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High-Density Peptide Arrays Uncover Specifics of Chagas Disease Immune Response

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NEW YORK (GenomeWeb) – An Argentinean and Danish research team has demonstrated in a new study the feasibility and effectiveness of using high-density peptide arrays to profile and characterize the antibody specificities of the human immune response to Chagas disease.

According to the authors, the results, published recently in Molecular and Cellular Proteomics, illustrate the power of these peptide chips for the discovery of pathogen-specific epitopes from clinical samples, and their potential as a tool for high-throughput biomarker discovery across a variety of pathogens.

In the study, the team, led by Fernán Agüero of the National University of San Martín, designed a high-density microarray containing more than 175,000 overlapping 15mer peptides derived from the proteome of Trypanosoma cruzi, the protozoan that causes Chagas.

According to the study authors, while whole-protein arrays have been used to successfully identify antigens in the context of the immune response to infectious disease, they are not able to provide information on the nature and precise location of the epitope, or antigenic determinants in a protein, and are more likely to suffer from non-specific antibody binding due to the exposure of a large number of potentially antigenic regions.

In contrast, peptide arrays can provide "exquisite detail of epitope localization," the group wrote, but have been limited, mainly by a reduced capacity, in their ability to scan large numbers of candidate proteins.

With more recent advances in array technologies, it has become possible to create much higher density peptide microarrays.

To create their arrays, the San Martín-led group used a high-density peptide array technique that was developed and published previously by one of the current study authors located at the University of Copenhagen.

"It's a technology where the peptides are synthesized directly in situ on the glass side, and it's driven by the same type of processor as in a projector where a computer directs which pixel is being illuminated. The synthesis of peptides at each location is being guided by light," said Agüero, the study's lead author.

"There are other technologies and manufacturers, but this is so far the [method] we see producing the highest density per slide, which is key to being able to map and discover lots of biomarkers in each assay," he added.

For their Chagas study, he and his colleagues synthesized peptides spanning the complete length of 457 T. cruzi parasite proteins onto microarray slides, with each peptide having only one residue shift difference from the previous peptide so as to fully overlap.

The peptides used in the arrays were derived from 381 serologically uncharacterized protein sequences from the T. cruzi proteome, along with 68 T. cruzi protein sequences from antigens that did have previous serological evidence in infected humans, and finally 54 neo-proteins of random sequence as a negative set used to define the signal baseline in all experiments.

The use of random sequence synthetic peptides to investigate antibody responses to disease is something several other groups have adopted. For example, a group from Arizona State University's Biodesign Institute recently demonstrated the ability of an immunosignaturing diagnostics platform using random-sequence peptide microarrays to detect a wide range of diseases, including early stage cancers.

But Agüero explained that approaches using only random peptides do not allow the characterization of the actual pathogen proteins that are being recognized in an immune response.

"You can see a pattern in disease versus control or in one disease versus another, but you don't know what the molecules are from the pathogen that are causing the response," he said.

The researchers believe they are the first to demonstrate a method of arraying peptides derived directly from a known pathogen genome, and exposing them to a complex mixture of antibodies from a natural infection.

Agüero added that including some random-sequence peptides was essential for the group to define a non-specific antibody-binding baseline. "Using the random peptides as a way to get an idea of where the baseline is, you then see that the pathogen-specific signal is way higher," he explained.

In the study, the group created and screened their slides with antibodies purified from infected Chagas patients and healthy donors. Comparing the two signals, that they were able to achieve both a high technical reproducibility as well as demonstrate highly similar epitope mapping compared with known results from earlier lower-throughput analyses.

With only 20 µg of purified immunoglobulin obtained directly from clinical samples, these high-density peptide microarrays allowed the recognition of specificities against approximately 180,000 distinct pathogen-specific 15-mer peptides, the investigators noted.

The results showed that the team's HD peptide chips had a very strong epitope mapping performance compared to known epitopes from earlier testing with an area under the receiver operating curve of 0.96 for a single microarray and an average AUC of 0.972 after combining data from multiple samples.

Overall, using a conservative signal threshold to classify positive peptides, the team identified 2,031 disease-specific peptides and 97 novel parasite antigens, "effectively doubling the number of known antigens and providing a tenfold increase in the number of fine mapped antigenic determinants for [Chagas] disease" — this despite the fact that only about three percent of the T. cruzi proteome was represented in their initial protein target set, the authors wrote.

According to Agüero, the team also calculated in computation experiments after the main study that by changing the amount of sequence overlap of displayed peptides — allowing larger residue shifts between each peptide on the chip —  they should be able to increase the protein space covered in a single chip by at least three-fold without sacrificing sensitivity.

Based on that data, the investigators believe that they can go as far as a three-residue shift from one 15-mer peptide to the next without a significant loss of epitope-mapping performance. Doing this, the complete proteome could be covered with only four or five chips, without sacrificing significant sensitivity, Agüero said.

Moving forward, the researchers are now working through the whole T. cruzi proteome to uncover all the antibody specificities of the immune response to the pathogen. They are also interested, Agüero said, in whether they can find signatures of the immune response that correlate with different clinical manifestations of the disease.

Another hope is that their results may impact the development of new diagnostic strategies or improvement of existing diagnostics for Chagas.

Interestingly, even in just their initial study, the researchers saw that in healthy donor samples there was antibody binding to known pathogen protein regions, indicating that these particular antigenic determinants may be shared with other pathogens.

This information could be important in improving the specificity and utility of recombinant antigens for diagnostic Chagas assays, the authors wrote.

"All the kits available currently for Chagas have some problems with sensitivity or specificity," Agüero said. "We saw in our experiments that there are lots of proteins from known antigens that react to [samples] from healthy people … so that information could be used potentially to remove those cross-reactive determinants from diagnostic kits."

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