097–2102 (2004).

    Article  Google Scholar 

  27. Kensil, C. R. Saponins as vaccine adjuvants. Crit. Rev. Ther. Drug Carrier Syst. 13, 1–55 (1996).

    CAS  Google Scholar 

  28. Marciani, D. J. Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity. Drug Discov. Today 8, 934–943 (2003).

    CAS  Article  Google Scholar 

  29. Nimmerjahn, F. & Ravetch, J. V. Divergent immunoglobulin G subclass activity through selective Fc receptor binding. Science 310, 1510–1512 (2005).

    CAS  Article  Google Scholar 

  30. Kensil, C. R. & Kammer, R. QS-21: a water-soluble triterpene glycoside adjuvant. Exp. Opin. Invest. Drugs 7, 1475–1482 (1998).

    CAS  Article  Google Scholar 

  31. Bomford, R. Studies on the cellular site of action of the adjuvant activity of saponin for sheep erythrocytes. Int. Arch. Allergy Appl. Immunol. 67, 127–131 (1982).

    CAS  Article  Google Scholar 

  32. Calabro, S. et al. Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes. Vaccine 29, 1812–1823 (2011).

    CAS  Article  Google Scholar 

  33. Dupuis, M., McDonald, D. M. & Ott, G. Distribution of adjuvant MF59 and antigen gD2 after intramuscular injection in mice. Vaccine 18, 434–439 (1999).

    CAS  Article  Google Scholar 

  34. Dupuis, M. et al. Dendritic cells internalize vaccine adjuvant after intramuscular injection. Cell. Immunol. 186, 18–27 (1998).

    CAS  Article  Google Scholar 

  35. Kim, S-K., Ragupathi, G., Cappello, S., Kagan, E. & Livingston, P. O. Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1–KLH and GD3–KLH conjugates. Vaccine 19, 530–537 (2000).

    CAS  Article  Google Scholar 

Download references


Dedicated to the memory of our mentor and colleague, David Y. Gin (1967–2011). We thank S. J. Danishefsky, M. M. Adams and W. E. Walkowicz for helpful discussions, G. Sukenick, H. Liu, H. Fang and S. Rusli for expert NMR and mass spectral support and K. K. Sevak, N. Ramos and C. B. Davis for technical support with biodistribution and radiometric studies. Generous financial support was provided by the European Commission (Marie Curie International Outgoing Fellowship to A.F-T.), the National Institutes of Health (R01 AI085622 to J.S.L. and D.Y.G., R01 GM058833 to D.S.T. and D.Y.G., CCSG P30 CA008748 in support of the Center of Comparative Medicine and Pathology and the Radiochemistry and Molecular Imaging Probe Core), William and Alice Goodwin and the Commonwealth Foundation for Cancer Research and the Experimental Therapeutics Center of MSKCC.

Author information

Authors and Affiliations



A.F-T., E.K.C., N.P., J.R.G., G.R., J.S.L., D.S.T. and D.Y.G. conceived and designed the experiments. A.F-T. and E.K.C. performed the syntheses. C.G. performed the preclinical mouse-vaccination experiments. C.G. and N.P. performed the biodistribution experiments. J.R.G. performed the fluorescence imaging experiments. A.F-T., E.K.C., N.P., J.R.G., P.O.L., G.R., J.S.L., D.S.T. and D.Y.G. analysed the data. A.F-T. and D.S.T. wrote the manuscript.

Corresponding authors

Correspondence to Govind Ragupathi, Jason S. Lewis or Derek S. Tan.

Ethics declarations

Competing interests

J.R.G., P.O.L., G.R. and D.Y.G are founders of Adjuvance Technologies Inc. and have financial interests in the company.

Supplementary information

Supplementary information

Supplementary information (PDF 24985 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fernández-Tejada, A., Chea, E., George, C. et al. Development of a minimal saponin vaccine adjuvant based on QS-21. Nature Chem 6, 635–643 (2014).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing