Harnessing helminth-driven immunoregulation in the search for novel therapeutic modalities.

Parasitic helminths have coevolved with humans over millennia, intricately refining and developing an array of mechanisms to suppress or skew the host's immune system, thereby promoting their long-term survival. Some helminths, such as hookworms, cause little to no overt pathology when present in modest numbers and may even confer benefits to their human host. To exploit this evolutionary phenomenon, clinical trials of human helminth infection have been established and assessed for safety and efficacy for a range of immune dysfunction diseases and have yielded mixed outcomes. Studies of live helminth therapy in mice and larger animals have convincingly shown that helminths and their excretory/secretory products possess anti-inflammatory drug-like properties and represent an untapped pharmacopeia. These anti-inflammatory moieties include extracellular vesicles, proteins, glycans, post-translational modifications, and various metabolites. Although the concept of helminth-inspired therapies holds promise, it also presents a challenge to the drug development community, which is generally unfamiliar with foreign biologics that do not behave like antibodies. Identification and characterization of helminth molecules and vesicles and the molecular pathways they target in the host present a unique opportunity to develop tailored drugs inspired by nature that are efficacious, safe, and have minimal immunogenicity. Even so, much work remains to mine and assess this out-of-the-box therapeutic modality. Industry-based organizations need to consider long-haul investments aimed at unraveling and exploiting unique and differentiated mechanisms of action as opposed to toe-dipping entries with an eye on rapid and profitable turnarounds.

Novel cholinesterase paralogs of Schistosoma mansoni have perceived roles in cholinergic signalling and drug detoxification and are essential for parasite survival.

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 -smp_154600 and Smache2 -smp_136690) and a butyrylcholinesterase (BChE) (Smbche1 -smp_125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor 2,2- dichlorovinyl dimethyl phosphate-dichlorvos (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP.

Inflammasome-Independent Role for NLRP3 in Controlling Innate Antihelminth Immunity and Tissue Repair in the Lung.

Alternatively activated macrophages are essential effector cells during type 2 immunity and tissue repair following helminth infections. We previously showed that Ym1, an alternative activation marker, can drive innate IL-1R-dependent neutrophil recruitment during infection with the lung-migrating nematode, Nippostrongylus brasiliensis, suggesting a potential role for the inflammasome in the IL-1-mediated innate response to infection. Although inflammasome proteins such as NLRP3 have important proinflammatory functions in macrophages, their role during type 2 responses and repair are less defined. We therefore infected Nlrp3 -/- mice with N. brasiliensis Unexpectedly, compared with wild-type (WT) mice, infected Nlrp3 -/- mice had increased neutrophilia and eosinophilia, correlating with enhanced worm killing but at the expense of increased tissue damage and delayed lung repair. Transcriptional profiling showed that infected Nlrp3 -/- mice exhibited elevated type 2 gene expression compared with WT mice. Notably, inflammasome activation was not evident early postinfection with N. brasiliensis, and in contrast to Nlrp3 -/- mice, antihelminth responses were unaffected in caspase-1/11-deficient or WT mice treated with the NLRP3-specific inhibitor MCC950. Together these data suggest that NLRP3 has a role in constraining lung neutrophilia, helminth killing, and type 2 immune responses in an inflammasome-independent manner.

Lipopeptide-Based Oral Vaccine Against Hookworm Infection.

BACKGROUND: The human hookworm, Necator americanus, is a parasite that infects almost half a billion people worldwide. Although treatment is available, vaccination is favorable to combat the spread of this parasite due to its wide distribution and continuous reinfection cycle in endemic communities. METHODS: We have designed a lipopeptide oral delivery system using a B-cell epitope derived from the aspartic protease Na-APR-1 from N americanus, attached to a T-helper epitope. Lipopeptides were self-assembled into nanoparticles or entrapped in liposomes that were electrostatically coated with alginate and trimethyl chitosan polymer shields. The adjuvant-free vaccine candidates were orally administered to mice and generated a humoral immune response against both peptide antigen, and the parent protein in the hookworm gut. RESULTS: The vaccine candidates were evaluated in a rodent hookworm challenge model, resulting in up to 98% and 99% decreases in mean intestinal worm and egg burdens in immunized mice, respectively. CONCLUSIONS: Lipopeptide survived the gastrointestinal conditions, induced humoral immune responses and drived protection against parasite challenge infection.

Metabolomic profiling of the excretory-secretory products of hookworm and whipworm.

INTRODUCTION: Soil-transmitted helminths infect billions of people, livestock and companion animals worldwide, and chronic infections with these nematodes represent a major health burden in many developing countries. On the other hand, complete elimination of parasitic helminths and other infectious pathogens has been implicated with rising rates of autoimmune and allergic disorders in developed countries. Given the enormous health impact of these parasites, it is surprising how little is known about the non-protein small metabolites of the excretory-secretory products (ESP), including their composition and pharmacological properties. OBJECTIVES: We sought proof-of-concept that Nippostrongylus brasiliensis and Trichuris muris, rodent models of two of the most important human soil-transmitted helminths, secrete small metabolites and that some of these metabolites may have specific pharmacological functions. METHODS: N. brasiliensis and T. muris ESP were collected from adult worms and filtered using a 10 kDa cut-off membrane to produce excretory-secretory metabolites (ESM). The ESM were analysed using targeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry for polar and non-polar small metabolites. RESULTS: ESM from both N. brasiliensis and T. muris contained small molecules. A total of 54 small molecules (38 polar metabolites and 16 fatty acids) were identified, 36 known polar metabolites from N. brasiliensis and 35 from T. muris. A literature review of the identified compounds revealed that 17 of them have various demonstrated pharmacological activities. CONCLUSION: N. brasiliensis and T. muris secrete polar and non-polar small molecules with as many as 17 metabolites known to exhibit various pharmacological activities.

Characterization of Tapeworm Metabolites and Their Reported Biological Activities.

Parasitic helminths infect billions of people, livestock, and companion animals worldwide. Recently, they have been explored as a novel therapeutic modality to treat autoimmune diseases due to their potent immunoregulatory properties. While feeding in the gut/organs/tissues, the parasitic helminths actively release excretory-secretory products (ESP) to modify their environment and promote their survival. The ESP proteins of helminths have been widely studied. However, there are only limited studies characterizing the non-protein small molecule (SM) components of helminth ESP. In this study, using GC-MS and LC-MS, we have investigated the SM ESP of tapeworm Dipylidium caninum (isolated from dogs) which accidentally infects humans via ingestion of infected cat and dog fleas that harbor the larval stage of the parasite. From this D. caninum ESP, we have identified a total of 49 SM (35 polar metabolites and 14 fatty acids) belonging to 12 different chemotaxonomic groups including amino acids, amino sugars, amino acid lactams, organic acids, sugars, sugar alcohols, sugar phosphates, glycerophosphates, phosphate esters, disaccharides, fatty acids, and fatty acid derivatives. Succinic acid was the major small molecule present in the D. caninum ESP. Based on the literature and databases searches, we found that of 49 metabolites identified, only 12 possessed known bioactivities.

Immunobiology of parasitic worm extracellular vesicles.

Helminth parasites (worms) have evolved a vast array of strategies to manipulate their vertebrate hosts. Extracellular vesicles (EVs) are secreted by all helminth species investigated thus far, and their salient roles in parasite-host interactions are being revealed. Parasite EVs directly interact with various cell types from their hosts, including immune cells, and roles for their molecular cargo in both regulation and promotion of inflammation in the host have been reported. Despite the growing body of literature on helminth EVs, limited availability of genetic manipulation tools for helminth research has precluded detailed investigation of specific molecular interactions between parasite EVs and host target cells. Here, we review the current state of the field and discuss innovative strategies targeting helminth EVs for the discovery and development of new therapeutic strategies, placing particular emphasis on both anti-helminth vaccines and EV small RNAs for treating noninfectious inflammatory diseases.

RNA Seq analysis of the Eimeria tenella gametocyte transcriptome reveals clues about the molecular basis for sexual reproduction and oocyst biogenesis.

BACKGROUND: The protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite's sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission. RESULTS: Comparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered. CONCLUSIONS: The need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.

Small extracellular vesicles but not microvesicles from Opisthorchis viverrini promote cell proliferation in human cholangiocytes.

Chronic infection with O. viverrini has been linked to the development of cholangiocarcinoma (CCA), which is a major public health burden in the Lower Mekong River Basin countries, including Thailand, Lao PDR, Vietnam and Cambodia. Despite its importance, the exact mechanisms by which O. viverrini promotes CCA are largely unknown. In this study, we characterized different extracellular vesicle populations released by O. viverrini (OvEVs) using proteomic and transcriptomic analyses and investigated their potential role in host-parasite interactions. While 120k OvEVs promoted cell proliferation in H69 cells at different concentrations, 15k OvEVs did not produce any effect compared to controls. The proteomic analysis of both populations showed differences in their composition that could contribute to this differential effect. Furthermore, the miRNAs present in 120k EVs were analysed and their potential interactions with human host genes was explored by computational target prediction. Different pathways involved in inflammation, immune response and apoptosis were identified as potentially targeted by the miRNAs present in this population of EVs. This is the first study showing specific roles for different EV populations in the pathogenesis of a parasitic helminth, and more importantly, an important advance towards deciphering the mechanisms used in establishment of opisthorchiasis and liver fluke infection-associated malignancy.

Administration of Hookworm Excretory/Secretory Proteins Improves Glucose Tolerance in a Mouse Model of Type 2 Diabetes.

Diabetes is recognised as the world's fastest growing chronic condition globally. Helminth infections have been shown to be associated with a lower prevalence of type 2 diabetes (T2D), in part due to their ability to induce a type 2 immune response. Therefore, to understand the molecular mechanisms that underlie the development of T2D-induced insulin resistance, we treated mice fed on normal or diabetes-promoting diets with excretory/secretory products (ES) from the gastrointestinal helminth Nippostrongylus brasiliensis. We demonstrated that treatment with crude ES products from adult worms (AES) or infective third-stage larvae (L3ES) from N. brasiliensis improved glucose tolerance and attenuated body weight gain in mice fed on a high glycaemic index diet. N. brasiliensis ES administration to mice was associated with a type 2 immune response measured by increased eosinophils and IL-5 in peripheral tissues but not IL-4, and with a decrease in the level of IL-6 in adipose tissue and corresponding increase in IL-6 levels in the liver. Moreover, treatment with AES or L3ES was associated with significant changes in the community composition of the gut microbiota at the phylum and order levels. These data highlight a role for N. brasiliensis ES in modulating the immune response associated with T2D, and suggest that N. brasiliensis ES contain molecules with therapeutic potential for treating metabolic syndrome and T2D.