Antigenic epitope targets of rhesus macaques self-curing from Schistosoma mansoni infection

The self-cure of rhesus macaques from a schistosome infection and their subsequent strong immunity to a cercarial challenge should provide novel insights into the way these parasites can be eliminated by immunological attack. High-density arrays comprising overlapping 15-mer peptides from target proteins printed on glass slides can be used to screen sera from host species to determine antibody reactivity at the single epitope level. Careful selection of proteins, based on compositional studies, is crucial to encompass only those exposed on or secreted from the intra-mammalian stages and is intended to focus the analysis solely on targets mediating protection. We report the results of this approach using two pools of sera from hi- and lo-responder macaques undergoing self-cure, to screen arrays comprising tegument, esophageal gland, and gastrodermis proteins. We show that, overall, the target epitopes are the same in both groups, but the intensity of response is twice as strong in the high responders. In addition, apart from Sm25, tegument proteins elicit much weaker responses than those originating in the alimentary tract, as was apparent in IFNγR KO mice. We also highlight the most reactive epitopes in key proteins. Armed with this knowledge, we intend to use multi-epitope constructs in vaccination experiments, which seek to emulate the self-cure process in experimental animals and potentially in humans.

Models of Protective Immunity against Schistosomes: Implications for Vaccine Development.

After many decades of research, a schistosome vaccine still looks to be a distant prospect. These helminths can live in the human bloodstream for years, even decades, surrounded by and feeding on the components of the immune response they provoke. The original idea of a vaccine based on the killing of invading cercariae in the skin has proven to be illusory. There has also been a realisation that even if humans develop some protection against infection over a protracted period, it very likely involves IgE-mediated responses that cannot provide the basis for a vaccine. However, it has also become clear that both invasive migrating larvae and adult worms must expose proteins and release secretions into the host environment as part of their normal biological activities. The application of modern 'omics approaches means that we now have a much better idea of the identity of these potential immune targets. This review looks at three animal models in which acquired immunity has been demonstrated and asks whether the mechanisms might inform our vaccine strategies to achieve protection in model hosts and humans. Eliciting responses, either humoral or cellular, that can persist for many months is a challenge. Arming of the lungs with effector T cells, as occurs in mice exposed to the radiation-attenuated cercarial vaccine, is one avenue. Generating IgG antibody titres that reach levels at which they can exert sustained immune pressure to cause worm elimination, as occurs in rhesus macaques, is another. The induction of memory cell populations that can detect trickle invasions of larval stages remains to be explored. One promising approach is the analysis of protective antibodies using high-density peptide arrays of target proteins to identify reactive regions. These can be combined in multi-epitope constructs to immunise a host against many targets simultaneously and cheaply.

Dissection of schistosome tissues under LC-MS compatible preservative conditions for quantitative proteomics.

Schistosomes are blood flukes with specialised tissues and organs, each one playing a pivotal role in perpetuating the parasite life cycle. Herein, we describe a detailed methodology for preserving the proteome of adult Schistosoma mansoni worms during manual dissection for enrichment of tissues associated with the parasite's alimentary tract. We provide step-by-step directions for specimen storage and dissection while in preservative solution, tissue homogenisation, protein extraction and digestion using a methodology fully compatible with downstream quantitative liquid chromatography-mass spectrometry analysis. Our methodology uses label-free and QconCAT-based absolute quantification for detection of S. mansoni oesophageal gland products proposed as vaccine candidates. Through stabilisation of the proteome and minimising sample degradation during dissection our approach has allowed us to access the hidden proteome of target tissues not readily available from total lysates because of their small volume. This protocol can be replicated or adapted to other Schistosoma species lacking quantitative proteomics characterisation of specialised tissues for discovery of proteins with potential diagnostic and therapeutic utility.

Metabolic reprogramming of hepatocytes by Schistosoma mansoni eggs.

Background & Aims: Schistosomiasis is a parasitic infection which affects more than 200 million people globally. Schistosome eggs, but not the adult worms, are mainly responsible for schistosomiasis-specific morbidity in the liver. It is unclear if S. mansoni eggs consume host metabolites, and how this compromises the host parenchyma. Methods: Metabolic reprogramming was analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging, liquid chromatography with high-resolution mass spectrometry, metabolite quantification, confocal laser scanning microscopy, live cell imaging, quantitative real-time PCR, western blotting, assessment of DNA damage, and immunohistology in hamster models and functional experiments in human cell lines. Major results were validated in human biopsies. Results: The infection with S. mansoni provokes hepatic exhaustion of neutral lipids and glycogen. Furthermore, the distribution of distinct lipid species and the regulation of rate-limiting metabolic enzymes is disrupted in the liver of S. mansoni infected animals. Notably, eggs mobilize, incorporate, and store host lipids, while the associated metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes. Administration of reactive oxygen species scavengers ameliorates these deleterious effects. Conclusions: Our findings indicate that S. mansoni eggs completely reprogram lipid and carbohydrate metabolism via soluble factors, which results in oxidative stress-induced cell damage in the host parenchyma. Impact and implications: The authors demonstrate that soluble egg products of the parasite S. mansoni induce hepatocellular reprogramming, causing metabolic exhaustion and a strong redox imbalance. Notably, eggs mobilize, incorporate, and store host lipids, while the metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes, independent of the host's immune response. S. mansoni eggs take advantage of the host environment through metabolic reprogramming of hepatocytes and enterocytes. By inducing DNA damage, this neglected tropical disease might promote hepatocellular damage and thus influence international health efforts.

Antigenic epitope targets of rhesus macaques self-curing from Schistosoma mansoni infection.

The self-cure of rhesus macaques from a schistosome infection and their subsequent strong immunity to a cercarial challenge should provide novel insights into the way these parasites can be eliminated by immunological attack. High-density arrays comprising overlapping 15-mer peptides from target proteins printed on glass slides can be used to screen sera from host species to determine antibody reactivity at the single epitope level. Careful selection of proteins, based on compositional studies, is crucial to encompass only those exposed on or secreted from the intra-mammalian stages and is intended to focus the analysis solely on targets mediating protection. We report the results of this approach using two pools of sera from hi- and lo-responder macaques undergoing self-cure, to screen arrays comprising tegument, esophageal gland, and gastrodermis proteins. We show that, overall, the target epitopes are the same in both groups, but the intensity of response is twice as strong in the high responders. In addition, apart from Sm25, tegument proteins elicit much weaker responses than those originating in the alimentary tract, as was apparent in IFNγR KO mice. We also highlight the most reactive epitopes in key proteins. Armed with this knowledge, we intend to use multi-epitope constructs in vaccination experiments, which seek to emulate the self-cure process in experimental animals and potentially in humans.

Schistosome proteomics: updates and clinical implications.

INTRODUCTION: Schistosomes are long-lived blood dwelling helminth parasites using intricate mechanisms to invade, mature, and reproduce inside their vertebrate hosts, whilst simultaneously deploying immune evasion strategies. Their multi-tissue organization and solid body plan presents particular problems for the definition of sub-proteomes. AREAS COVERED: Here, we focus on the two host-parasite interfaces of the adult worm accessible to the immune system, namely the tegument and the alimentary tract, but also on the secretions of the infective cercaria, the migrating schistosomulum and the mature egg. In parallel, we introduce the concepts of "leakyome' and 'disintegrome' to emphasize the importance of interpreting data in the context of schistosome biology so that misleading conclusions about the distinct proteome compositions are avoided. Lastly, we highlight the possible clinical implications of the reviewed proteomic findings for pathogenesis, vaccine design and diagnostics. EXPERT OPINION: Proteomics has provided considerable insights into the biology of schistosomes, most importantly for rational selection of novel vaccine candidates that might confer protective immunity, but also into the pathogenesis of schistosomiasis. However, given the increasing sensitivity of mass spectrometric instrumentation, we stress the need for care in data interpretation since schistosomes do not deviate from the fundamental rules of eukaryotic cell biology.

An atlas of the germ ball-cercaria-schistosomulum transition in Schistosoma mansoni, using confocal microscopy and in situ hybridisation.

Schistosomes are complex platyhelminth parasites with a genome comprising ∼12,000 protein-coding genes, three distinct generations, and at least seven distinct phenotypes. We chart here cellular and gene expression changes associated with development of the cercaria, in the intramolluscan daughter sporocyst, and its transformation into the skin stage schistosomulum upon infection of the mammalian host. We describe the morphology of the early daughter sporocyst and the increasing complexity of cellular organisation in germ balls as they rapidly develop into cercariae. We show how individual myocytes differentiate and combine to create the complex musculature of the head capsule and body wall. In situ hybridisation reveals that some transcripts encoding the secretory proteins, released during skin penetration, are expressed in gland-cell precursors very early in germ ball development. However, those for the projected anti-inflammatory protein Sm16-stathmin are widely expressed in germ ball tissues, suggesting the protein has intracellular functions. Transcripts for smkk7 are expressed in six cells of the larval body, while the KK7 protein is present throughout the peripheral nerve net, including sensory nerve bulbs, providing a marker for the nerve net in adult worms. We also note that the cercaria-schistosomulum transformation is accompanied by tissue remodelling without growth.

Fifty years of the schistosome tegument: discoveries, controversies, and outstanding questions.

The unique multilaminate appearance of the tegument surface of schistosomes was first described in 1973, in one of the earliest volumes of the International Journal for Parasitology. The present review, published almost 50 years later, traces the development of our knowledge of the tegument, starting with those earliest cytological advances, particularly the surface plasma membrane-membranocalyx complex, through an era of protein discovery to the modern age of protein characterization, aided by proteomics. More recently, analysis of single cell transcriptomes of schistosomes is providing insight into the organisation of the cell bodies that support the surface syncytium. Our understanding of the tegument, notably the nature of the proteins present within the plasma membrane and membranocalyx, has provided insights into how the schistosomes interact with their hosts but many aspects of how the tegument functions remain unanswered. Among the unresolved aspects are those concerned with maintenance and renewal of the surface membrane complex, and whether surface proteins and membrane components are recycled. Current controversies arising from investigations about whether the tegument is a source of extracellular vesicles during parasitism, and if it is covered with glycolytic enzymes, are evaluated in the light of cytological and proteomic knowledge of the layer.

Rhesus macaques self-curing from a schistosome infection can display complete immunity to challenge.

The rhesus macaque provides a unique model of acquired immunity against schistosomes, which afflict >200 million people worldwide. By monitoring bloodstream levels of parasite-gut-derived antigen, we show that from week 10 onwards an established infection with Schistosoma mansoni is cleared in an exponential manner, eliciting resistance to reinfection. Secondary challenge at week 42 demonstrates that protection is strong in all animals and complete in some. Antibody profiles suggest that antigens mediating protection are the released products of developing schistosomula. In culture they are killed by addition of rhesus plasma, collected from week 8 post-infection onwards, and even more efficiently with post-challenge plasma. Furthermore, cultured schistosomula lose chromatin activating marks at the transcription start site of genes related to worm development and show decreased expression of genes related to lysosomes and lytic vacuoles involved with autophagy. Overall, our results indicate that enhanced antibody responses against the challenge migrating larvae mediate the naturally acquired protective immunity and will inform the route to an effective vaccine.

Rhesus macaques self-curing from a schistosome infection can display complete immunity to challenge

The rhesus macaque provides a unique model of acquired immunity against schistosomes, which afflict >200 million people worldwide. By monitoring bloodstream levels of parasite-gut-derived antigen, we show that from week 10 onwards an established infection with Schistosoma mansoni is cleared in an exponential manner, eliciting resistance to reinfection. Secondary challenge at week 42 demonstrates that protection is strong in all animals and complete in some. Antibody profiles suggest that antigens mediating protection are the released products of developing schistosomula. In culture they are killed by addition of rhesus plasma, collected from week 8 post-infection onwards, and even more efficiently with post-challenge plasma. Furthermore, cultured schistosomula lose chromatin activating marks at the transcription start site of genes related to worm development and show decreased expression of genes related to lysosomes and lytic vacuoles involved with autophagy. Overall, our results indicate that enhanced antibody responses against the challenge migrating larvae mediate the naturally acquired protective immunity and will inform the route to an effective vaccine.

Epitope mapping of exposed tegument and alimentary tract proteins identifies putative antigenic targets of the attenuated schistosome vaccine

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

Mapping the epitopes of Schistosoma japonicum esophageal gland proteins for incorporation into vaccine constructs.

BACKGROUND: The development of a schistosome vaccine has proved challenging but we have suggested that characterisation of the self-cure mechanism in rhesus macaques might provide a route to an effective product. The schistosome esophagus is a complex structure where blood processing is initiated by secretions from anterior and posterior glands, achieved by a mixture of ~40 unique proteins. The mechanism of self-cure in macaques involves cessation of feeding, after which worms slowly starve to death. Antibody coats the esophagus lumen and disrupts the secretory processes from the glands, potentially making their secretions ideal vaccine targets. METHODOLOGY/PRINCIPAL FINDINGS: We have designed three peptide arrays comprising overlapping 15-mer peptides encompassing 32 esophageal gland proteins, and screened them for reactivity against 22-week infection serum from macaques versus permissive rabbit and mouse hosts. There was considerable intra- and inter-species variation in response and no obvious unique target was associated with self-cure status, which suggests that self-cure is achieved by antibodies reacting with multiple targets. Some immuno-dominant sequences/regions were evident across species, notably including: MEGs 4.1C, 4.2, and 11 (Array 1); MEG-12 and Aspartyl protease (Array 2); a Tetraspanin 1 loop and MEG-n2 (Array 3). Responses to MEGs 8.1C and 8.2C were largely confined to macaques. As proof of principle, three synthetic genes were designed, comprising several key targets from each array. One of these was expressed as a recombinant protein and used to vaccinate rabbits. Higher antibody titres were obtained to the majority of reactive regions than those elicited after prolonged infection. CONCLUSIONS/SIGNIFICANCE: It is feasible to test simultaneously the additive potential of multiple esophageal proteins to induce protection by combining their most reactive regions in artificial constructs that can be used to vaccinate suitable hosts. The efficacy of the approach to disrupt esophageal function now needs to be tested by a parasite challenge.

Schistosomiasis then and now: what has changed in the last 100 years?

Only with the completion of the life cycles of Fasciola hepatica in 1883 and 30 years later those of Schistosoma japonicum (1913), Schistosoma haematobium and Schistosoma mansoni (1915) did research on schistosomiasis really get underway. One of the first papers by Cawston in 1918, describing attempts to establish the means of transmission of S. haematobium in Natal, South Africa, forms the historical perspective against which to judge where we are now. Molecular biology techniques have produced a much better definition of the complexity of the schistosome species and their snail hosts, but also revealed the extent of hybridization between human and animal schistosomes that may impact on parasite adaptability. While diagnostics have greatly improved, the ability to detect single worm pair infections routinely, still falls short of its goal. The introduction of praziquantel ~1982 has revolutionized the treatment of infected individuals and led directly to the mass drug administration programmes. In turn, the severe pathological consequences of high worm burdens have been minimized, and for S. haematobium infections the incidence of associated squamous cell carcinoma has been reduced. In comparison, the development of effective vaccines has yet to come to fruition. The elimination of schistosomiasis japonica from Japan shows what is possible, using multiple lines of approach, but the clear and present danger is that the whole edifice of schistosome control is balanced on the monotherapy of praziquantel, and the development of drug resistance could topple that.

Epitope Mapping of Exposed Tegument and Alimentary Tract Proteins Identifies Putative Antigenic Targets of the Attenuated Schistosome Vaccine.

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

Quantitative Proteomics of Enriched Esophageal and Gut Tissues from the Human Blood Fluke Schistosoma mansoni Pinpoints Secreted Proteins for Vaccine Development.

Schistosomes are blood-dwelling helminth parasites that cause schistosomiasis, a debilitating disease resulting in inflammation and, in extreme cases, multiple organ damage. Major challenges to control the transmission persist, and the discovery of protective antigens remains of critical importance for vaccine development. Rhesus macaques can self-cure following schistosome infection, generating antibodies that target proteins from the tegument, gut, and esophagus, the last of which is the least investigated. We developed a dissection technique that permitted increased sensitivity in a comparative proteomics profiling of schistosome esophagus and gut. Proteome analysis of the male schistosome esophagus identified 13 proteins encoded by microexon genes (MEGs), 11 of which were uniquely located in the esophageal glands. Based on this and transcriptome information, a QconCAT was designed for the absolute quantification of selected targets. MEGs 12, 4.2, and 4.1 and venom allergen-like protein 7 were the most abundant, spanning over 245 million to 6 million copies per cell, while aspartyl protease, palmitoyl thioesterase, and galactosyl transferase were present at <1 million copies. Antigenic variation by alternative splicing of MEG proteins was confirmed together with a specialized machinery for protein glycosylation/secretion in the esophagus. Moreover, some gastrodermal secretions were highly enriched in the gut, while others were more uniformly distributed throughout the parasite, potentially indicating lysosomal activity. Collectively, our findings provide a more rational, better-oriented selection of schistosome vaccine candidates in the context of a proven model of protective immunity.

Micro Array-Assisted Analysis of Anti-Schistosome Glycan Antibodies Elicited by Protective Vaccination With Irradiated Cercariae.

Baboons vaccinated with radiation-attenuated cercariae develop high levels of protection against schistosome infection, correlating to high antibody titres towards schistosome antigens with unknown molecular identity. Using a microarray consisting of glycans isolated from different life-stages of schistosomes, we studied the anti-glycan immunoglobulin (Ig) G and IgM responses in vaccinated and challenged baboons over a time course of 25 weeks. Anti-glycan IgM responses developed early after vaccination, but did not rise in response to later vaccinations. In contrast, anti-glycan IgG developed more slowly, but was boosted by all five subsequent vaccinations. High IgM and IgG levels against O-glycans and glycosphingolipid glycans of cercariae were observed. At the time of challenge, while most antibody levels decreased in the absence of vaccination, IgG towards a subset of glycans containing multiple-fucosylated motifs remained high until 6 weeks post-challenge during challenge parasite elimination, suggesting a possible role of this IgG in protection.

Diagnosis of schistosomiasis mansoni: an evaluation of existing methods and research towards single worm pair detection.

The inadequacy of current diagnostics for the detection of low worm burdens in humans means that schistosomiasis mansoni is more widespread than previously acknowledged. With the inception of mass drug treatment programmes aimed at disease elimination and the advent of human vaccine trials, the need for more sensitive diagnostics is evident. In this review, we evaluate the merits and limitations of the principal diagnostic methods, namely detection of eggs in faeces; anti-schistosome antibodies in serum; parasite-derived proteins and glycans in serum or urine; parasite DNA in blood, faeces or urine. Only in the baboon model, where actual worm burden is determined by portal perfusion, have faecal smear and circulating antigen methods been calibrated, and shown to have thresholds of detection of 10-19 worm pairs. There is scope for improvement in all the four methods of detection, e.g. the identification of single targets for host antibodies to improve the specificity of enzyme linked immunosorbent assay. Despite recent advances in the definition of the schistosome secretome, there have been no comprehensive biomarker investigations of parasite products in the urine of infected patients. Certainly, the admirable goal of eliminating schistosomiasis will not be achieved unless individuals with low worm burdens can be diagnosed.

Microexon gene transcriptional profiles and evolution provide insights into blood processing by the Schistosoma japonicum esophagus.

BACKGROUND: Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a Schistosoma japonicum infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of S. japonicum esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process. METHODOLOGY/PRINCIPAL FINDINGS: The heads and a small portion of the rear end of male and female S. japonicum worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled de novo using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome Trichobilharzia regenti, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood. SIGNIFICANCE/CONCLUSIONS: The mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates.

Nitric oxide blocks the development of the human parasite Schistosoma japonicum.

Human schistosomiasis, caused by Schistosoma species, is a major public health problem affecting more than 700 million people in 78 countries, with over 40 mammalian host reservoir species complicating the transmission ecosystem. The primary cause of morbidity is considered to be granulomas induced by fertilized eggs of schistosomes in the liver and intestines. Some host species, like rats (Rattus norvegicus), are naturally intolerant to Schistosoma japonicum infection, and do not produce granulomas or pose a threat to transmission, while others, like mice and hamsters, are highly susceptible. The reasons behind these differences are still a mystery. Using inducible nitric oxide synthase knockout (iNOS-/-) Sprague-Dawley rats, we found that inherent high expression levels of iNOS in wild-type (WT) rats play an important role in blocking growth, reproductive organ formation, and egg development in S. japonicum, resulting in production of nonfertilized eggs. Granuloma formation, induced by fertilized eggs in the liver, was considerably exacerbated in the iNOS-/- rats compared with the WT rats. This inhibition by nitric oxide acts by affecting mitochondrial respiration and energy production in the parasite. Our work not only elucidates the innate mechanism that blocks the development and production of fertilized eggs in S. japonicum but also offers insights into a better understanding of host-parasite interactions and drug development strategies against schistosomiasis.