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Delivery of foreign antigens by engineered outer membrane vesicle vaccines

  1. David J. Chena,
  2. Nikolaus Osterriederb,c,
  3. Stephan M. Metzgerb,
  4. Elizabeth Bucklesd,
  5. Anne M. Doodye,
  6. Matthew P. DeLisaa, and
  7. David Putnama,e,1
  1. aSchool of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853;
  2. bDepartment of Microbiology and Immunology, Cornell University, Ithaca, NY 14853;
  3. cInstitut für Virologie, Freie Üniversitat Berlin, 10015 Berlin, Germany;
  4. dDepartment of Biomedical Sciences, Cornell University, Ithaca, NY 14853; and
  5. eDepartment of Biomedical Engineering, Cornell University, Ithaca, NY 14853
  1. Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved December 15, 2009 (received for review June 6, 2008)


As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines.


  • 1To whom correspondence should be addressed. E-mail: dap43{at}cornell.edu.
  • Author contributions: N.O., E.B., M.P.D., and D.P. designed research; D.J.C., S.M.M., E.B., and A.M.D. performed research; and D.J.C. and D.P. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0805532107/DCSupplemental.