Abstract
Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.
Highlights
Coxiella burnetii is an obligate intracellular bacterium and etiological agent of Q fever
Construction and Profiling IgG and IgM Seroreactivity in Longitudinal Samples—C. burnetii open reading frames (ORFs) cloned into the pXT7 vector were expressed under the T7 promoter in a 5 h cell-free in vitro transcription-translation (IVTT) reaction according to manufacturer’s instructions
Signal intensities were quantified and the seroreactivity for each antigen was recorded for each patient individually for both IgG and IgM. As such the total IgG antibody response to C. burnetii of the longitudinal sera collection was determined to seroreact with 49 antigens or 2.5% of all of the antigens printed on the array
Summary
Coxiella burnetii is an obligate intracellular bacterium and etiological agent of Q fever. Identification of IgG Seroreactive Antigens in Acute and Chronic Fever Patients—We profiled the antibody repertoire of a large sera collection in order to identify differentially reactive proteins between healthy, acute, and chronic Q fever patients. All proteins with individual patient seroreactivity greater than 2.5 times the standard deviation of the IVTT reaction without plasmid were considered reactive and were included on a down-selected microarray containing the previously identified reactive IgG and highly reactive IgM antigens, in addition to 68 (of 106) newly cloned antigens, for a total of 322 proteins.
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