Development of a T7 Phage Display Library to Detect Sarcoidosis and Tuberculosis by a Panel of Novel Antigens

Abstract

Sarcoidosis is a granulomatous inflammatory disease, diagnosed through tissue biopsy of involved organs in the absence of other causes such as tuberculosis (TB). No specific serologic test is available to diagnose and differentiate sarcoidosis from TB. Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL) cells and leukocytes of sarcoidosis patients. This complex cDNA library was biopanned to obtain 1152 potential sarcoidosis antigens and a microarray was constructed to immunoscreen two different sets of sera from healthy controls and sarcoidosis. Meta-analysis identified 259 discriminating sarcoidosis antigens, and multivariate analysis identified 32 antigens with a sensitivity of 89% and a specificity of 83% to classify sarcoidosis from healthy controls. Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls. The top 10 sarcoidosis and TB specific clones were sequenced and homologies were searched in the public database revealing unique epitopes and mimotopes in each group. Here, we show for the first time that immunoscreenings of a library derived from sarcoidosis tissue differentiates between sarcoidosis and tuberculosis antigens. These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

Keywords: IgG; Immunoscreening; Microarray; Sarcoidosis; T7phage library; Tuberculosis.

Fig. 1

Schematic diagram of discovery of sarcoidosis antigens. The process of combining phage-display technology, protein microarray and bioinformatic tools to select a panel of novel clones for the diagnosis of sarcoidosis. A cDNA library was constructed from a pool of total RNA isolated from 20 BAL samples and 36 WBC samples from sarcoid patients, and then combined with RNA extracts from cultured human monocytes and human embryonic fibroblasts. After digestion, the cDNA library was inserted into the T7 phage vector and packaged into T7 phages to generate a sarcoid cDNA–phage-display library. Several rounds of biopannings of the library were performed with pooled control sera for negative selection, and with sarcoid sera for positive enrichments. After four rounds of biopanning, enriched sarcoid specific peptide clones were cultured onto LB agar plates. A total of 1152 single colonies, including positive and negative clones, were randomly picked and propagated into 96-well plates. Phage–clone lysates were then printed robotically onto coated glass slides to create a sarcoid–phage–protein microarray. Cy5 (red fluorescent dye)-labeled antihuman antibody was used to detect IgGs in human serum that were reactive to peptide clones, and a Cy3 (green fluorescent dye)-labeled antibody was used to detect the phage capsid protein in order to normalize for spotting. Thus, if a phage clone carries a peptide that is non-reactive to human IgG, the spot will remain green suggesting an unreactive clone, whereas clones reactive to human IgG give raise to a red signal. A total of 115 sarcoid sera, 64 healthy control sera and 17 TB sera were tested on the 1152 phage peptide microarray. Bioinformatically analyzed data identified 259 antigens with the highest level of differentiation between sarcoidosis and healthy controls.

Fig. 2

A) Heatmap generated by applying meta-analysis using microarray analysis of 2 separate datasets derived from 115 sarcoidosis patients and 64 healthy controls. Data reflecting 259 antigens expressed significantly differently between healthy controls and sarcoidosis subjects in immunoscreening using sera. The 259 antigens are further divided into three categories according to the AW-OC method. I: 78 antigens are consistently over- or under-expressed in sarcoidosis in both datasets; II: 115 antigens are over- or under-expressed in sarcoidosis in the second dataset only; III: 66 antigens are over- or under-expressed in sarcoidosis in the first dataset only. B) Receiver operating characteristics (ROC) curve demonstrating the performance of 32 classifiers to discriminate between healthy controls and sarcoidosis subjects.

Fig. 3

ROC for top 10 sarcoidosis clones.

Fig. 4

Venn diagram depicts differential phage clone significances among sarcoidosis, TB and healthy controls (q < 0.01). Venn diagram shows the overlap between 259 sarcoidosis clones and 238 TB clones as compared to healthy controls, as well as 380 TB clones versus sarcoidosis. Forty seven clones could differentiate both sarcoidosis and TB from healthy controls. Five clones could not discriminate between TB and sarcoidosis.

Fig. 5

Heatmap generated from the microarray analysis using 3 datasets derived from 115 sarcoidosis patients, 64 control subjects and 17 TB patients. Fifty antigens showed significant differential expression among the three groups.