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Clinical and Vaccine Immunology, December 2008, p. 1771-1779, Vol. 15, No. 12
1071-412X/08/$08.00+0     doi:10.1128/CVI.00300-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray{triangledown}

Paul A. Beare,1 Chen Chen,3 Timo Bouman,3 Jozelyn Pablo,4 Berkay Unal,4 Diane C. Cockrell,1 Wendy C. Brown,5 Kent D. Barbian,2 Stephen F. Porcella,2 James E. Samuel,3 Philip L. Felgner,4 and Robert A. Heinzen1*

Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites,1 Genomics Unit, Research Technology Section, Research Technology Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840,2 Department of Microbial and Molecular Pathogenesis, Texas A&M Health Sciences Center, College Station, Texas 77843,3 Department of Medicine, University of California Irvine, Irvine, California 92697,4 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 991645

Received 18 August 2008/ Returned for modification 10 September 2008/ Accepted 19 September 2008

Q fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for cross-reactivity with other pathogens; (ii) an inability to distinguish between C. burnetii strains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetii protein(s). Here we describe the use of a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.

* Corresponding author. Mailing address: Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, 903 S. 4th St., Hamilton, MT 59840. Phone: (406) 375-9695. Fax: (406) 363-9380. E-mail: rheinzen{at}niaid.nih.gov

{triangledown} Published ahead of print on 8 October 2008.

Clinical and Vaccine Immunology, December 2008, p. 1771-1779, Vol. 15, No. 12
1071-412X/08/$08.00+0     doi:10.1128/CVI.00300-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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