Analysis of recombinant proteins for Q fever diagnostics

Abstract

Serology is essential for Q fever diagnostics, a disease caused by the bacterial pathogen Coxiella burnetii. The gold standard test is an immunofluorescence assay utilizing whole cell antigens, which are both dangerous and laborious to produce. Complexities of the antigen coupled with the subjective nature of the assay lead to decreased uniformity of test results and underscore the need for improved methodologies. Thirty-three C. burnetii proteins, previously identified as immunoreactive, were screened for reactivity to naturally infected goat serum. Based on reactivity, 10 proteins were analyzed in a secondary screen against human serum from healthy donors. Assay sensitivity and specificity ranged from 21 to 71% and 90 to 100%, respectively. Three promising antigens were identified based on receiver operating characteristic curve analysis (CBU_1718, CBU_0307, and CBU_1398). Five multiplex assays failed to outperform the individual proteins, with sensitivities and specificities ranging from 29 to 57% and 90 to 100%, respectively. Truncating the top antigen, CBU_1718, had no effect on specificity (90%); yet sensitivity decreased dramatically (71% to 21%). Through this study, we have expanded the subset of C. burnetii immunoreactive proteins validated by enzyme-linked immunosorbent assay and demonstrate the effect of novel antigen combinations and protein truncations on assay performance.

Development of High-Throughput Multiplex Serology to Detect Serum Antibodies against Coxiella burnetii

“…GroEL and DnaK were also persistent and accurate molecular markers described by Xiong et al [32,37,44,[46][47][48]. Using multiplex serology, they achieved specificities of 84% and 79%, respectively, and sensitivities of 79% and 68%, respectively.…”

Detection of Coxiella antibodies in ruminants using a SucB recombinant antigen

“…12 Other immunodominant antigens have been evaluated in ruminants obtaining good specificity but again low sensitivity. 26 To date, the recombinant antigen that has obtained the best performances is HspB (Se: 80-90%; Sp: 97%), which, however, has been evaluated exclusively in experimentally infected goats. 11 SucB has been identified as one of the main immunodominant antigens in humans and as one of the main candidates for future application in serodetection and vaccinology.…”

“…33 In a study including 33 Coxiella proteins, SucB also ranked in the top 3 for diagnostic performance, having reacted with ~70% of positive goat sera in a primary screen and with ~60% sensitivity and ~90% specificity using human sera. 26 The reason for the reduced diagnostic performance must first be sought in the complex immune response against C. burnetii, which is characterized in humans by an initial response against phase II antigens and a subsequent increase in antibodies against phase I. 3,5 SucB is described in the literature as a phase II antigen, therefore, it should react mainly with antibodies produced in the acute form, not identifying, instead, antibodies produced in the chronic form.…”

“…12 Other immunodominant antigens have been evaluated in ruminants obtaining good specificity but again low sensitivity. 26 To date, the recombinant antigen that has obtained the best performances is HspB (Se: 80–90%; Sp: 97%), which, however, has been evaluated exclusively in experimentally infected goats. 11…”

Proteome-wide Epitope Prediction: Leveraging Bioinformatic Technologies in Rational Vaccine Design

“…Antibodies are important during the immune response as they mediate opsonization for complement and innate immune cells; however, they can also inactivate circulating viruses [8]. Identification of B-cell immunogens typically relies on antibody responses seen in patients that have survived previous infection with the agent of interest [9][10][11].…”