Pharmaceutical Research

, Volume 31, Issue 4, pp 1015–1031 | Cite as

Comparison of PLA Microparticles and Alum as Adjuvants for H5N1 Influenza Split Vaccine: Adjuvanticity Evaluation and Preliminary Action Mode Analysis

  • Weifeng Zhang
  • Lianyan Wang
  • Yuan Liu
  • Xiaoming Chen
  • Jiahui Li
  • Tingyuan Yang
  • Wenqi An
  • Xiaowei Ma
  • Ruowen Pan
  • Guanghui Ma
Research Paper



To compare the adjuvanticity of polymeric particles (new-generation adjuvant) and alum (the traditional and FDA-approved adjuvant) for H5N1 influenza split vaccine, and to investigate respective action mode.


Vaccine formulations were prepared by incubating lyophilized poly(lactic acid) (PLA) microparticles or alum within antigen solution. Antigen-specific immune responses in mice were evaluated using ELISA, ELISpot, and flow cytometry assay. Adjuvants’ action modes were investigated by determining antigen persistence at injection sites, local inflammation response, antigen transport into draining lymph node, and activation of DCs in secondary lymphoid organs (SLOs).


Alum promoted antigen-specific humoral immune response. PLA microparticles augmented both humoral immune response and cell-mediated-immunity which might enhance cross-protection of influenza vaccine. With regard to action mode, alum adjuvant functions by improving antigen persistence at injection sites, inducing severe local inflammation, slightly improving antigen transport into draining lymph nodes, and improving the expression of MHC II on DCs in SLOs. PLA microparticles function by slightly improving antigen transport into draining lymph nodes, and promoting the expression of both MHC molecules and co-stimulatory molecules on DCs in SLOs.


Considering the adjuvanticity and side effects (local inflammation) of both adjuvants, we conclude that PLA microparticles are promising alternative adjuvant for H5N1 influenza split vaccine.

Key words

adjuvanticity alum influenza vaccine microparticles mode of action 





Aluminum hydroxide


Antigen presenting cell


Bovine serum albumin


Dendritic cells


Enzyme-linked immunosorbant assay


Enzyme-linked immunospot




Hemagglutination inhibition


Major histocompatibility complex


Oil in water


Poly(lactic acid)


Standard errors of mean


Secondary lymphoid organs




Acknowledgments and Disclosures

The authors gratefully thank Dr Wei Wei for assistance with CLSM characterization of PLA microparticles-adjuvanted vaccine formulation. This work was financially supported by the 973 Program (Grant No. 2013CB531500), the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KSCX2-EW-R-19), and the 863 Program (Grant No. 2012AA02A406).

Supplementary material

11095_2013_1224_MOESM1_ESM.docx (272 kb)
ESM 1 (DOCX 271 kb)


  1. 1.
    Subbarao K, Joseph T. Scientific barriers to developing vaccines against avian influenza viruses. Nat Rev Immunol. 2007;7(4):267–78.PubMedCrossRefGoogle Scholar
  2. 2.
    Horimoto T, Kawaoka Y. Strategies for developing vaccines against H5N1 influenza A viruses. Trends Mol Med. 2006;12(11):506–14.PubMedCrossRefGoogle Scholar
  3. 3.
    Geeraedts F, Goutagny N, Hornung V, Severa M, de Haan A, Pool J, et al. Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by toll-like receptor signalling. PLoS Pathog. 2008;4(8):e1000138.PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Stropkovska A, Janulikova J, Vareckova E. Trends in development of the influenza vaccine with broader cross-protection. Acta Virol. 2010;54(1):7–19.PubMedCrossRefGoogle Scholar
  5. 5.
    Brown LE, Kelso A. Prospects for an influenza vaccine that induces cross-protective cytotoxic T lymphocytes. Immunol Cell Biol. 2009;87(4):300–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Carrat F, Flahault A. Influenza vaccine: the challenge of antigenic drift. Vaccine. 2007;25(39–40):6852–62.PubMedCrossRefGoogle Scholar
  7. 7.
    Tamura SI, Tanimoto T, Kurata T. Mechanisms of broad cross-protection provided by influenza virus infection and their application to vaccines. Jpn J Infect Dis. 2005;58(4):195–207.PubMedGoogle Scholar
  8. 8.
    Doherty PC, Kelso A. Toward a broadly protective influenza vaccine. J Clin Invest. 2008;118(10):3273–5.PubMedCentralPubMedGoogle Scholar
  9. 9.
    Marrack P, McKee AS, Munks MW. Towards an understanding of the adjuvant action of aluminium. Nat Rev Immunol. 2009;9(4):287–93.PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Mbow ML, De Gregorio E, Valiante NM, Rappuoli R. New adjuvants for human vaccines. Curr Opin Immunol. 2010;22(3):411–6.PubMedCrossRefGoogle Scholar
  11. 11.
    Exley C, Siesjö P, Eriksson H. The immunobiology of aluminium adjuvants: how do they really work? Trends Immunol. 2010;31(3):103–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Sharp FA, Ruane D, Claass B, Creagh E, Harris J, Malyala P, et al. Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome. Proc Natl Acad Sci U S A. 2009;106(3):870–5.PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Harris J, Sharp FA, Lavelle EC. The role of inflammasomes in the immunostimulatory effects of particulate vaccine adjuvants. Eur J Immunol. 2010;40(3):634–8.PubMedCrossRefGoogle Scholar
  14. 14.
    Broaders KE, Cohen JA, Beaudette TT, Bachelder EM, Fréchet JMJ. Acetalated dextran is a chemically and biologically tunable material for particulate immunotherapy. Proc Natl Acad Sci U S A. 2009;106(14):5497–502.PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Yanasarn N, Sloat BR, Cui Z. Negatively charged liposomes show potent adjuvant activity when simply admixed with protein antigens. Mol Pharm. 2011;8(4):11741185.CrossRefGoogle Scholar
  16. 16.
    Zaharoff DA, Rogers CJ, Hance KW, Schlom J, Greiner JW. Chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination. Vaccine. 2007;25(11):2085–94.PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Wei Q, Wei W, Lai B, Wang L-Y, Wang Y-x, Su Z-G, et al. Uniform-sized PLA nanoparticles: preparation by premix membrane emulsification. Int J Pharm. 2008;359(1–2):294–7.PubMedCrossRefGoogle Scholar
  18. 18.
    Amidi M, Romeijn SG, Verhoef JC, Junginger HE, Bungener L, Huckriede A, et al. N-Trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model. Vaccine. 2007;25(1):144–53.PubMedCrossRefGoogle Scholar
  19. 19.
    Bright RA, Carter DM, Crevar CJ, Toapanta FR, Steckbeck JD, Cole KS, et al. Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle. PLoS One. 2008;3(1):e1501.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Hagenaars N, Mastrobattista E, Verheul R, Mooren I, Glansbeek H, Heldens J, et al. Physicochemical and immunological characterization of N, N, N-Trimethyl Chitosan-coated whole inactivated influenza virus vaccine for intranasal administration. Pharmaceut Res. 2009;26(6):1353–64.CrossRefGoogle Scholar
  21. 21.
    Chesko J, Kazzaz J, Ugozzoli M, O’Hagan DT, Singh M. An investigation of the factors controlling the adsorption of protein antigens to anionic PLG microparticles. J Pharm Sci. 2005;94(11):2510–9.PubMedCrossRefGoogle Scholar
  22. 22.
    Horimoto T, Kawaoka Y. Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Micro. 2005;3(8):591–600.CrossRefGoogle Scholar
  23. 23.
    Ma Y, Zhuang Y, Xie X, Wang C, Wang F, Zhou D, et al. The role of surface charge density in cationic liposome-promoted dendritic cell maturation and vaccine-induced immune responses. Nanoscale. 2011;3(5):2307–14.PubMedCrossRefGoogle Scholar
  24. 24.
    Marzio R, Mauël J, Betz-Corradin S. CD69 and regulation of the immune function. Immunopharm Immunot. 1999;21(3):565–82.CrossRefGoogle Scholar
  25. 25.
    Nygaard UC, Ormstad H, Aase A, Løvik M. The IgE adjuvant effect of particles: characterisation of the primary cellular response in the draining lymph node. Toxicology. 2005;206(2):181–93.PubMedCrossRefGoogle Scholar
  26. 26.
    Lindsey W, Lowdell M, Marti G, Abbasi F, Zenger V, King K, et al. CD69 expression as an index of T-cell function: assay standardization, validation and use in monitoring immune recovery. Cytotherapy. 2007;9(2):123–32.PubMedCrossRefGoogle Scholar
  27. 27.
    Kaech SM, Wherry EJ, Ahmed R. Effector and memory T-cell differentiation: implications for vaccine development. Nat Rev Immunol. 2002;2(4):251–62.PubMedCrossRefGoogle Scholar
  28. 28.
    Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, Antia R, et al. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol. 2003;4(3):225–34.PubMedCrossRefGoogle Scholar
  29. 29.
    Thakur A, Pedersen LE, Jungersen G. Immune markers and correlates of protection for vaccine induced immune responses. Vaccine. 2012;30(33):4907–20.PubMedCrossRefGoogle Scholar
  30. 30.
    Patel A, Zhang Y, Croyle M, Tran K, Gray M, Strong J, et al. Mucosal delivery of adenovirus-based vaccine protects against ebola virus infection in mice. J Infect Dis. 2007;196(2):S413–20.PubMedCrossRefGoogle Scholar
  31. 31.
    Trimnell A, Takagi A, Gupta M, Richie TL, Kappe SH, Wang R. Genetically attenuated parasite vaccines induce contact-dependent CD8+ T cell killing of plasmodium yoelii liver stage-infected hepatocytes. J Immunol. 2009;183(9):5870–8.PubMedCrossRefGoogle Scholar
  32. 32.
    Yue H, Wei W, Fan B, Yue Z, Wang L, Ma G, et al. The orchestration of cellular and humoral responses is facilitated by divergent intracellular antigen trafficking in nanoparticle-based therapeutic vaccine. Pharmacol Res. 2012;65(2):189–97.PubMedCrossRefGoogle Scholar
  33. 33.
    Singh M, Chakrapani A, O’Hagan D. Nanoparticles and microparticles as vaccine-delivery systems. Expert Rev Vaccines. 2007;6(5):797–808.PubMedCrossRefGoogle Scholar
  34. 34.
    Han RL, Zhu JM, Yang XL, Xu HB. Surface modification of poly(D, L-lactic-co-glycolic acid) nanoparticles with protamine enhanced cross-presentation of encapsulated ovalbumin by bone marrow-derived dendritic cells. J Biomed Mater Res Part A. 2011;96A(1):142–9.CrossRefGoogle Scholar
  35. 35.
    Klimovich V. IgM and its receptors: structural and functional aspects. Biochem Mosc. 2011;76(5):534–49.CrossRefGoogle Scholar
  36. 36.
    Saini V, Jain V, Sudheesh MS, Jaganathan KS, Murthy PK, Kohli DV. Comparison of humoral and cell-mediated immune responses to cationic PLGA microspheres containing recombinant hepatitis B antigen. Int J Pharm. 2011;408(1–2):50–7.PubMedCrossRefGoogle Scholar
  37. 37.
    Nimmerjahn F, Ravetch JV. Divergent immunoglobulin G subclass activity through selective Fc receptor binding. Science. 2005;310(5753):1510–2.PubMedCrossRefGoogle Scholar
  38. 38.
    Woof JM. Tipping the scales toward more effective antibodies. Science. 2005;310(5753):1442–3.PubMedCrossRefGoogle Scholar
  39. 39.
    Zhang S, Cubas R, Li M, Chen C, Yao Q. Virus-like particle vaccine activates conventional B2 cells and promotes B cell differentiation to IgG2a producing plasma cells. Mol Immunol. 2009;46(10):1988–2001.PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Nimmerjahn F, Ravetch JV. Fcγ receptors: old friends and new family members. Immunity. 2006;24(1):19–28.PubMedCrossRefGoogle Scholar
  41. 41.
    Bian G, Cheng Y, Wang Z, Hu Y, Zhang X, Wu M, et al. Whole recombinant Hansenula polymorpha expressing hepatitis B virus surface antigen (yeast-HBsAg) induces potent HBsAg-specific Th1 and Th2 immune responses. Vaccine. 2009;28(1):187–94.PubMedCrossRefGoogle Scholar
  42. 42.
    Kanchan V, Panda AK. Interactions of antigen-loaded polylactide particles with macrophages and their correlation with the immune response. Biomaterials. 2007;28(35):5344–57.PubMedCrossRefGoogle Scholar
  43. 43.
    Chen X, Kim P, Farinelli B, Doukas A, Yun S-H, Gelfand JA, et al. A novel laser vaccine adjuvant increases the motility of antigen presenting cells. PLoS One. 2010;5(10):e13776.PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Singh M, O’Hagan DT. Recent advances in vaccine adjuvants. Pharmaceut Res. 2002;19(6):715–28.CrossRefGoogle Scholar
  45. 45.
    Hagan DT, Singh M. Microparticles as vaccine adjuvants and delivery systems. Expert Rev Vaccines. 2003;2(2):269–83.CrossRefGoogle Scholar
  46. 46.
    Tritto E, Mosca F, De Gregorio E. Mechanism of action of licensed vaccine adjuvants. Vaccine. 2009;27(25–26):3331–4.PubMedCrossRefGoogle Scholar
  47. 47.
    Calabro S, Tortoli M, Baudner BC, Pacitto A, Cortese M, O’Hagan DT, et al. Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes. Vaccine. 2011;29(9):1812–23.PubMedCrossRefGoogle Scholar
  48. 48.
    Mohanan D, Slütter B, Henriksen-Lacey M, Jiskoot W, Bouwstra JA, Perrie Y, et al. Administration routes affect the quality of immune responses: a cross-sectional evaluation of particulate antigen-delivery systems. J Control Release. 2010;147(3):342–9.PubMedCrossRefGoogle Scholar
  49. 49.
    Shen H, Ackerman AL, Cody V, Giodini A, Hinson ER, Cresswell P, et al. Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles. Immunology. 2006;117(1):78–88.PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    PANYAM J, W-Z ZHOU, PRABHA S, SAHOO SK, LABHASETWAR V. Rapid endo-lysosomal escape of poly(dl-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. FASEB J. 2002;16(10):1217–26.PubMedCrossRefGoogle Scholar
  51. 51.
    De Koker S, Lambrecht BN, Willart MA, van Kooyk Y, Grooten J, Vervaet C, et al. Designing polymeric particles for antigen delivery. Chem Soc Rev. 2011;40(1):320–39.PubMedCrossRefGoogle Scholar
  52. 52.
    Zhou S, Liao X, Li X, Deng X, Li H. Poly-d, l-lactide–co-poly(ethylene glycol) microspheres as potential vaccine delivery systems. J Control Release. 2003;86(2–3):195–205.PubMedCrossRefGoogle Scholar
  53. 53.
    Huntimer L, Ramer-Tait AE, Petersen LK, Ross KA, Walz KA, Wang C, et al. Evaluation of biocompatibility and administration site reactogenicity of polyanhydride-particle-based platform for vaccine delivery. Adv Healthc Mater. 2013;2(2):369–78.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Weifeng Zhang
    • 1
    • 2
  • Lianyan Wang
    • 1
  • Yuan Liu
    • 1
    • 2
  • Xiaoming Chen
    • 1
    • 2
  • Jiahui Li
    • 1
  • Tingyuan Yang
    • 1
  • Wenqi An
    • 3
  • Xiaowei Ma
    • 3
  • Ruowen Pan
    • 3
  • Guanghui Ma
    • 1
  1. 1.National Key Laboratory of Biochemical Engineering PLA Key Laboratory of Biopharmaceutical Production & Formulation EngineeringInstitute of Process Engineering Chinese Academy of SciencesHaidian DistrictPeople’s Republic of China
  2. 2.University of Chinese Academy of SciencesBeijingPeople’s Republic of China
  3. 3.Hualan Biological Engineering, Inc.XinxiangPeople’s Republic of China

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