A Pneumococcal Protein Array as a Platform to Discover Serodiagnostic Antigens Against Infection

Abstract

Pneumonia is one of the most common and severe diseases associated with Streptococcus pneumoniae infections in children and adults. Etiological diagnosis of pneumococcal pneumonia in children is generally challenging because of limitations of diagnostic tests and interference with nasopharyngeal colonizing strains. Serological assays have recently gained interest to overcome some problems found with current diagnostic tests in pediatric pneumococcal pneumonia. To provide insight into this field, we have developed a protein array to screen the antibody response to many antigens simultaneously. Proteins were selected by experimental identification from a collection of 24 highly prevalent pediatric clinical isolates in Spain, using a proteomics approach consisting of "shaving" the cell surface with proteases and further LC/MS/MS analysis. Ninety-five proteins were recombinantly produced and printed on an array. We probed it with a collection of sera from children with pneumococcal pneumonia. From the set of the most seroprevalent antigens, we obtained a clear discriminant response for a group of three proteins (PblB, PulA, and PrtA) in children under 4 years old. We validated the results by ELISA and an immunostrip assay showed the translation to easy-to-use, affordable tests. Thus, the protein array here developed presents a tool for broad use in serodiagnostics.

Fig. 1.

Pneumococcal protein array and serological profiles of cohorts of sera samples. A, Construction of protein array. Arrays were printed containing the 95 pneumococcal recombinant proteins and the negative controls, according to positions indicated in Table II. Proteins were printed in quintuplicate and grouped in sectors which represented different subcellular localizations. Two positions of buffers (B) were printed in the right-above corners of each sector. B, Representative image of a chip, divided in the different sectors, after incubation with a human serum, followed by Cy3-labeled anti-human IgG; C, Representative image of a chip, divided in the different sectors, after incubation with a human serum, followed by Cy5-labeled anti-human IgM. D, IgG serological profile of all the studied sera of the test set (n = 71; 33 controls aged 49.8 ± 40.3 months, and 38 pneumococcal-infected patients aged 48.2 ± 30.6 months) displayed as a heatmap of seroreactivity. The antigens are listed in rows and the sera grouped in columns (C1, controls <4 years old; C2, controls >4 years old; P1, patients <4 years old; P2, patients >4 years old). The reaction intensity is visualized according to a color scale, with green being the weakest, red being the strongest and black in between.

Fig. 2.

Response of serum IgG antibodies against 6 pneumococcal proteins in the control sera using the protein array. A, Heatmap of the sera belonging to the G3 group, with reaction intensities visualized according to a color scale, with green being the weakest, red being the strongest and black in between. The antigens are listed in rows and the sera in columns, grouped in C1 (controls <4 years old) and C2 (controls >4 years old). B, Kinetics of IgG levels for the six selected proteins in control sera rearranged in 12-month intervals.

Fig. 3.

Discovery of diagnostic candidates in children sera using the protein array. The figure shows the heatmap, receiver operating characteristics (ROC) curve and interactive dot diagram of the best protein biomarkers chosen fom the protein array analysis using sera from the ALL group (A) and from the G1 group (B), i.e. children <4 years old. For the interpretation of heatmaps, reaction intensities were visualized according to a color scale, with green being the weakest, red being the strongest, and black in between.

Fig. 4.

Validation of biomarker discovery from protein array results by ELISA using an independent validation set of sera. A, For each protein (PrtA, PulA, PblB) and the 1:1:1 combination of them, it is represented the receiver operating characteristics (ROC) curve and interactive dot diagram to show the diagnostic capacity of the assay (C: controls; P: patients). B, Statistical parameters of the Area Under the ROC Curve (AUC) of the three single proteins and their combination in the ELISA test.

Fig. 5.

Immunostrip test using the three best protein biomarkers discovered with the protein array. On the upper panel, the dot blot assay using the three individual antigens (PrtA, PulA, PblB) and their 1:1:1 combination is shown. As negative controls, the irrelevant yeast protein Lys9 (NC-1) and commercial trypsin (NC-2) were used. As positive controls, pneumococcal serotype 8 strain total protein extract (PC-1) and commercial anti-human IgG produced in goat (PC-2) were used. The 24 sera of the independent validation set (12 controls and 12 pneumococcal-infected children) were used at a 1:200 dilution to probe the nitrocellulose membranes. Down, the receiver operating characteristics (ROC) curve and interactive dot diagram of the combination of antigens are represented to show the diagnostic capacity of the assay (C: controls; P: patients).