Protein array profiling of tic patient sera reveals a broad range and enhanced immune response against Group A Streptococcus antigens

Abstract

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders.

Conflict of interest statement

Competing Interests: M.M, V.R., R.P., F.F., A.M., I.M., G.G. and G.B. are employed by the Company that funded the work. G.G. and I.M. are holders of company stock options. All other authors report no potential conflicts.

Figures

Figure 1. Protein micro array set-up and validation.

A, SDS-PAGE analysis of purified recombinant GAS proteins stained with Coomassie. Molecular weight markers in lane 1. B, Representative image of a chip after incubation with a human serum and with Cy3-labelled anti-human IgG and Cy5-labelled anti-human IgM. Replicates of tested antigens and of negative and positive IgG and IgM controls are highlighted. C, graphic representation of the control human IgG curve. Orange dots correspond to the different IgG concentrations measured on the x-axis, while the continuous line corresponds to the interpolated resulting curve. MFIs values are reported on the y-axis. The chip image of different IgG concentration revealed by incubation with anti-human IgG-Cy3 is shown below the graph. D, Sigmoid-derived data normalization method. Data are normalized using the sigmoid control curve (black) referred to a reference sigmoid curve (red). IgG control concentrations and MFIs are reported on the x- and the y-axis respectively. HL, high level signal area (normalized MFI value >40,000); LL, low level signal area (normalized MFI value

Figure 2. Tic, No Tic and Pharyngitis sera analysis.

A, Unsupervised hierarchical clustering of human Tic (n = 61), No Tic (n = 35) and Pharyngitis (n = 239) sera versus the 102 selected GAS antigens. Antigens/sera interactions resulting in signals with high or low FI are visualized in yellow and blue respectively. Color scale of signal intensity is reported on top-left. Clusters group antigens and/or sera showing similar reactivity profiles. Bars and roman numbers on the right of the dendrogram identify the sera clusters considered for the analysis. Light green lines next to cluster bars highlight how the 61 tic sera distribute within the dendrogram. Colored bars at the bottom of the dendrogram highlight the distribution of group 1, group 2 and group 3 antigens, as defined in Tables I, II and III. B, Distribution of sera in the different clusters. The histogram shows the percent of positive sera (FI>15,000) for each class in relation to the five defined clusters. The number of positive sera in each cluster calculated on the total number of sera tested for each group is shown in the table below the histogram.

Figure 3. Correlation between sera ELISA titers and MFI.

Black circles and triangles represent the ELISA Titers (geometric means, x-axis) and Mean Fluorescent Intensities (arithmetic means, y-axis) obtained using 8 sera from either no tic or tic patients with each of the antigens Spy0843, M5005_Spy0249, Spy1306 and Spy1939. Standard errors of the means are reported. Both ELISA titers and MFIs differences between the two sera classes were statistically significant with P values

Figure 4. Relative reactivity of the three sera classes.

A, Percentages of tested GAS antigens recognized by at least 30% of sera for each patient group. B, Percentages of antigens recognized by at least 30% of the sera of each class with FI>40,000. C, Percentages of sera for each class reacting with at least 30% of the antigens and with FI>40,000. Numbers above histograms bars indicate P values calculated with the two-tailed χ2 test.