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.     

Molecular characterization of an ancient Hepatozoon species parasitizing the 'living fossil' marsupial 'Monito del Monte' Dromiciops gliroides from Chile

The Microbiotheriid Dromiciops gliroides , also known as 'Monito del Monte', is considered to be a threatened species and the only living representative of this group of South American marsupials. During the last few years, several blood samples from specimens of 'Monito del Monte' captured at Chiloé island in Chile have been investigated for blood parasites. Inspection of blood smears detected a Hepatozoon species infecting red blood cells. The sequences of DNA fragments corresponding to small subunit ribosomal RNA gene revealed two parasitic lineages belonging to Hepatozoon genus. These parasite lineages showed a basal position with respect to Hepatozoon species infecting rodents, reptiles, and amphibians but are phylogenetically distinct from Hepatozoon species infecting the order Carnivora. In addition, the Hepatozoon lineages infecting D. gliroides are also different from those infecting other micro-mammals living in sympatry, as well as from some that have been described to infect an Australian species of bandicoot. The potential vector of this parasite appears to be the host-specific tick Ixodes neuquenensis because the sequencing of a long amplicon determined the presence of one of the two lineages found in the marsupial.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 568–576.     

Integrative taxonomy reveals cryptic Amazonian species of Pristimantis (Anura: Strabomantidae)

Hypotheses on the taxonomic status of two Bolivian Pristimantis with taxonomic problems are assessed by an integrative taxonomic approach that integrates three independent lines of evidence: external morphology, prezygotic reproductive barriers (advertisement calls) and reciprocal monophyly (phylogenetic analyses of partial 16S mtDNA sequences). Central Andean Bolivian populations previously assigned to either P. peruvianus or P. dundeei, and lowland Amazonian populations from southern Peru and northern Bolivia previously considered P. peruvianus do not correspond to these species. Indeed, multivariate analyses of qualitative and quantitative morphological and bioacoustic characters, and phylogenetic analyses support the hypothesis that they represent different, previously unknown, cryptic lineages. They are herein described as new species. The former is a sibling species of P. fenestratus that inhabits the Amazonian and semideciduous forests of the Andean foothills in central Bolivia. The latter is sibling to the Andean species P. danae and is parapatric to it in the Amazonian lowland forests and adjacent foothills of northern Bolivia, southern Peru and adjacent Brazil. Most species of Neotropical frogs, and especially Pristimantis, have been described by using external qualitative morphological characters only. An extended integrative taxonomic approach, as exemplified herein, may lead to the discovery of many other cryptic and sibling lineages that would increase the species numbers of tropical areas. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 97–122.     

Is Cupressus sempervirens native in Italy? An answer from genetic and palaeobotanical data

This study represents the first large-scale analysis using nuclear molecular markers to assess genetic diversity and structure of Cupressus sempervirens L.. Genetic and fossil data were combined to infer the possible role of human activity and evolutionary history in shaping the diversity of cypress populations. We analysed 30 populations with six polymorphic nuclear microsatellite markers. Dramatic reductions in heterozygosity and allelic richness were observed from east to west across the species range. Structure analysis assigned individuals to two main groups separating central Mediterranean and eastern populations. The two main groups could be further divided into five subgroups which showed the following geographical distributions: Turkey with the Greek islands Rhodes and Samos, Greece (Crete), Southern Italy, Northern Italy, Tunisia with Central Italy. This pattern of genetic structure is also supported by samova and Barrier analyses. Palaeobotanical data indicated that Cupressus was present in Italy in the Pliocene, Pleistocene and Holocene. Furthermore, our molecular survey showed that Italian cypress populations experienced bottlenecks that resulted in reduced genetic diversity and allelic richness and greater genetic differentiation. Recent colonization or introduction may also have influenced levels of diversity detected in the Italian populations, as most individuals found in this range today have multilocus genotypes that are also present in the eastern range of the species. The data reveal a new interpretation of the history of cypress distribution characterized by ancient eastern populations (Turkey and Greek islands) and a mosaic of recently introduced trees and remnants of ancient, depauperate populations in the central Mediterranean range.     

Similar breakdown rates and benthic macroinvertebrate assemblages on native and Eucalyptus globulus leaf litter in Californian streams

1. Eucalyptus globulus, a tree species planted worldwide in many riparian zones, has been reported to affect benthic macroinvertebrates negatively. Although there is no consensus about the effects of Eucalyptus on aquatic macrobenthos, its removal is sometimes proposed as a means of ecological restoration. 2. We combined the sampling of macroinvertebrates with measurement of the colonisation of leaf packs in mesh bags, to examine the effects of riparian Eucalyptus and its litter on benthic macroinvertebrates in three small streams in California, U.S.A. Each stream included one reach bordered by Eucalyptus (E‐site) and a second bordered by native vegetation (N‐site). 3. The macrobenthos was sampled and two sets of litter bags were deployed at each site: one set with Eucalyptus litter (Euc‐bags) and one with mixed native tree litter (Nat‐bags) containing Quercus, Umbellularia, Acer and Alnus. Bags were exposed for 28, 56 and 90 days and this experiment was repeated in the autumn, winter and spring to account for effects of changing stream flow and insect phenology. 4. Litter input (average dry mass: 950 g m^?2 year^?1 in E‐sites versus 669 g m^?2 year^?1 in N‐sites) was similar, although in‐stream litter composition differed between E‐ and N‐sites. Litter broke down at similar rates in Euc‐bags and Nat‐bags (0.0193 day^?1 versus 0.0134 day^?1), perhaps reflecting the refractory nature of some of the leaves of the native trees (Quercus agrifolia). 5. Summary metrics for macroinvertebrates (taxon richness, Shannon diversity, pollution tolerance index) did not differ significantly between the E and N sites, or between Euc‐bags and Nat‐bags. No effect of exposure time or site was detected by ordination of the taxa sampled. However, distinct seasonal ordination clusters were observed in winter, spring and autumn, and one of the three streams formed a separate cluster. 6. The presence of Eucalyptus was less important in explaining the taxonomic composition of the macrobenthos than either ‘season’ or ‘stream’. Similarly, these same two factors (but not litter species) also helped explain the variation in leaf breakdown. We conclude that patches of riparian Eucalyptus and its litter have little effect on stream macrobenthos in this region.     

Small artery elasticity is decreased in patients with systemic lupus erythematosus without increased intima media thickness

Introduction  

The objectives of this study were to determine small arterial elasticity (SAE) in systemic lupus erythematosus (SLE) and to investigate its relationship with intima media thickness (IMT), accumulation of advanced glycation end products (AGEs), endothelial activation and inflammation.     

Analysis of the 10q11 cancer risk locus implicates MSMB and NCOA4 in human prostate tumorigenesis

Genome-wide association studies (GWAS) have established a variant, rs10993994, on chromosome 10q11 as being associated with prostate cancer risk. Since the variant is located outside of a protein-coding region, the target genes driving tumorigenesis are not readily apparent. Two genes nearest to this variant, MSMB and NCOA4, are strong candidates for mediating the effects of rs109939934. In a cohort of 180 individuals, we demonstrate that the rs10993994 risk allele is associated with decreased expression of two MSMB isoforms in histologically normal and malignant prostate tissue. In addition, the risk allele is associated with increased expression of five NCOA4 isoforms in histologically normal prostate tissue only. No consistent association with either gene is observed in breast or colon tissue. In conjunction with these findings, suppression of MSMB expression or NCOA4 overexpression promotes anchorage-independent growth of prostate epithelial cells, but not growth of breast epithelial cells. These data suggest that germline variation at chromosome 10q11 contributes to prostate cancer risk by influencing expression of at least two genes. More broadly, the findings demonstrate that disease risk alleles may influence multiple genes, and associations between genotype and expression may only be observed in the context of specific tissue and disease states.