Large scale immune profiling of infected humans and goats reveals differential recognition of Brucella melitensis antigens

Abstract

Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

Figure 1. Construction of a B. melitensis Protein Microarray.

Arrays were printed containing 1406 B. melitensis proteins, positive and negative control spots. Proteins were printed in duplicates. Each array contains positive control spots printed from 6 serial dilutions of human and mouse IgG, 6 serial dilutions of EBNA1 protein, and “No DNA” negative control spots. (A) The array was probed with anti-His antibody as described in Materials and Methods, to confirm the expression and printing of over 95% proteins. (B) Comparison of arrays probed with Peruvian naïve serum and Culture positive serum. The arrays were read in a laser confocal scanner, analyzed, and the data normalized as described in Materials and Methods. The signal intensity of each antigen is represented by rainbow palette of blue, green, red and white by increasing signal intensity.

Figure 2. Probing a collection of B. melitensis infected, uninfected, and SPF control goat sera and discovery of goat serodiagnostic antigens.

Arrays containing 1406 B. melitensis proteins were probed with goat sera organized into 3 groups as described in the text. (A). Heatmap showing normalized intensity with red strongest, bright green weakest and black in between. The antigens are in rows and are grouped to serodiagnostic and cross-reactive antigens. The goat samples are in columns and sorted left to right by increasing average intensity to serodiagnostic antigens. (B) The mean sera reactivity of the 1406 antigens was compared between the Infected and SPF Naive groups. Antigens with Benjamini Hochberg corrected p-value less than 0.05 are organized to the left and cross-reactive antigens to the right. The 18 most reactive serodiagnostic and 31 of the most reactive cross-reactive antigens are shown.

Figure 3. Probing a collection of B. melitensis human sera and discovery of human serodiagnostic antigens.

Arrays were probed with human sera organized into 5 groups: Culture Positive, Culture Negative/Rose Bengal Positive, Rose Bengal Negative, USA Naïve, and Peruvian Naïve, as described in the text. (A). Heatmap showing normalized intensity with red strongest, bright green weakest and black in between. The antigens are in rows and are grouped to serodiagnostic and cross-reactive antigens. The human samples are in columns and sorted left to right by increasing average intensity to serodiagnostic antigens. (B) The mean sera reactivity of the 1406 antigens was compared between the Culture Positive and Peruvian Naive groups. Antigens with Benjamini Hochberg corrected p-value less than 0.05 are organized to the left and cross-reactive antigens to the right. The 13 most reactive serodiagnostic and 31 of the most reactive cross-reactive antigens are shown. C−/RB+, Culture Positive and Rose Bengal negative; RB−, Rose Bengal negative. Numbers in () are case numbers from each group.

Figure 4. Multiple Antigen LOOCV ROC curves.

The LOOCV ROC graphs show classifiers with increasing number of human serodiagnostic antigens. Overall, the sensitivity and specificity for array test is over 95%.

Figure 5. Immunostrips probing.

(a)Thirteen serodiagnostic antigens were printed onto nitrocellulose paper in adjacent stripes using a BioDot jet dispenser as described in Materials and Methods. Strips were probed with Culture Positive or Peruvian naive sera diluted 1/200 followed by alkaline phosphatase conjugated secondary antibody and enzyme substrate. Weak reactivity in the naïve healthy controls can be distinguished from the strong reactivity in infected group. (b). The LOOCV ROC curve was generated and sensitivity and specificity of immunostrips probing test is 94% and 89%, respectively.

Figure 6. Serodiagnostic and cross-reactive antigens for humans and goats.