Summary
In our previous work, we used high-density peptide arrays displaying short peptides to interrogate the specificity of the antibody repertoires of Chagas Disease patients across the Americas, creating the Chagas Antigen and Epitope Atlas. By using the information obtained in the Atlas, we now analyze serum samples from Chagas Disease patients at different stages of Chagas cardiomyopathy, study which can be seen in detail in this paper.
Introduction
Chagas disease
Chagas Disease (American trypanosomiasis) is a lifelong infection caused by the protozoan parasite Trypanosoma cruzi. Despite being discovered ~100 years ago, the condition remains a major social and public health problem in the Americas and is regarded as a neglected tropical disease by the World Health Organization.
Trypanosoma cruzi
Trypanosoma cruzi is a unicellular eukaryote that infects and replicates within cells. There are 6-7 evolutionary lineages of the parasite across the Americas, circulating in different ecoepidemiological cycles (domestic, peridomestic, sylvatic). All cause human infections, with a diversity of clinical manifestations.
Chagas cardiomyopathy
The majority (60%–70%) of infected individuals remain asymptomatic throughout life. Although some develop only conduction defects and mild segmental wall motion abnormalities, others develop severe symptoms of heart failure, thromboembolic phenomena, and life-threatening ventricular arrhythmias. In Pereira Nunes et al (2018) the authors mention several stages of Chagas cardiomyopathy. In this study we’ll focus on 3 of them: Chagas indeterminate form (CHIF, Stage A), Chagas cardiomyopathy (CCM, Stage B1), and Chagas dilated cardiomyopathy (CDCM, Stages B2 and C).
High Density Peptide Arrays
Peptide arrays display short peptides at addressable positions. A very high-density of probes is achieved by in situ peptide synthesis driven by digital light processors. Such arrays contain hundreds of thousands to millions of peptides.
Antibody Repertoires
The term “antibody repertoire” refers to the entire set of antibodies produced by an individual as part of the adaptive immune response to infections or vaccines.
Assays
We assayed high density arrays displaying immobilized short peptides (candidate antigens and epitopes) with the same methodology used in indirect immunoassays. Arrays were first incubated with a primary antibody (human serum sample or pool of samples), then washed to remove unbound immunoglobulins. Arrays were next incubated with a secondary antibody, fluorescently labeled. This secondary antibody binds to human immunoglobulin G (total IgG). After washing to remove unbound antibodies, fluorescence was read in a scanner.
Proteome-wide analysis of pooled serum samples
Our first step was to analyze pooled serum samples from individuals at different stages of Chagas cardiomyopathy using the CHAGASTOPE-v1 microarray design created in the Chagas Antigen and Epitope Atlas. This design contains the complete proteomes of two strains of T. cruzi, CL-Brener and Sylvio X10, which are representative of lineages TcI and TcVI respectively. This resulted in a microarray with 2.84 million peptides.
Visual Summary
Visual Summary of the first analysis. Schematic representation of the steps followed to analyze two T. cruzi proteomes (CL-Brener and Sylvio X10) using pooled serum samples of individuals at different stages of Chagas cardiomyopathy, as well as healthy controls.
Experiment Design
Proteomes, Proteins & Microarrays
We used high-density peptide arrays to perform high-resolution antigen discovery. We designed an array that includes protein sequences encoded in the genomes of two T. cruzi strains: the genome reference from the CL Brener strain (19,668 proteins, Discrete Typing Unit (DTU) TcVI, hybrid), and the Sylvio X10 strain (10,832 proteins, DTU TcI, non-hybrid). A total of 30,500 proteins were displayed in our microarrays.
To create the microarray slides, each protein was split in peptides of 16 amino acids. The overlap between consecutive (neighboring peptides) was of 12 amino acid residues (e.g. the offset between one peptide and the next was 4 residues, see Visual Summary). A total of 2,441,908 unique peptides were used in this design to display all proteins in these two proteomes. We called this design CHAGASTOPE-v1.
Serum Samples
We assayed CHAGASTOPE-v1 whole-proteome microarray slides with pooled serum samples. These included samples from Chagas positive donors (infected with T. cruzi), as well as negative sample pools from healthy subjects. All serum samples were from Argentina.
Chagas-positive pools were labeled as: AR_CHIF (Chagas indeterminate form, Stage A), AR_CCM (Chagas cardiomyopathy, Stage B1), and AR_CDCM (Chagas dilated cardiomyopathy, Stages B2 and C). Each pool was also tagged as either Positive (for Chagas/Leishmania-positive individuals) or Negative for healthy individuals. The data for the healthy subjects is the same for the three serum samples (since they are all from Argentina).
Data Processing
Data processing of Chagastope arrays produces the following types of numerical values for peptides:
Raw Signal data were those obtained from scanning each microarray slide. From the experiments we obtained 2 raw signal values per peptide per sample (because they were assayed in duplicate). When browsing “All Peptide Data” these are shown under the “Replica” column.
Smoothed Signals were obtained after placing all peptides from the same protein in order, and smoothing the raw signal data using a rolling median followed by averaging the values obtained for each replica. This was done to remove outliers, and uses neighboring peptides in a protein sequence as pseudo-replicates (a given peptide shares 75% of its sequence with the previous/next peptide in the protein sequence). This resulted in a signal value for each peptide in a specific position inside each protein.The standard deviation (SD) in this case summarizes the dispersion between smoothed values between the two replicates.
Antigenicity threshold
We established an arbitrary but very conservative antigenicity threshold to classify peptides as antigenic. We calculated the mode and the standard deviation for all peptides in the arrays using Raw Signals. The antigenicity threshold was defined as the statistical mode + 4 standard deviations (this equals a normalized signal value of 9,383.82 arbitrary fluorescence units). The antigenicity (signal) threshold of dynamic plots can be adjusted manually for visualization purposes using the options in the “Plot options” tab.
Individual patient analysis
Our second experiment used a smaller but more detailed microarray design that focused on the antigenic regions found previously in the Chagas Antigen and Epitope Atlas. We used this design to analyze individual serum samples of the same patient at two stages of Chagas cardiomyopathy, CCM and CDCM.
Experiment Design
Antigenic Regions, Antigenic Peaks & Microarrays
All assays were performed using sectorized high-density peptide arrays. These arrays can assay up to 12 samples (primary antibodies) in parallel in the same slide. All 12 sectors in each slide contained the same set of peptides representative of 9,547 antigenic regions found in the Chagas Antigen and Epitope Atlas.
Same as before, each protein was split in peptides of 16 amino acids. However, in this microarray design neighboring peptides are maximally overlapped, meaning, there is a sliding offset of 1 amino acid residue between neighboring peptides. A total of 241,772 unique peptides were used in this design to display all proteins in the 9,547 antigenic regions. We called this design CHAGASTOPE-v2.
Serum Samples
Array sectors were assayed with individual serum samples from patient CH060 at two stages of Chagas cardiomyopathy (in duplicate).
At the Chagastope web resource, plots and tables refer to these samples as CH060_1_CCM for a serum sample extracted when the patient show signs of Chagas cardiomyopathy without dilatation, and CH060_2_CDCM for a serum sample from the same patient extracted one year later which show dilatation. These serum samples were all tagged as Positive serum for Chagas.
Data Processing
Data was processed and analyzed in a similar manner as in the discovery screening.
Antigenicity threshold
A separate arbitrary threshold was calculated and defined for these experiments. After calculating the statistical mode and the standard deviation for all Normalized Signals in these experiments, we defined an antigenicity threshold equal to the statistical mode plus 4 standard deviations, which resulted in 9,538.69 arbitrary fluorescence units.