Evaluation of copromicroscopy, multiplex-qPCR and antibody serology for monitoring of human ascariasis in endemic settings.

BACKGROUND: The standard diagnosis of Ascaris lumbricoides and other soil-transmitted helminth (STH) infections relies on the detection of worm eggs by copromicroscopy. However, this method is dependent on worm patency and shows only limited accuracy in low-intensity infection settings. We aimed to decipher the diagnostic accuracy of different antibodies using various Ascaris antigens in reference to copromicroscopy and quantitative PCR (qPCR), four months after national STH preventative chemotherapy among school children in western Kenya. METHODOLOGY: STH infection status of 390 school children was evaluated via copromicroscopy (Kato-Katz and mini-FLOTAC) and qPCR. In parallel, Ascaris-specific antibody profiles against larval and adult worm lysates, and adult worm excretory-secretory (ES) products were determined by enzyme-linked immunosorbent assay. Antibody cross-reactivity was evaluated using the closely related zoonotic roundworm species Toxocara cati and Toxocara canis. The diagnostic accuracy of each antibody was evaluated using receiver operating curve analysis and the correspondent area under the curve (AUC). PRINCIPAL FINDINGS: Ascaris was the predominant helminth infection with an overall prevalence of 14.9% (58/390). The sensitivity of mini-FLOTAC and Kato-Katz for Ascaris diagnosis reached only 53.5% and 63.8%, respectively compared to qPCR. Although being more sensitive, qPCR values correlated with microscopic egg counts (R = -0.71, P<0.001), in contrast to antibody levels. Strikingly, IgG antibodies recognizing the ES products of adult Ascaris worms reliably diagnosed active Ascaris infection as determined by qPCR and microscopy, with IgG1 displaying the highest accuracy (AUC = 0.83, 95% CI: 0.75-0.91). CONCLUSION: IgG1 antibody responses against adult Ascaris-ES products hold a promising potential for complementing the standard fecal and molecular techniques employed for monitoring Ascaris infections. This is of particular importance in the context of deworming programs as the antibody diagnostic accuracy was independent of egg counts.

Ascaris suum infection in juvenile pigs elicits a local Th2 response in a setting of ongoing Th1 expansion.

Ascaris spp. undergo extensive migration within the body before establishing patent infections in the small intestinal tract of humans and pigs. However, whether larval migration is critical for inducing efficient type 2 responses remains poorly understood. Therefore, we investigated systemic versus local adaptive immune responses along the hepato-tracheal migration of Ascaris suum during primary, single infections in conventionally raised pigs. Neither the initial invasion of gut tissue nor migration through the liver resulted in discernable Th2 cell responses. In contrast, lung-stage larvae elicited a Th2-biased pulmonary response, which declined after the larvae had left the lungs. In the small intestine, we observed an accumulation of Th2 cells upon the arrival of fourth-stage larvae (L4) to the small intestinal lumen. In parallel, we noticed robust and increasing Th1 responses in circulation, migration-affected organs, and draining lymph nodes. Phenotypic analysis of CD4+ T cells specifically recognizing A. suum antigens in the circulation and lung tissue of infected pigs confirmed that the majority of Ascaris-specific T cells produced IL-4 (Th2) and, to a much lesser extent, IL-4/IFN-g (Th2/1 hybrids) or IFN-g alone (Th1). These data demonstrate that lung-stage but not the early liver-stage larvae lead to a locally restricted Th2 response. Significant Th2 cell accumulation in the small intestine occurs only when L4 complete the body migration. In addition, Th2 immunity seems to be hampered by the concurrent, nonspecific Th1 bias in growing pigs. Together, the late onset of Th2 immunity at the site of infection and the Th1-biased systemic immunity likely enable the establishment of intestinal infections by sufficiently large L4 stages and pre-adult worms, some of which resist expulsion mechanisms.

Getting around the roundworms: Identifying knowledge gaps and research priorities for the ascarids.

The ascarids are a large group of parasitic nematodes that infect a wide range of animal species. In humans, they cause neglected diseases of poverty; many animal parasites also cause zoonotic infections in people. Control measures include hygiene and anthelmintic treatments, but they are not always appropriate or effective and this creates a continuing need to search for better ways to reduce the human, welfare and economic costs of these infections. To this end, Le Studium Institute of Advanced Studies organized a two-day conference to identify major gaps in our understanding of ascarid parasites with a view to setting research priorities that would allow for improved control. The participants identified several key areas for future focus, comprising of advances in genomic analysis and the use of model organisms, especially Caenorhabditis elegans, a more thorough appreciation of the complexity of host-parasite (and parasite-parasite) communications, a search for novel anthelmintic drugs and the development of effective vaccines. The participants agreed to try and maintain informal links in the future that could form the basis for collaborative projects, and to co-operate to organize future meetings and workshops to promote ascarid research.

Quantifying metabolic activity of Ascaris suum L3 using resazurin reduction.

BACKGROUND: Helminth infections are an important public health problem in humans and have an even greater impact on domestic animal and livestock welfare. Current readouts for anthelmintic drug screening assays are stage development, migration, or motility that can be subjective, laborious, and low in throughput. The aim of this study was to apply and optimize a fluorometric technique using resazurin for evaluating changes in the metabolic activity of Ascaris suum third-stage larvae (L3), a parasite of high economic relevance in swine. METHODS: Ascaris suum L3 were mechanically hatched from 6- to 8-week embryonated and sucrose-gradient-enriched eggs. Resazurin dye and A. suum L3 were titrated in 96-well microtiter plates, and resazurin reduction activity was assessed by fluorometry after 24 h of incubation. Fluorescence microscopy was used to localize the resazurin reduction site within the larvae. Finally, we exposed A. suum L3 to various stress conditions including heat, methanol, and anthelmintics, and investigated their impact on larval metabolism through resazurin reduction activity. RESULTS: We show that the non-fluorescent dye resazurin is reduced inside vital A. suum L3 to fluorescent resorufin and released into the culture media. Optimal assay parameters are 100-1000 L3 per well, a resazurin concentration of 7.5 µg/ml, and incubation at 37 °C/5% CO2 for 24 h. An intact L2 sheath around the L3 of A. suum completely prevents the uptake of resazurin, while in unsheathed L3, the most intense fluorescence signal is observed along the larval midgut. L3 exposed to methanol or heat show a gradually decreased resazurin reduction activity. In addition, 24 h exposure to ivermectin at 0.625 µM, mebendazole at 5 µM, and thiabendazole from 10 to 100 µM significantly decreased larval metabolic activity by 55%, 73%, and 70% to 89%, respectively. CONCLUSIONS: Together, our results show that both metabolic stressors and anthelmintic drugs significantly and reproducibly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy-to-use method to evaluate metabolic activity of A. suum L3 in vitro.

CD4+ T Cell Epitope Identification from Complex Parasite Antigen Mixtures.

Antigen complexity represents a major challenge for scoring CD4+ T cell immunogenicity, a key hallmark of immunity and with great potential to improve vaccine development. In this chapter, we provide a comprehensive picture of a pipeline that can be applied to virtually any complex antigen to overcome different limitations. Antigens are characterized by Mass Spectrometry to determine the available protein sources and their abundances. A reconstituted in vitro antigen processing system is applied along with bioinformatics tools to prioritize the list of candidates. Finally, the immunogenicity of candidate peptides is validated ex vivo using PBMCs from HLA-typed individuals. This protocol compiles the essential information for executing the whole pipeline while focusing on the candidate epitope prioritizing scheme.

Guts within guts: the microbiome of the intestinal helminth parasite Ascaris suum is derived but distinct from its host.

BACKGROUND: Intestinal helminths are extremely prevalent among humans and animals. In particular, intestinal roundworms affect more than 1 billion people around the globe and are a major issue in animal husbandry. These pathogens live in intimate contact with the host gut microbiota and harbor bacteria within their own intestines. Knowledge of the bacterial host microbiome at the site of infection is limited, and data on the parasite microbiome is, to the best of our knowledge, non-existent. RESULTS: The intestinal microbiome of the natural parasite and zoonotic macropathogen, Ascaris suum was analyzed in contrast to the diversity and composition of the infected host gut. 16S sequencing of the parasite intestine and host intestinal compartments showed that the parasite gut has a significantly less diverse microbiome than its host, and the host gut exhibits a reduced microbiome diversity at the site of parasite infection in the jejunum. While the host's microbiome composition at the site of infection significantly determines the microbiome composition of its parasite, microbial signatures differentiate the nematodes from their hosts as the Ascaris intestine supports the growth of microbes that are otherwise under-represented in the host gut. CONCLUSION: Our data clearly indicate that a nematode infection reduces the microbiome diversity of the host gut, and that the nematode gut represents a selective bacterial niche harboring bacteria that are derived but distinct from the host gut. Video Abstract.

Ascaris suum excretory/secretory products differentially modulate porcine dendritic cell subsets.

Helminths produce excretory/secretory products (E/S) which can modulate the immune responses of their hosts. Dendritic cells (DC) are essential for initiating the host T cell response and are thus potential targets for modulation by helminth E/S. Here we study immunomodulation of porcine peripheral blood DC subsets following ex vivo stimulation with E/S from Ascaris suum, a common helminth of pigs with considerable public health and economic importance. Our data showed that the relative frequencies of DC subsets in porcine blood differ, with plasmacytoid DC (pDC) being the most prominent in healthy 6-month-old pigs. pDC are an important cytokine source, and we found that A. suum E/S suppressed production of the type 1 cytokines IL-12p40 and TNF-α by this subset following toll-like receptor (TLR) ligation. In contrast, conventional DC (cDC) are more efficient antigen presenters, and the expression of CD80/86, costimulatory molecules essential for efficient antigen presentation, were modulated differentially by A. suum E/S between cDC subsets. CD80/86 expression by type 1 cDC (cDC1) following TLR ligation was greatly suppressed by the addition of A. suum E/S, while CD80/86 expression by type 2 cDC (cDC2) was upregulated by A. suum E/S. Further, we found that IFN-γ production by natural killer (NK) cells following IL-12 and IL-18 stimulation was suppressed by A. suum E/S. Finally, in the presence of E/S, IFN-γ production by CD4+ T cells co-cultured with autologous blood-derived DC was significantly impaired. Together, these data provide a coherent picture regarding the regulation of type 1 responses by A. suum E/S. Responsiveness of pDC and cDC1 to microbial ligands is reduced in the presence of E/S, effector functions of Th1 cells are impaired, and cytokine-driven IFN-γ release by NK cells is limited.

Age-dependent rise in IFN-γ competence undermines effective type 2 responses to nematode infection.

The efficient induction of type 2 immune responses is central to the control of helminth infections. Previous studies demonstrated that strong Th1 responses driven by intracellular pathogens as well as a bias for type 1 activity in senescent mice impedes the generation of Th2 responses and the control of intestinal nematode infections. Here, we show that the spontaneous differentiation of Th1 cells and their expansion with age restrains type 2 immunity to infection with the small intestinal nematode H. polygyrus much earlier in life than previously anticipated. This includes the more extensive induction of IFN-γ competent, nematode-specific Th2/1 hybrid cells in BALB/c mice older than three months compared to younger animals. In C57BL/6 mice, Th1 cells accumulate more rapidly at steady state, translating to elevated Th2/1 differentiation and poor control of parasite fitness in primary infections experienced at a young age. Blocking of early IFN-γ and IL-12 signals during the first week of nematode infection leads to sharply decreased Th2/1 differentiation and promotes resistance in both mouse lines. Together, these data suggest that IFN-γ competent, type 1 like effector cells spontaneously accumulating in the vertebrate host progressively curtail the effectiveness of anti-nematode type 2 responses with rising host age.

A semisynthetic glycoconjugate provides expanded cross-serotype protection against Streptococcus pneumoniae.

Streptococcus pneumoniae (S. pneumoniae)infections are the leading cause of child mortality globally. Currentvaccines fail to induceaprotective immune response towards a conserved part of the pathogen,resulting in newserotypescausing disease. Therefore, new vaccinestrategies are urgently needed.Described is atwo-pronged approach combiningS. pneumoniaeproteins, pneumolysin (Ply) and pneumococcal surface protein A (PspA),with aprecisely defined synthetic oligosaccharide,wherebythe carrier protein actsas a serotype-independent antigen to provideadditional protection. Proof of concept in mice and swine modelsrevealed thatthe conjugatesinhibited colonization of the nasopharynx, decreased the bacterial load and reduced disease severity in the bacteria challenge model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model.Acombination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective ("universal") pneumococcal vaccines.

Novel protocol for the isolation of highly purified neonatal murine microglia and astrocytes.

BACKGROUND: The crosstalk and reactivity of the cell type glia, especially microglia and astrocytes, have progressively gathered research attention in understanding proper brain function regulated by the innate immune response. Therefore, methods to isolate highly viable and pure glia for the analysis on a cell-specific level are indispensable. NEW METHOD: We modified previously established techniques: Animal numbers were reduced by multiple microglial harvests from the same mixed glial culture, thereby maximizing microglial yields following the principles of the 3Rs (replacement, reduction, and refinement). We optimized Magnetic-activated cell sorting (MACS®) of microglia and astrocytes by applying cultivated primary glial cell suspensions instead of directly sorting dissociated single cell suspension. RESULTS: We generated highly viable and pure microglia and astrocytes derived from a single mixed culture with a purity of ~99%, as confirmed by FACS analysis. Field emission scanning electron microscopy (FESEM) demonstrated integrity of the MACS-purified glial cells. Tumor necrosis factor (TNF) and Interleukin-10 (IL-10) ELISA confirmed pro- and anti-inflammatory responses to be functional in purified glia, but significantly weakened compared to non-purified cells, further highlighting the importance of cellular crosstalk for proper immune activation. COMPARISON WITH EXISTING METHOD(S): Unlike previous studies that either isolated a single type of glia or displayed a substantial proportion of contamination with other cell types, we achieved isolation of both microglia and astrocytes at an increased purity (99-100%). CONCLUSIONS: We have created an optimized protocol for the efficient purification of both primary microglia and astrocytes. Our results clearly demonstrate the importance of purity in glial cell cultivation in order to examine immune responses, which particularly holds true for astrocytes. We propose the novel protocol as a tool to investigate the cell type-specific crosstalk between microglia and astrocytes in the frame of CNS diseases.

A Helminth-Derived Chitinase Structurally Similar to Mammalian Chitinase Displays Immunomodulatory Properties in Inflammatory Lung Disease.

Immunomodulation of airway hyperreactivity by excretory-secretory (ES) products of the first larval stage (L1) of the gastrointestinal nematode Trichuris suis is reported by us and others. Here, we aimed to identify the proteins accounting for the modulatory effects of the T. suis L1 ES proteins and studied six selected T. suis L1 proteins for their immunomodulatory efficacy in a murine OVA-induced allergic airway disease model. In particular, an enzymatically active T. suis chitinase mediated amelioration of clinical signs of airway hyperreactivity, primarily associated with suppression of eosinophil recruitment into the lung, the associated chemokines, and increased numbers of RELMα + interstitial lung macrophages. While there is no indication of T. suis chitinase directly interfering with dendritic cell activation or antigen presentation to CD4 T cells, treatment of allergic mice with the worm chitinase influenced the hosts' own chitinase activity in the inflamed lung. The three-dimensional structure of the T. suis chitinase as determined by high-resolution X-ray crystallography revealed high similarities to mouse acidic mammalian chitinase (AMCase) but a unique ability of T. suis chitinase to form dimers. Our data indicate that the structural similarities between the parasite and host chitinase contribute to the disease-ameliorating effect of the helminth-derived chitinase on allergic lung inflammation.

Influence of Nutrition and Maternal Bonding on Postnatal Lung Development in the Newborn Pig.

Background: Microbial colonization and immune cell maturation coincide at mucosal sites and are decisive for postnatal lung development. How external factors influence neonatal pulmonary immune development is poorly understood. Objective: To elucidate the impact of key determinants in early life, nutrition, and maternal bonding, on postnatal lung maturation in a human-relevant animal model. To investigate the underlying immunological changes of impaired lung maturation and study the mechanisms of conversion. Methods: Newborn piglets were kept with or without isolation from their mothers and fed bovine milk-based infant formula or received milk of sow. Lung growth, histomorphology, respiratory immune responses, and lung microbiota were analyzed. Mother- and sow-milk-deprived piglets received maternal material or were reintroduced to the maternal environment at varying intervals to study options for reversal. Results: Formula feeding combined with isolation of newborn piglets resulted in disturbed postnatal lung maturation. Reduced lung growth correlated with dampened IL-33 expression, impaired lung myeloid cell activation, and decreased Th1 differentiation, along with diminished richness and diversity of the lung microbiota. Transfer of bacteria-enriched maternal material reversed the negative effects on pulmonary immune maturation. Early (within 3 days) but not late (within 7 days) reintroduction to the mother allowed restoration of normal lung development. Conclusion: Our findings reveal that lung growth, respiratory immunity, and microbial lung colonization in newborns depend on postnatal diet and maternal contact, and targeting these key regulators could promote lung development during this critical life stage. Summary: Disturbances in natural diet and reduced maternal contact during the neonatal period impair postnatal lung maturation. In pediatrics, timely breast milk feeding and intensive maternal bonding represent valuable intervention measures to promote early postnatal lung development.

Early Immune Initiation by Porcine Cells following Toxoplasma gondii Infection versus TLR Ligation.

Containment of acute Toxoplasma gondii infection is dependent on an efficient interferon gamma response. However, the earliest steps of immune response initiation immediately following exposure to the parasite have not been previously characterized in pigs. Murine and human myeloid cells produce large quantities of interleukin (IL)-12 during early T. gondii infection. We therefore examined IL-12 expression by porcine peripheral blood monocytes and dendritic cell (DC) subsets following toll-like receptor (TLR) ligation and controlled T. gondii tachyzoite infection. We detected IL-12p40 expression by porcine plasmacytoid DC, but not conventional or monocyte-derived DC following TLR ligation. Unexpectedly, we also observed considerable IL-12p40 production by porcine CD3- NKp46+ cells-a classical natural killer cell phenotype-following TLR ligation. However, in response to T. gondii exposure, no IL-12 production was observed by either DC or CD3- NKp46+ cells. Despite this, IL-18 production by DC-enriched peripheral blood mononuclear cells was detected following live T. gondii tachyzoite exposure. Only combined stimulation of porcine peripheral blood mononuclear cells with recombinant IL-12p70 and IL-18 induced innate interferon gamma production by natural killer cells, while T cells and myeloid cells did not respond. Therefore, porcine CD3- NKp46+ cells serve as important IL-12 producers following TLR ligation, while IL-18 likely plays a prominent role in early immune response initiation in the pig following T. gondii infection.

The Worm-Specific Immune Response in Multiple Sclerosis Patients Receiving Controlled Trichuris suis Ova Immunotherapy.

Considering their potent immunomodulatory properties, therapeutic applications of Trichuris suis ova (TSO) are studied as potential alternative treatment of autoimmune disorders like multiple sclerosis (MS), rheumatoid arthritis (RA), or inflammatory bowel disease (IBD). Clinical phase 1 and 2 studies have demonstrated TSO treatment to be safe and well tolerated in MS patients, however, they reported only modest clinical efficacy. We therefore addressed the cellular and humoral immune responses directed against parasite antigens in individual MS patients receiving controlled TSO treatment (2500 TSO p.o. every 2 weeks for 12 month). Peripheral blood mononuclear cells (PBMC) of MS patients treated with TSO (n = 5) or placebo (n = 6) were analyzed. A continuous increase of serum IgG and IgE antibodies specific for T. suis excretory/secretory antigens was observed up to 12 months post-treatment. This was consistent with mass cytometry analysis identifying an increase of activated HLA-DRhigh plasmablast frequencies in TSO-treated patients. While stable and comparable frequencies of total CD4+ and CD8+ T cells were detected in placebo and TSO-treated patients over time, we observed an increase of activated HLA-DR+CD4+ T cells in TSO-treated patients only. Frequencies of Gata3+ Th2 cells and Th1/Th2 ratios remained stable during TSO treatment, while Foxp3+ Treg frequencies varied greatly between individuals. Using a T. suis antigen-specific T cell expansion assay, we also detected patient-to-patient variation of antigen-specific T cell recall responses and cytokine production. In summary, MS patients receiving TSO treatment established a T. suis-specific T- and B-cell response, however, with varying degrees of T cell responses and cellular functionality across individuals, which might account for the overall miscellaneous clinical efficacy in the studied patients.

Trilateral Relationship: Ascaris, Microbiota, and Host Cells.

Ascariasis is a globally spread intestinal nematode infection of humans and a considerable concern in pig husbandry. Ascaris accomplishes a complex body migration from the intestine via the liver and lung before returning to the intestine. Tissue migration and the habitat shared with a complex microbial community pose the question of how the nematode interacts with microbes and host cells from various tissues. This review addresses the current knowledge of the trilateral relationship between Ascaris, its microbial environment, and host cells, and discusses novel approaches targeting these interactions to combat this widespread infection of livestock and man.

Toward a porcine in vivo model to analyze the pathogenesis of TLR5-dependent enteropathies.

Non-communicable diseases, such as the metabolic syndrome and inflammatory bowel disease, constitute serious public health threats in developed countries. Besides environmental factors, genetic predispositions contribute to the onset and progression of the disease. State-of-the-art mouse models recently highlight the involvement of Toll-like receptor 5 (TLR5)-driven microbiota composition in the development of metabolic disorders. Here, we discuss the causes and consequences of an altered enteric microbiota and provide information on a similar mechanism in another species, the pig. We show for the first time that a single nucleotide polymorphism in the porcine TLR5 gene conferring impaired functionality is associated with changes in the intestinal microbiota in adult sows and neonatal piglets. Changes in the developing adaptive cellular immune response support the concept of TLR5-driven changes of the microbe-host interplay also in the pig. Together, these findings suggest that pigs with impaired TLR-functionality might represent a model for TLR5-driven diseases in humans.

The domestic pig as human-relevant large animal model to study adaptive antifungal immune responses against airborne Aspergillus fumigatus.

Pulmonary mucosal immune response is critical for preventing opportunistic Aspergillus fumigatus infections. Although fungus-specific CD4+ T cells in blood are described to reflect the actual host-pathogen interaction status, little is known about Aspergillus-specific pulmonary T-cell responses. Here, we exploit the domestic pig as human-relevant large animal model and introduce antigen-specific T-cell enrichment in pigs to address Aspergillus-specific T cells in the lung compared to peripheral blood. In healthy, environmentally Aspergillus-exposed pigs, the fungus-specific T cells are detectable in blood in similar frequencies as observed in healthy humans and exhibit a Th1 phenotype. Exposing pigs to 106 cfu/m3 conidia induces a long-lasting accumulation of Aspergillus-specific Th1 cells locally in the lung and also systemically. Temporary immunosuppression during Aspergillus-exposure showed a drastic reduction in the lung-infiltrating antifungal T-cell responses more than 2 weeks after abrogation of the suppressive treatment. This was reflected in blood, but to a much lesser extent. In conclusion, by using the human-relevant large animal model the pig, this study highlights that the blood clearly reflects the mucosal fungal-specific T-cell reactivity in environmentally exposed as well as experimentally exposed healthy pigs. But, immunosuppression significantly impacts the mucosal site in contrast to the initial systemic immune response.

Influence of Immune Status on the Airborne Colonization of Piglets with Methicillin-Resistant Staphylococcus aureus (MRSA) Clonal Complex (CC) 398.

Colonized vertebrates including humans and pigs are to date the main reservoirs of livestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA). Currently, the mechanisms underlying colonization of pigs are not fully understood. We investigated the influence of piglet pre-immune status on airborne MRSA colonization. Three groups of MRSA-negative piglets were primed and exposed to airborne LA-MRSA (104 colony forming units (cfu)/m3) in an aerosol chamber for 24 h. One group was treated intramuscularly with dexamethasone (1 mg/kg body weight) to imitate weaning stress. The second group was exposed to bacterial endotoxin containing MRSA aerosol. Both conditions play a role in the development of multifactorial diseases and may promote MRSA colonization success. The third group served as control. The piglets' MRSA status was monitored for 21 days via swab samples. At necropsy, specific tissues and organs were analyzed. Blood was collected to examine specific immunological parameters. The duration of MRSA colonization was not extended in both treated groups compared to the control group, indicating the two immune-status influencing factors do not promote MRSA colonization. Blood sample analysis confirmed a mild dexamethasone-induced immune suppression and typical endotoxin-related changes in peripheral blood. Of note, the low-dose dexamethasone treatment showed a trend of increased MRSA clearance.

CD4+ Th immunogenicity of the Ascaris spp. secreted products.

Ascaris spp. is a major health problem of humans and animals alike, and understanding the immunogenicity of its antigens is required for developing urgently needed vaccines. The parasite-secreted products represent the most relevant, yet complex (>250 proteins) antigens of Ascaris spp. as defining the pathogen-host interplay. We applied an in vitro antigen processing system coupled to quantitative proteomics to identify potential CD4+ Th cell epitopes in Ascaris-secreted products. This approach considerably restricts the theoretical list of epitopes using conventional CD4+ Th cell epitope prediction tools. We demonstrate the specificity and utility of our approach on two sets of candidate lists, allowing us identifying hits excluded by either one or both computational methods. More importantly, one of the candidates identified experimentally, clearly demonstrates the presence of pathogen-reactive T cells in healthy human individuals against these antigens. Thus, our work pipeline identifies the first human T cell epitope against Ascaris spp. and represents an easily adaptable platform for characterization of complex antigens, in particular for those pathogens that are not easily amenable for in vivo experimental validation.

Distinct editing functions of natural HLA-DM allotypes impact antigen presentation and CD4+ T cell activation.

Classical human leukocyte antigen (HLA) molecules of the major histocompatibility class II (MHCII) complex present peptides for the development, surveillance and activation of CD4+ T cells. The nonclassical MHCII-like protein HLA-DM (DM) catalyzes the exchange and loading of peptides onto MHCII molecules, thereby shaping MHCII immunopeptidomes. Natural variations of DM in both chains of the protein (DMA and DMB) have been hypothesized to impact peptide presentation, but no evidence for altered function has been reported. Here we define the presence of DM allotypes in human populations covered by the 1000 Genomes Project and probe their activity. The functional properties of several allotypes are investigated and show strong enhancement of peptide-induced T cell activation for a particular combination of DMA and DMB. Biochemical evidence suggests a broader pH activity profile for the new variant relative to that of the most commonly expressed DM allotype. Immunopeptidome analysis indicates that the compartmental activity of the new DM heterodimer extends beyond the late endosome and suggests that the natural variation of DM has profound effects on adaptive immunity when antigens bypass the canonical processing pathway.