Brazilian Journal of Microbiology

, Volume 50, Issue 1, pp 67–77 | Cite as

Nano-multilamellar lipid vesicles (NMVs) enhance protective antibody responses against Shiga toxin (Stx2a) produced by enterohemorrhagic Escherichia coli strains (EHEC)

  • M. J. Rodrigues-Jesus
  • W. L. Fotoran
  • R. M. Cardoso
  • K. Araki
  • G. Wunderlich
  • Luís C. S. FerreiraEmail author
Biotechnology and Industrial Microbiology - Research Paper


Microlipid vesicles (MLV) have a broad spectrum of applications for the delivery of molecules, ranging from chemical compounds to proteins, in both in vitro and in vivo conditions. In the present study, we developed a new set of nanosize multilayer lipid vesicles (NMVs) containing a unique combination of lipids. The NMVs enable the adsorption of histidine-tagged proteins at the vesicle surface and were demonstrated to be suitable for the in vivo delivery of antigens. The NMVs contained a combination of neutral (DOPC) and anionic (DPPG) lipids in the inner membrane and an external layer composed of DOPC, cholesterol, and a nickel-containing lipid (DGS-NTA [Ni]). NMVs combined with a recombinant form of the B subunit of the Shiga toxin (rStx2B) produced by certain enterohemorragic Escherichia coli (EHEC) strains enhanced the immunogenicity of the antigen after parenteral administration to mice. Mice immunized with rStx2B-loaded NMVs elicited serum antibodies capable of neutralizing the toxic activities of the native toxin; this result was demonstrated both in vitro and in vivo. Taken together, these results demonstrated that the proposed NMVs represent an alternative for the delivery of antigens, including recombinant proteins, generated in different expression systems.


Nanoparticles Delivery system Multilamellar vesicles Lipids vesicles Shiga toxin 



The authors would like to thank Eduardo Gimenez and Carolina Bertelli Ferreira for their technical support, Leticia Barbosa and Roxane Piazza of the Butantan Institute for the supply of the native Stx2a toxin used in this study, and Marina S. Palermo from the Faculty of Medicine at the University of Buenos Aires for supplying the pGEM-stx2 construction. All the data needed to evaluate the conclusions made in this paper are present within the data presented in the paper and/or the Supplemental Materials. Additional data may be requested from the authors.

Authors’ contributions

Jesus-Rodrigues, M.J. performed the rStx2B expression and purification assays, preparation of the NMVs, immunization and challenge of the mice, cell assays, antibody titers, data analysis, and wrote the manuscript. Fotoran, W.L. developed and assisted in the preparation of NMVs. Cardoso, R.M. carried out the physico-chemical characterization tests of NMVs. Araki, K. and Wunderlich, G. contributed scientific and technical assistance to the preparation and characterization of NMVs. Ferreira, L.C.S. supervised the study and the writing of the manuscript. All authors reviewed and commented on the manuscript.

Funding information

This work was carried out at the Institute of Biomedical Sciences of the University of São Paulo and with financial support of the Research Support Foundation of the States of São Paulo (FAPESP—Process: 2014/21141-4 and 2015/17174-7).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

42770_2018_35_MOESM1_ESM.docx (1.3 mb)
ESM 1 (DOCX 1.34 MB)


  1. 1.
    Allen TM, Cullis PR (2013) Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev. Elsevier B.V.; 65(1):36–48.
  2. 2.
    Watson DS, Endsley AN, Huang L (2012) Design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens. Vaccine. Elsevier Ltd; 30(13):2256–72. Available from:
  3. 3.
    Smith DM, Simon JK, Baker JR (2013) Applications of nanotechnology for immunology. Nat Rev Immunol 13(8):592–605 Available from: CrossRefGoogle Scholar
  4. 4.
    Torres-Sangiao E, Holban AM, Gestal MC (2016) Advanced nanobiomaterials: vaccines, diagnosis and treatment of infectious diseases. Molecules 21(7):1–22CrossRefGoogle Scholar
  5. 5.
    Yang L, Li W, Kirberger M, Liao W, Ren J (2016) Design of nanomaterial based systems for novel vaccine development. Biomater Sci. [cited 2018 Jan 18];4(5):785–802. Available from:
  6. 6.
    Afrin F, Anam K, Ali N (2000) Induction of partial protection against Leishmania donovani by promastigote antigens in negatively charged liposomes. J Parasitol 86(4):730–735 Available from: CrossRefGoogle Scholar
  7. 7.
    Migliaccio V, Santos FR, Ciancaglini P, Ramalho-Pinto FJ (2008) Use of proteoliposome as a vaccine against Trypanosoma cruzi in mice. Chem Phys Lipids 152(2):86–94CrossRefGoogle Scholar
  8. 8.
    Malam Y, Loizidou M, Seifalian AM (2009) Liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer. Trends Pharmacol Sci 30(11):592–599CrossRefGoogle Scholar
  9. 9.
    Schmidt ST, Foged C, Korsholm KS, Rades T, Christensen D (2016) Liposome-based adjuvants for subunit vaccines: formulation strategies for subunit antigens and immunostimulators. Pharmaceutics 8(1):1–22CrossRefGoogle Scholar
  10. 10.
    Fotoran WL, Colhone MC, Ciancaglini P, Stabeli RG, Wunderlich G (2016) Merozoite-protein loaded liposomes protect against challenge in two murine models of Plasmodium infection. ACS Biomater Sci Eng [Internet]. American Chemical Society; [cited 2018 Feb 25];2(12):2276–86.
  11. 11.
    Fotoran WL, Santangelo RM, Medeiros MM, Colhone M, Ciancaglini P, Barboza R, et al (2015) Liposomes loaded with P. falciparum merozoite-derived proteins are highly immunogenic and produce invasion-inhibiting and anti-toxin antibodies. J Control Release. Elsevier [cited 2018 Feb 25];217:121–7. Available from:
  12. 12.
    Fotoran WL, Santangelo R, de Miranda BNM, Irvine DJ, Wunderlich G (2017) DNA-loaded cationic liposomes efficiently function as a vaccine against malarial proteins. Mol Ther Methods Clin Dev. American Society of Gene & Cell Therapy; [cited 2018 Feb 25];7:1–10. Available from:
  13. 13.
    Torchilin VP (2005) Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 4(2):145–160CrossRefGoogle Scholar
  14. 14.
    Brewer JM, Tetley L, Richmond J, Liew FY, Alexander J (1998) Lipid vesicle size determines the Th1 or Th2 response to entrapped antigen. J Immunol 161(8):4000–4007Google Scholar
  15. 15.
    Brewer JM, Pollock KGJ, Tetley L, Russell DG (2004) Vesicle size influences the trafficking, processing, and presentation of antigens in lipid vesicles. J Immunol 173(10):6143–6150CrossRefGoogle Scholar
  16. 16.
    Papahadjopoulos D, Nir S, Düzgünes N (1990) Molecular mechanisms of calcium-induced membrane fusion. J Bioenerg Biomembr 22(2):157–179CrossRefGoogle Scholar
  17. 17.
    DeMuth PC, Moon JJ, Suh H, Hammond PT, Irvine DJ (2012) Releasable layer-by-layer assembly of stabilized lipid nanocapsules on microneedles for enhanced transcutaneous vaccine delivery. ACS Nano. NIH Public Access; [cited 2016 Oct 13];6(9):8041–51. Available from:
  18. 18.
    Pejawar-Gaddy S, Kovacs JM, Barouch DH, Chen B, Irvine DJ (2014) Design of lipid nanocapsule delivery vehicles for multivalent display of recombinant Env trimers in HIV vaccination. Bioconjug Chem. [cited 2016 Oct 13];25(8):1470–8. Available from:
  19. 19.
    Foged C, Arigita C, Sundblad A, Jiskoot W, Storm G, Frokjaer S (2004) Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition. Vaccine 22(15–16):1903–1913CrossRefGoogle Scholar
  20. 20.
    Genito CJ, Beck Z, Phares TW, Kalle F, Limbach KJ, Stefaniak ME et al (2017) Liposomes containing monophosphoryl lipid A and QS-21 serve as an effective adjuvant for soluble circumsporozoite protein malaria vaccine FMP013. Vaccine 35(31):3865–3874. CrossRefGoogle Scholar
  21. 21.
    Joo K-I, Xiao L, Liu S, Liu Y, Lee C-L, Conti PS, et al (2013) Crosslinked multilamellar liposomes for controlled delivery of anticancer drugs. Biomaterials. [cited 2018 Feb 13];34(12):3098–109. Available from:
  22. 22.
    Alinaghi A, Rouini MR, Johari Daha F, Moghimi HR (2014) The influence of lipid composition and surface charge on biodistribution of intact liposomes releasing from hydrogel-embedded vesicles. Int J Pharm. Elsevier B.V.; 459(1–2):30–9.
  23. 23.
    Luo Y, Liu Z, Zhang X, Huang J, Yu X, Li J et al (2016) Effect of a controlled-release drug delivery system made of oleanolic acid formulated into multivesicular liposomes on hepatocellular carcinoma in vitro and in vivo. Int J Nanomedicine. [cited 2016 Oct 4];11:3111–29. Available from:
  24. 24.
    Giddam AK, Zaman M, Skwarczynski M, Toth I (2012) Liposome-based delivery system for vaccine candidates: constructing an effective formulation. Nanomedicine [Internet]. Future Medicine Ltd London, UK; [cited 2018 Jan 17];7(12):1877–93. Available from:
  25. 25.
    Mazumder S, Maji M, Ali N (2011) Potentiating effects of MPL on DSPC bearing cationic liposomes promote recombinant GP63 vaccine efficacy: high immunogenicity and protection. PLoS Negl Trop Dis 5(12)Google Scholar
  26. 26.
    Mukherjee J, Chios K, Fishwild D, Hudson D, O’Donnell S, Rich SM et al (2002) Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection. Infect Immun. American Society for Microbiology (ASM); [cited 2016 Oct 19];70(2):612–9. Available from:
  27. 27.
    Smith JL, Fratamico PM, Gunther NW (2014) Shiga toxin-producing Escherichia coli, 1st edn. Adv Appl Microbiol. Copyright © 2014 Elsevier Inc. All rights reserved, pp 145–197.
  28. 28.
    Luz D, Chen G, Maranhão AQ, Rocha LB, Sidhu S, Piazza RMF (2015) Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro Stx2 toxin from Shiga toxin-producing Escherichia coli. PLoS One. [cited 2016 Oct 19];10(3):e0120481. Available from:
  29. 29.
    Bernedo-Navarro RA, Miyachiro MM, Da Silva MJ, Reis CF, Conceição RA, Gatti MSV et al (2014) Peptides derived from phage display libraries as potential neutralizers of Shiga toxin-induced cytotoxicity in vitro and in vivo. J Appl Microbiol 116(5):1322–1333CrossRefGoogle Scholar
  30. 30.
    Mejias MP, Hiriart Y, Lauche C, Fernandez-Brando RJ, Pardo R, Bruballa A et al (2016) Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic uremic syndrome (HUS). Sci Rep. Nature Publishing Group; 6(April):24913. Available from:
  31. 31.
    Bentancor LV, Bilen M, Brando RJF, Ramos MV, Ferreira LCS, Ghiringhelli PD et al (2009) A DNA vaccine encoding the enterohemorragic Escherichia coli Shiga-like toxin 2 A2 and B subunits confers protective immunity to Shiga toxin challenge in the murine model. Clin Vaccine Immunol 16(5):712–718CrossRefGoogle Scholar
  32. 32.
    Mejias MP, Ghersi G, Craig PO, Panek CA, Bentancor LV, Baschkier A et al (2013) Immunization with a chimera consisting of the B subunit of Shiga toxin type 2 and Brucella lumazine synthase confers total protection against Shiga toxins in mice. J Immunol 191(5):2403–2411 Available from: CrossRefGoogle Scholar
  33. 33.
    Rojas RLG, Gomes PADP, Bentancor LV, Sbrogio-Almeida ME, Costa SOP, Massis LM et al (2010) Salmonella enterica serovar typhimurium vaccine strains expressing a nontoxic Shiga-like toxin 2 derivative induce partial protective immunity to the toxin expressed by enterohemorrhagic Escherichia coli. Clin Vaccine Immunol 17(4):529–536CrossRefGoogle Scholar
  34. 34.
    Gomes PAD, Bentancor LV, Paccez JD, Sbrogio-Almeida ME, Palermo MS, RCC F et al (2009) Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7. Brazilian J Microbiol 40:333–338CrossRefGoogle Scholar
  35. 35.
    Garcia-Angulo VA, Kalita A, Torres AG. Advances in the development of enterohemorrhagic Escherichia coli vaccines using murine models of infectionGoogle Scholar
  36. 36.
    Reviakine I, Simon A, Brisson A (2000) Effect of Ca 2+ on the morphology of mixed DPPC-DOPS supported phospholipid bilayers. Langmuir. [cited 2018 Feb?13];16(4):1473–7. Available from:
  37. 52.
    Casal HL, Martin A, Mantsch HH, Paltauf F, Hauser H (1987) Infrared studies of fully hydrated unsaturated phosphatidylserine bilayers. Effect of lithium and calcium. Biochemistry. [cited 2018 Feb?13];26(23):7395–401. Available from:
  38. 37.
    Moon JJ, Suh H, Li AV, Ockenhouse CF, Yadava A, Irvine DJ (2012) Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction. Proc Natl Acad Sci U S A 109:1080–1085CrossRefGoogle Scholar
  39. 38.
    Capozzo AVE, Creydt VP, Dran G, Ferna G, Bentancor LV, Rubel C et?al (2003) Development of DNA vaccines against hemolytic-uremic syndrome in a murine model. Society 71(7):3971–3978Google Scholar
  40. 39.
    Schwendener RA (2014) Liposomes as vaccine delivery systems: a review of the recent advances. Ther Adv Vaccines 2(6):159–182 Available from: CrossRefGoogle Scholar
  41. 40.
    Casal HL, Martin A, Mantsch HH, Paltauf F, Hauser H (1987) Infrared studies of fully hydrated unsaturated phosphatidylserine bilayers. Effect of lithium and calcium. Biochemistry. [cited 2018 Feb?13];26(23):7395–401. Available from:
  42. 41.
    Tah B, Pal P, Mishra S, Talapatra GB (2014) Interaction of insulin with anionic phospholipid (DPPG) vesicles. Phys Chem Chem Phys. The Royal Society of Chemistry; [cited 2018 Feb?13];16(39):21657–63. Available from:
  43. 42.
    Oussoren C, Zuidema J, Crommelin DJA, Storm G (1997) Lymphatic uptake and biodistribution of liposomes after subcutaneous injection. II. Influence of liposomal size, lipid composition and lipid dose. Biochim Biophys Acta Biomembr 1328(2):261–272CrossRefGoogle Scholar
  44. 43.
    Patel JD, O’Carra R, Jones J, Woodward JG, Mumper RJ (2007) Preparation and characterization of nickel nanoparticles for binding to His-tag proteins and antigens. Pharm Res. Springer US; [cited 2018 Jan 16];24(2):343–52. Available from:
  45. 44.
    Smith MJ, Teel LD, Carvalho HM, Melton-Celsa AR, O’Brien AD (2006) Development of a hybrid Shiga holotoxoid vaccine to elicit heterologous protection against Shiga toxins types 1 and 2. Vaccine 24(19):4122–4129CrossRefGoogle Scholar
  46. 45.
    Tsuji T, Shimizu T, Sasaki K, Tsukamoto K, Arimitsu H, Ochi S et al (2008) A nasal vaccine comprising B-subunit derivative of Shiga toxin 2 for cross-protection against Shiga toxin types 1 and 2. Vaccine 26(17):2092–2099CrossRefGoogle Scholar
  47. 46.
    Arimitsu H, Sasaki K, Iba Y, Kurosawa Y, Shimizu T, Tsuji T (2015) Isolation of B subunit-specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2. Microbiol Immunol 59(2):71–81CrossRefGoogle Scholar
  48. 47.
    Melton-Celsa AR (2014) Shiga toxin (Stx) classification, structure, and function. Microbiol Spectr. NIH Public Access; [cited 2018 Jan 17];2(4):EHEC – 0024–2013. Available from:
  49. 48.
    Olavesen KK, Lindstedt B-A, Løbersli I, Brandal LT (2016) Expression of Shiga toxin 2 (Stx2) in highly virulent Stx-producing Escherichia coli (STEC) carrying different anti-terminator (q) genes. Microb Pathog. Academic Press; [cited 2018 Jan 17];97:1–8. Available from:
  50. 49.
    Mejias MP, Cabrera G, Fernández-Brando RJ, Baschkier A, Ghersi G, Abrey-Recalde MJ et al (2014) Protection of mice against Shiga toxin 2 (Stx2)-associated damage by maternal immunization with a Brucella lumazine synthase-Stx2 B subunit chimera. Infect Immun 82(4):1491–1499CrossRefGoogle Scholar
  51. 50.
    Fukuda T, Kimiya T, Takahashi M, Arakawa Y, Ami Y, Suzaki Y et al (1998) Induction of protection against oral infection with cytotoxin-producing Escherichia coli O157:H7 in mice by Shiga-like toxin-liposome conjugate. Int Arch Allergy Immunol 116(4):313–317 Available from: CrossRefGoogle Scholar
  52. 51.
    Suzaki Y, Ami Y, Nagata N, Naito S, Kato H, Taneichi M et al (2002) Protection of monkeys against Shiga toxin induced by Shiga toxin-liposome conjugates. Int Arch Allergy Immunol 127(4):294–298 Avrailable from: Scholar

Copyright information

© Sociedade Brasileira de Microbiologia 2018

Authors and Affiliations

  • M. J. Rodrigues-Jesus
    • 1
  • W. L. Fotoran
    • 2
  • R. M. Cardoso
    • 3
  • K. Araki
    • 3
  • G. Wunderlich
    • 2
  • Luís C. S. Ferreira
    • 1
    Email author
  1. 1.Vaccine Development Laboratory, Department of MicrobiologyInstitute of Biomedical Sciences, University of São PauloSão PauloBrazil
  2. 2.Unit for Drug Development and Plasmodium Molecular Biology, Department of ParasitologyInstitute of Biomedical Sciences, University of São PauloSão PauloBrazil
  3. 3.Supramolecular Chemistry and Nanotechnology Laboratory, Department of Fundamental ChemistryInstitute of Chemistry, University of São PauloSão PauloBrazil

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