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A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens

  1. Philip L. Felgnera,b,c,1,
  2. Matthew A. Kayalab,d,
  3. Adam Vigila,
  4. Chad Burka,
  5. Rie Nakajima-Sasakia,
  6. Jozelyn Pabloa,
  7. Douglas M. Molinac,
  8. Siddiqua Hirsta,
  9. Janet S. W. Chewe,
  10. Dongling Wange,
  11. Gladys Tane,
  12. Melanie Duffieldf,
  13. Ron Yangg,
  14. Julien Neelb,d,
  15. Narisara Chantratitah,
  16. Greg Bancrofti,
  17. Ganjana Lertmemongkolchaij,
  18. D. Huw Daviesa,
  19. Pierre Baldib,d,k,
  20. Sharon Peacockh,l and
  21. Richard W. Titballg,i
  1. aDepartment of Medicine, Division of Infectious Diseases, University of California, Irvine, CA 92697;
  2. bInstitute for Genomics and Bioinformatics,
  3. dDepartment of Computer Science, and
  4. kDepartment of Biological Chemistry, University of California, Irvine, CA 92067;
  5. cAntigen Discovery, Inc., Irvine, CA 92618;
  6. eDefence Medical & Environmental Research Institute, DSO National Laboratories, 27 Medical Drive, #13-01, Singapore 117510;
  7. fDefence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, United Kingdom;
  8. gSchool of Biosciences, Geoffrey Pope Building, University of Exeter, Exeter EX4 4QD, United Kingdom;
  9. iDepartment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom;
  10. jDepartment of Clinical Immunology, Khon Kaen University, Khon Kaen 40002, Thailand;
  11. hMahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand; and
  12. lCenter for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford CB2 0QQ, United Kingdom

Abstract

Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.

Footnotes

  • 1To whom correspondence should be addressed at:
    Department of Medicine, Division of Infectious Diseases, University of California, 3501 Hewitt Hall, Irvine, CA 92697.
    E-mail: pfelgner{at}uci.edu

  • Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved June 19, 2009

  • Author contributions: P.L.F., A.V., G.T., G.B., G.L., D.H.D., P.B., S.P., and R.W.T. designed research; M.A.K., A.V., R.N.-S., J.P., D.M.M., S.H., J.S.W.C., D.W., and N.C. performed research; P.L.F., M.A.K., A.V., C.B., G.T., M.D., R.Y., J.N., G.L., D.H.D., P.B., S.P., and R.W.T. analyzed data; and P.L.F., A.V., P.B., S.P., and R.W.T. wrote the paper.

  • Conflict of interest statement: P.L.F. and D.H.D. have patent applications related to protein microarray fabrication and have stock positions with Antigen Discovery, Inc. D.M.M. is an employee with Antigen Discovery, Inc.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0812080106/DCSupplemental.

  • Freely available online through the PNAS open access option.

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