Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.Detailed knowledge of antigens and epitopes recognized in the context of naturally acquired human infections has important implications for our understanding of immune system responses against pathogens, and of the immunopathogenesis of infectious diseases. This knowledge is also important for practical clinical applications such as the development of improved vaccines, intervention strategies, and diagnostics.In the last decades, significant progress has been made in the discovery of antigens and epitopes thanks to a number of methodologies such as cDNA expression libraries (1), combinatorial peptide libraries (2), and peptide and protein microarrays (3, 4). However, current knowledge of the B-cell antigens and the epitope repertoire recognized by the immune system in human infections is still scarce. Indeed, the Immune Epitope Database (5) currently contains an average of only 10 antigens with mapped B-cell epitopes recognized from naturally acquired human infections for bacterial or eukaryotic pathogens. The reasons for this are many, but can be largely attributed to different limitations in the mentioned screening technologies. Heterologous expression of cDNA libraries has been used to guide antigen discovery, but mapping of epitopes most often lags behind as it is a much more costly exercise. Similarly, combinatorial peptide libraries greatly facilitate the identification of peptides that are specifically recognized by antibodies, but these peptides have sequences that can greatly differ from those of the native epitopes (they are mimotopes), thus making it difficult to identify the original antigens. As a result, we currently have only limited detailed information on the fine specificities of the antibody response against complex pathogens.The number of tools for studying immune responses has recently expanded with the inclusion of peptide and protein microarrays, which have been used to identify pathogen-specific antigens and linear epitopes (6–13). Although whole-protein arrays can successfully identify antigens recognized by antibodies, they present the typical difficulties associated with the production of recombinant proteins in heterologous or in vitro systems, do not provide information on the nature and precise location of the epitope(s) in a protein, and are more likely to suffer from nonspecific antibody binding because of the exposure of a large number of potentially antigenic regions. In contrast, peptide arrays can provide exquisite detail of epitope localization, but until now had other limitations mostly associated with their reduced capacity, preventing the complete scanning of large numbers of candidate proteins.Recent advances in computerized photolithography and photochemistry have led to the development of a novel high-density peptide microarray technology, where individual peptides can be synthesized in situ on a glass slide at high densities (14–17). This technology makes the production of high-density peptide arrays highly cost effective compared with previous approaches, while allowing the interrogation of complex immune responses with unprecedented throughput and mapping precision. Previous applications of this technology were limited to the fine mapping of epitopes in single proteins, using monoclonal antibodies, or using immunized animal sera as the source of polyclonal antibodies (16–18).Using these high-density peptide arrays, we here describe the first large-scale study of fine antibody specificities associated with Chagas Disease, which is an exemplar of a chronic human infectious disease. Chagas Disease, caused by the protozoan Trypanosoma cruzi, is an endemic disease of the Americas, affecting ∼8 million people (19). The parasite invades and replicates within host cells, and briefly enters the bloodstream to reach other target tissues. Initially, the disease goes through an acute stage, characterized by patent parasitaemia and the appearance of antibodies against acute-phase antigens, such as SAPA (20), followed by a delayed specific humoral response. In general, the parasite-specific immune response mounted during T. cruzi infections is insufficient to completely eradicate the pathogen, leading to chronic infection (19). In this chronic stage circulating parasites are difficult to detect, even by extremely sensitive methods such as PCR. Therefore, detection of antibodies against whole-parasite extracts or defined antigens (21, 22) remains the standard for diagnosis of Chagas Disease.In this work, we screened high-density microarray slides containing peptides derived from T. cruzi proteins with mixtures of immunoglobulins purified directly from blood samples of Chagas Disease patients. This led to the identification of novel antigens and the simultaneous mapping of their linear B-cell epitopes, thus demonstrating the capacity and performance of this platform for studying antibody specificities associated with human infectious diseases.