PLOS PATHOGENS

REVIEW

Harnessing helminth-driven
immunoregulation in the search for novel
therapeutic modalities
Stephanie M. Ryan, Ramon M. Eichenberger ID, Roland Ruscher ID, Paul R. Giacomin,
Alex Loukas ID*
Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook
University, Cairns, Queensland, Australia

* [email protected]

Abstract
Parasitic helminths have coevolved with humans over millennia, intricately refining and
a1111111111 developing an array of mechanisms to suppress or skew the host’s immune system, thereby
a1111111111 promoting their long-term survival. Some helminths, such as hookworms, cause little to no
a1111111111
overt pathology when present in modest numbers and may even confer benefits to their
a1111111111
a1111111111 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/secre-
OPEN ACCESS
tory products possess anti-inflammatory drug-like properties and represent an untapped
pharmacopeia. These anti-inflammatory moieties include extracellular vesicles, proteins,
Citation: Ryan SM, Eichenberger RM, Ruscher R,
Giacomin PR, Loukas A (2020) Harnessing glycans, post-translational modifications, and various metabolites. Although the concept of
helminth-driven immunoregulation in the search helminth-inspired therapies holds promise, it also presents a challenge to the drug develop-
for novel therapeutic modalities. PLoS Pathog 16 ment community, which is generally unfamiliar with foreign biologics that do not behave like
(5): e1008508. https://doi.org/10.1371/journal.
antibodies. Identification and characterization of helminth molecules and vesicles and the
ppat.1008508
molecular pathways they target in the host present a unique opportunity to develop tailored
Editor: James B. Lok, University of Pennsylvania,
drugs inspired by nature that are efficacious, safe, and have minimal immunogenicity. Even
UNITED STATES
so, much work remains to mine and assess this out-of-the-box therapeutic modality. Indus-
Published: May 14, 2020
try-based organizations need to consider long-haul investments aimed at unraveling and
Copyright: © 2020 Ryan et al. This is an open exploiting unique and differentiated mechanisms of action as opposed to toe-dipping entries
access article distributed under the terms of the
with an eye on rapid and profitable turnarounds.
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.

Funding: National Health and Medical Research Introduction

Council, grants APP1132975 and APP1117504.
The funders had no role in study design, data Parasitic worms (helminths) infect approximately 2 billion people worldwide, predominantly
collection and analysis, decision to publish, or children in rural subtropical and tropical areas with inadequate sanitation [1]. Helminths have
preparation of the manuscript. struck a balance with their hosts, refined by millennia of coevolution, to meet their needs for
Competing interests: The authors have declared propagation and transmission while minimizing pathology [2]. They promote wound healing
that no competing interests exist. and tissue repair and skew distinct immune processes to improve their long-term survival.

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 1 / 20

PLOS PATHOGENS

Arguably, the most masterful trait of parasitic helminths, from a drug development perspec-
tive, is their ability to potently regulate host inflammatory responses.

Helminth-mediated prevention of inflammatory and metabolic

disorders in people
In industrialized nations, there has been a reduction in exposure to infectious agents because
of vaccination, increased sanitation, improved hygienic standards, and widespread use of anti-
biotics. The vast decline in the prevalence of contagious diseases from these communities, hel-
minthiases, in particular, is inversely associated with an alarming increase in the incidence of
inflammatory and metabolic disorders [3]. For example, the Western world is experiencing an
increasing rate of inflammatory bowel disease (IBD), and at present, there is no available cure.
Compounding matters, there has been a sharp rise in the incidence of IBD and allergies in the
newly industrialized nations of Asia and Latin America [4]. This increase in noncommunic-
able diseases is, at least in part, a result of diets becoming westernized, decreased exposure to
infections, large scale deworming programs, and mass migration [5].
Although the association between helminths and inflammatory diseases is multifactorial,
selective pressure placed on the human genome by historically widespread helminth infection
has driven various polymorphisms, including at loci associated with predispositions to inflam-
matory diseases [6]. Moreover, minimal exposure to pathogens results in an underdeveloped
regulatory immune network, culminating in an increased prevalence of disorders that result
from immune dysfunction [7, 8]. Helminth-mediated protection is not, however, restricted to
autoimmune and allergic diseases. There is an inverse relationship observed between human
helminth infection, insulin resistance, and type 2 diabetes (T2D) [9, 10]. It has been proposed
that chronic helminth infection results in long-term beneficial effects on host metabolism,
especially on white adipose tissue (WAT), intestines, and liver [11]. Understanding the molec-
ular mechanisms of WAT inflammation is topical in drug development given the epidemic of
metabolic diseases, and we will touch upon this again later in the review.
The mechanisms by which parasitic helminths regulate inflammation and metabolism are
diverse and complex and have been reviewed extensively [11–15]. Helminths are potent driv-
ers of T helper type 2 (TH2) immune responses, characterized by eosinophilia, mast cell masto-
cytosis, type 2 innate lymphoid cells (ILC2s), tuft cells, and mucus production. Overlaid on
this TH2 response, however, is a predominant state of immune tolerance, characterized by an
abundance of IL-10 produced by regulatory cell populations such as regulatory T cells (Tregs),
regulatory B cells (Bregs), tolerogenic dendritic cells (DCs), and alternatively activated macro-
phages. Furthermore, the interactions between helminths, the microbiome and its attendant
metabolites [16–21], and the nervous system have become increasingly important (Fig 1).

Clinical trials of experimental human helminth infections for

treating inflammatory diseases
Experimental helminth infections have been used in the treatment of allergic and autoimmune
diseases in human clinical trials for over 15 years and have yielded promising results (Table 1).
Two helminth species have been used in clinical trials for treating inflammatory diseases—the pig
whipworm Trichuris suis and the human hookworm Necator americanus. The therapeutic poten-
tial of orally administered T. suis ova (TSO) has been assessed in phase 1 trials in patients with the
2 primary forms of IBD—Crohn’s disease and ulcerative colitis. After 12 weeks of therapy, signifi-
cant improvement according to the intent-to-treat principle occurred in patients receiving TSO
compared with those who received placebo [22]. TSO was also assessed in an open-label clinical
trial in Crohn’s disease patients, in which 72% of subjects were in remission after 24 weeks [23].

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 2 / 20

PLOS PATHOGENS

Fig 1. "Worm therapy" for immune dysregulation diseases. The range of host physiological factors impacted by gastrointestinal helminth infection could
alleviate inflammatory disease. (1) Parasite-derived factors drive an inclusive or exclusive polarized regulatory or type 2 response, which is responsible for (2)
direct secretion of anti-inflammatory molecules from the host immune system (3) and the promotion of barrier integrity, which is often compromised in the
pathophysiology of IBD and foodborne incompatibilities. Furthermore, (4) helminth colonization provides factors for a diverse bacterial environment that
protects against gut inflammation. IBD, inflammatory bowel disease; TH2, T helper type 2; Treg, regulatory T cell.
https://doi.org/10.1371/journal.ppat.1008508.g001

Disappointingly, although early trials with T. suis showed promise, subsequent phase 2 trials failed
to reach their clinical endpoints in both IBD [24] and multiple sclerosis [25, 26].
T. suis establishes chronic infections in pigs but is expelled from the human body within
weeks and therefore requires frequent dosing. On the other hand, N. americanus is primarily a
human parasite and can survive for years in infected individuals [27]. Experimental infection
with N. americanus is safe and well tolerated in human volunteers [28–30]. In a small phase 1
clinical trial for Crohn’s disease, percutaneous administration of N. americanus in relatively low
doses was well tolerated, and patients who remained in the trial for 1 year were in disease remis-
sion [31]. A subsequent trial targeted celiac disease because of its histopathological similarities
to IBD. Celiac subjects on a gluten-free diet who were experimentally infected with N. ameri-
canus displayed improved tolerance to escalating gluten micro-challenge, a decreased presence
of inflammatory cytokine-producing T cells in the gut, and corresponding increases in mucosal
Treg numbers [30]. Indeed, with the growing body of literature supporting a role for inflamma-
tion in driving T2D [32], a clinical trial using experimental N. americanus infection is currently
being conducted to investigate the therapeutic effect of helminth infections in metabolic disor-
ders (Fig 2) [33]. Most of these early-phase clinical trials were impaired by the absence of a

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 3 / 20

PLOS PATHOGENS

Table 1. Completed and ongoing therapeutic clinical trials using helminth products in humans in disease settings. These trials are rigorously assessing the safety and
tolerability of experimental helminth infection and therapeutic efficacy of infections in disease indications.
Trial/phase Helminth Status (year Study title and treatment Results outcome Reference
range)
Phase I TSO 2003 Crohn’s disease Patients displayed clinical improvements [128]
(complete) Initial safety studies of oral inoculation (2,500 and no serious adverse events.
ova) over 12 weeks (n = 7).
NCT01433471 TSO 2005 Ulcerative colitis Improvement in disease index by 43% in [23]
Phase 1 and 2 (complete) Open-label study randomized, double-blind, treatment cohort.
placebo-controlled oral inoculation (2,500 ova)
over 12 weeks (n = 30).
EUCTR2011- TSO 2008 to 2011 Rheumatoid arthritis Trial terminated and results unknown. Immanuel
006344-71-DE (terminated) Oral inoculation (2,500 ova) in 2-week intervals Hospital Berlin,
Phase 1 for 24 weeks (n = 50). Germany
NCT00645749 TSO 2008 to 2015 Multiple sclerosis Trend toward 35% diminution in active [129]
Phase 2 (complete) (HINT) Oral inoculation (2,500 ova) 2-week lesions. Increase in T regulatory
intervals for 12 weeks (n = 17). lymphocytes with treatment. Increase in
serum levels of IL-4 and IL-10 during
treatment. No serious adverse events.
NCT01006941 TSO 2009 to 2011 Multiple sclerosis No obvious benefit observed in infection [25]
Phase 2 (complete) Nonrandomized, open-labeled oral inoculation group. Mild to self-limiting adverse events.
(2,500 ova) 2-week intervals for 12 weeks (n =
10).
EudraCT no. TSO 2007 to 2010 Allergic rhinitis No therapeutic effect on allergic rhinitis of [130]
2007-006099-12 (complete) Randomized, double-blind, placebo-controlled, infection.
Phase I 2,500 ova administered (n = 49) and placebo (n
= 47).
NCT01413243 TSO 2011 to 2016 Multiple sclerosis Unknown [131]
Phase 2 (terminated) (TRIOMS) randomized control trial of oral
inoculation (2,500 ova) 2-week intervals for 12
weeks (n = 50).
NCT01434693 TSO 2011 to 2013 Crohn’s disease Placebo and treatment groups experience [132]
Phase 1 (complete) Randomized, double-blind, placebo-controlled minor adverse events. No obvious
sequential oral dose escalation (500, 2,500, improvement in pathology with infection.
7,500 ova) (n = 36).
NCT01576471 TSO 2013 Crohn’s disease Unknown results of study Coronado
Phase 2 (unknown) (TRUST-1) Randomized, double-blind, Biosciences,
placebo-controlled oral inoculation of 7,500 United Kingdom
ova in 2-week intervals. Placebo group
included.
NCT01279577 TSO 2011 to 2015 Crohn’s disease Unknown results of study Dr. Falk Pharma,
Phase 2 (complete) Randomized, double-blind, placebo-controlled, Germany
low, medium, high oral inoculation ova
(n = 254) participants.
NCT01836939 TSO 2013 to 2015 Plaque psoriasis Unknown results of study Icahn School of
Phase 1 (complete) Randomized, 2-arm trial of oral inoculation Medicine at Mount
(2,500 ova) in 2-week intervals for 10 weeks Sinai, US
and (7,500 ova) in 2-week intervals for 10
weeks (n = 8).
NCT01948271 TSO 2013 to 2016 Plaque psoriasis Trial terminated because of a lack of Tufts Medical
Phase 1 (terminated) Open-label, oral inoculation (7,500 ova) in efficacy Center, US
2-week intervals for 14-week duration (n = 3).
NCT02011269 TSO 2013 to 2016 Plaque psoriasis Trial withdrawn and results unknown. Coronado
Phase 2 (withdrawn) Randomized, blinded, placebo-controlled, Biosciences, UK
3-arm trial of oral inoculation (7,500 ova) in
2-week intervals for 10 weeks, (15,000 ova) in
2-week intervals for 10 weeks.
(Continued )

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 4 / 20

PLOS PATHOGENS

Table 1. (Continued)

Trial/phase Helminth Status (year Study title and treatment Results outcome Reference
range)
N. 2006 Crohn’s disease Remission at week 45 observed in 5 [31]
Phase 1 americanus (complete) Proof of concept: inoculation with larvae at patients inoculated in week 0. No serious
larvae week 0 (n = 9) and week between week 27 to 30 adverse events.
(n = 5)
N. 2009 Allergic rhinoconjunctivitis Hookworm infection did not induce [133]
americanus 30 individuals with allergic rhinoconjunctivitis clinically significant exacerbation of airway
larvae were randomized and inoculated with 10 larvae responsiveness.
or placebo and followed for 12 weeks.
NCT00469989 N. 2010 Asthma Hookworm infection did not significantly [28]
americanus Randomized, placebo-controlled, inoculation improve bronchial responsiveness nor
larvae with 10 larvae and followed for 16 weeks other measures of asthma control.
(n = 30).
NCT00671138 N. 2007 to 2011 Celiac disease Infection conferred no obvious benefit to [30, 134]
Phase 2 americanus 2011 to 2016 Randomized, double-blinded, placebo- pathology. Mucosa of hookworm-infected
larvae (completed) controlled trial. Part 1: inoculating celiac subject maintained healthy appearance. No
disease patients with the human hookworm N. serious adverse events.
americanus larvae at week 0 (n = 10) and week Duodenal biopsy culture of hookworm-
12 (n = 10), followed by oral gluten challenge at infected subjects had suppressed IL-17A
week 20 of 16 g gluten per day for 5 days. and IFN-γ and increased levels of IL-10,
Part 2: inoculating celiac disease patients with IL-5 and regulatory T cells.
the N. americanus larvae at week 0 (n = 7) and No serious adverse events.
week 12 (n = 7). At week 20, subjects were
given an oral gluten challenge at week 20 of 16
g gluten per day for 5 days.
NCT01661933 N. 2012 to 2014 Celiac disease Ten patients successfully tolerated gluten [135]
Phase 1 and 2 americanus (completed) Inoculating celiac disease patients with the challenge. No serious adverse events.
larvae human hookworm N. americanus larvae at
week 0 (n = 10) and week 4 (n = 10), followed
by incremental gluten challenge (n = 12).
NCT02754609 N. 2016 to 2020 Celiac disease Manuscript in preparation James Cook
Phase I americanus (completed) Hookworm therapy for celiac disease University,
larvae (NainCeD-3). Randomized, placebo (n = 10), Australia
inoculation week 0 and week 8 (n = 40) to
assess safety and dose-ranging clinical trial
examining sustained gluten challenge.
NCT00630383 N. 2008 to 2012 Multiple sclerosis Withdrawn—superseded by a similar study University of
Phase 2 americanus (withdrawn) Randomized, inoculation 25 larvae at week 0. Nottingham, UK
larvae Placebo group included.
NCT01470521 N. 2011 to 2016 Multiple sclerosis Unknown study results University of
Phase 2 americanus (complete) (WIRMS) Nottingham, UK
larvae Randomized, inoculation 25 larvae at week 0 (n
= 36). Placebo group (n = 36).

HINT, Helminth-induced Immunomodulation Therapy; IFN-γ, interferon gamma; IL, interleukin; TRIOMS, Trichuris Suis Ova in Recurrent Remittent Multiple
Sclerosis; TRUST-1, Treatment With Oral CNDO 201 Trichuris Suis Ova Suspension in Patients; TSO, Trichuris suis ova; WIRMS, Worms for Immune Regulation of
Multiple Sclerosis.

https://doi.org/10.1371/journal.ppat.1008508.t001

standardized manufacturing protocol for N. americanus. However, methods for the production
of cGMP human hookworms were recently described [34] and are essential advances if hel-
minth therapy is to receive widespread acceptance by the medical fraternity [35].
Further to therapeutic trials of helminth infections in inflammatory disease settings, dose-esca-
lation controlled helminth infections in healthy volunteers are currently ongoing (S1 Table), pri-
marily intending to develop a platform to test anti-helminth vaccines and drugs [34, 36].

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 5 / 20

PLOS PATHOGENS

Fig 2. Inflammation and metabolic imbalance versus glucose homeostasis and weight loss, in response to infection with gastrointestinal nematodes and
intravascular blood flukes and their ESPs. Chronic inflammation in adipose tissue is linked to a switch to M1 macrophages and the production of TNF-α and
IL-1β. Helminth infection and helminth ESPs induce changes in the gut that lead to a regulatory/TH2 milieu that results in reduced inflammation in adipose
tissue, enhanced glucose homeostasis, and decreased weight gain in obese animals. Furthermore, this regulatory/TH2 milieu increases IL-33 produced in adipose
tissue by stromal cells within the progenitors of both adipocytes and mesenchymal cells. The production of IL-33 induces resident ILC2 to produce IL-5, which
recruits eosinophils. Eosinophils in white adipose tissue secrete IL-4, which induces M2 macrophages. The production of IL-33 also induces regulatory T and B
cells to produce IL-10, which sustains M2 macrophage activity. ESPs, excretory/secretory product; IL, interleukin; ILC2, type 2 innate lymphoid cell; M1,
classically activated macrophage; M2, alternatively activated macrophage; TH2, T helper type 2; TNF, tumour necrosis factor.
https://doi.org/10.1371/journal.ppat.1008508.g002

Insights into parasite–host interactions from animal models

Excretory-secretory products
Helminths secrete bioactive molecules that can suppress or skew host immune responses; col-
lectively, this suite of molecules is referred to as excretory/secretory products (ESPs). ESPs are
a complex mixture of proteins, peptides, nucleic acids, lipids, glycans, and small organic mole-
cules. Administration of ESPs from many nematodes and platyhelminth species induces
immune responses that reflect the active infections. Moreover, ESPs have therapeutic proper-
ties in a range of animal models of autoimmunity, allergy, and metabolic disease (see recent
reviews [37–39]). Indeed, the use of ESPs instead of active helminth infection potentially
addresses some of the drawbacks and obstacles currently faced by experimental helminth ther-
apy [38].
ESPs from many helminths, including at least 2 hookworm species (Ancylostoma caninum
and A. ceylanicum), protected mice against T-cell-dependent trinitrobenzene sulfonic acid
(TNBS)-induced colitis [39] and T-cell-independent dextran sulfate sodium (DSS) colitis [40,
41]. Prevention of immunopathology with ESPs is not only restricted to diseases driven by
TH1/TH17 responses; both immuno-epidemiological observations [42] and mouse studies [43,
44] have shown that helminths and ESPs are also potent suppressors of TH2-driven allergic

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 6 / 20

PLOS PATHOGENS

inflammation. The ability of ESPs to regulate all major forms of immunopathology is primarily
attributed to its potent regulatory properties [45, 46]. Helminth ESPs drive a modified TH2
response that is different from the canonical TH2 response seen in allergies in which activated
CD4+ cells secrete TH2 cytokines, including interleukin (IL)-4, IL-5, and IL-13 [47]. ESPs
instead induce a modified TH2 response characterized by the secretion of regulatory cytokines
such as IL-10 and the immunosuppressive transforming growth factor-beta (TGF-β) to ensure
the containment of infection to manageable levels and that excessive TH2 inflammation does
not ensue [48].

ESPs target innate immune cell function

Parasitic helminths target several cell types that orchestrate a characteristic immune regulatory
phenotype, notably, antigen-presenting cells such as DCs and macrophages. DCs possess
intrinsic tolerance mechanisms that are integral initiators of the TH2 response [49]. Regulatory
or tolerogenic DCs expressing integrin alpha E (CD103) are located in the intestinal mucosa
and mesenteric lymph nodes [50]. Mucosal CD103+ DCs are capable of antagonizing TH2
induction in Schistosoma mansoni and Heligmosomoides polygyrus infections in mice. Mucosal
CD103+ DCs secrete TGF-β, retinoic acid, and IL-2 and prevent immune-mediated pathology
by promoting the expansion of Treg populations and maintaining intestinal homeostasis. Hel-
minth ESPs target DCs and macrophage activation through suppression of pattern recognition
receptor function, diminishing the ability of these innate sentinels to detect and respond to
pathogen-associated molecular patterns (reviewed in [37]). Hookworm ESPs induce CD103+
DCs to express retinoic acid, which, in turn, facilitated the expansion of Tregs in a mouse
model of asthma [51]. Moreover, helminth ESPs are known potent inducers of M2 macro-
phages [52], a cell population that drives TH2 responses and promotes wound repair [2].
Recent studies have shed light on the mechanisms by which helminths initiate TH2
responses by signaling through ILC2s (reviewed in [53]). ILC2s are enriched in the mucosa of
gastrointestinal nematode–infected mice and arise in response to secretion of the alarmin IL-
25 by intestinal tuft cells [54]. ILC2s respond rapidly to the presence of helminths by increas-
ing in number and secretion of type 2 cytokines [54]. This cascade, in turn, results in the repair
of epithelial barriers and recruitment of other innate cells, including eosinophils and culmi-
nates in the activation of TH2 cells and regulatory pathways.

Helminths induce Treg activity

Tregs constitute around 5% of circulating CD4+ T cells and are identified by the lineage
marker forkhead box P3 (FOXP3). Mutations in several genes that orchestrate Treg function,
including the IL-2 receptor alpha subunit CD25 and cytotoxic T-lymphocyte-associated pro-
tein 4 (CTLA-4) result in the development of severe autoimmune syndromes [55], so Tregs
constitute a significant target for new therapeutic strategies. Such research is focusing on the
ability of Tregs to mediate immune regulation via multiple mechanisms, including IL-2 depri-
vation, secretion of the regulatory cytokines IL-10 and TGF-β, and acquisition of costimula-
tory molecules from antigen-presenting cells through binding to CTLA-4.
Both nematodes and platyhelminths are potent drivers of Treg responses. In mice infected
with either Nippostrongylus brasiliensis or S. mansoni, IL-4 receptor alpha-mediated signaling
on Tregs was required in vivo for control of helminth-induced inflammation [56]. H. polygyrus
ESPs directly induces Tregs in vitro using FOXP3-green fluorescent protein reporter mice [49],
and more recently, several helminth recombinant proteins have been shown to drive Treg
expansion [37]. Indeed, from a clinical perspective, molecules that drive expansion, mobiliza-
tion, or increased mucosal homing of 9Tregs are the holy grail for many disorders that result

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 7 / 20

PLOS PATHOGENS

from immune dysregulation [57, 58], and we will provide defined examples later in this review.
Regulatory B cells (Bregs) are also a feature of helminth infections [59] and are the primary
source of IL-10 and other tissue-protective proteins, including (Resistin-like molecule-α)
RELMα [60, 61]. Indeed, in mice infected with H. polygyrus, IL-10+ Breg cells were able to pro-
mote expansion and maintenance of IL-10+ FOXP3+ Treg cell populations [61]. Fig 3 summa-
rizes the multitude of cellular immune pathways upon which helminths and their ESPs impact,
including the well-established influence of DCs, but also the emerging potential roles for factors
produced by microbes, sensory neurons, and specialized intestinal epithelial cells that initiate
the very early response to helminth exposure via ILC2s and innate granulocytes [62, 63]. These
recently identified biological pathways may, therefore, be potential therapeutic targets for
immunoregulation by helminths and their secreted products.

Helminth therapeutic moieties

Despite the wealth of literature supporting the therapeutic use of helminth ESPs for treating
inflammatory disorders, the capacity of a crude parasite-derived supernatant to be developed
as a drug is limited. ESP proteomes from many distinct species of helminths have been charac-
terized, and their annotation has been supported by the increasing number of draft genomes
[64]. Although a handful of individual proteins with immunoregulatory properties have been
identified from ESPs, helminth-secreted proteomes still present a relatively untapped pharma-
copeia [65, 66]. Next, we highlight a select group of molecules with proven immunoregulatory
roles and potential therapeutic properties.

Proteases and protease inhibitors

There are numerous examples of proteins from multicellular ecto- and endoparasites that have
evolved from a protease or protease inhibitor scaffold but no longer possess canonical activity
[65–67]. For example, 2 of the most abundant ESP proteins in A. caninum possess an ancestral
netrin domain and are structural homologs of the tissue inhibitor of metalloprotease (TIMP)
family [68]. Although A. caninum anti-inflammatory protein (Ac-AIP)-1 and Ac-AIP-2 pos-
sess a TIMP-like domain, they do not appear to have the ability to suppress matrix metallopro-
tease catalytic activity and instead have evolved a distinct anti-inflammatory function [67, 69].
Recombinant AIPs suppressed expression of DC activation and costimulation markers [52,
70] and drove the subsequent expansion and mucosal homing of Tregs, which protected
against inducible asthma [51]. Prophylactic treatment of mice with recombinant Ac-AIP-1 in
chemically induced colitis protected mice against weight loss, clinical disease, and intestinal
histopathology and significantly reduced the expression of hallmark TH1/TH2/ TH17 cytokines
that drive inflammation in human IBD [71].
In similar fashion to the plasticity of the TIMP-like domain in hookworms, there is growing
evidence that throughout their evolution, some cysteine protease inhibitor (cystatin) super-
family members have acquired novel roles that are independent of cysteine protease inhibition
[72]. Filarial nematodes secrete cystatins that possess the canonical papain-like enzyme inhibi-
tory activity but have also evolved a novel function that allows them to inhibit the catalytically
distinct asparaginyl endopeptidase activity. This dual function assists in the inhibition of anti-
gen processing in the major histocompatibility complex (MHC) class II pathway [73]. Cysta-
tins from various helminth species suppress the secretion of inflammatory cytokines and
promote IL-10 production by macrophages in particular [74, 75]. Cystatin from the filarial
nematode Acanthocheilonema viteae suppressed inducible colitis and asthma in mice and dis-
played ex vivo bioactivity with human peripheral blood mononuclear cells from atopic patients
with grass pollen allergy [76]. Moreover, oral delivery of A. viteae recombinant cystatin via

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 8 / 20

PLOS PATHOGENS

Fig 3. Helminths and their ESPs manipulate the host immune system. Helminth infection promotes TH2 cell differentiation, Treg responses, macrophage
polarization, and mucus production, which are regulated by multiple upstream events and stimulated by signals from the worm (ESPs), but also signals from the
microbiome (metabolites) and tissue damage (alarmins). DCs are central to these processes and respond to alarmins, ESPs, and metabolites to adopt a regulatory
phenotype that promotes Treg, Breg, and TH2 cell development and suppress TH17 and TH1 cell responses. In addition, helminth-induced damage to the
epithelium causes the release of alarmins such as TSLP, IL-25, and IL-33 from tuft cells and other epithelial cells, which can act on ILC2s and granulocytes to
augment production of type 2 cytokines. This network is also influenced by sensory neurons within the gut that sense signals from helminths and microbes and
elicit production of neuropeptides such as NMU and CGRP to regulate ILC2 responses directly. Breg, regulatory B cell; CGRP, calcitonin gene-related protein;
DC, dendritic cell; ESPs, excretory/secretory product; ILC2, type 2 innate lymphoid cell; NMU, neuromedin U; TH2, T helper type 2, TLSP, thymic stromal
lymphopoietin; Treg, regulatory T cell.
https://doi.org/10.1371/journal.ppat.1008508.g003

continual dosing of transgenic Lactococcus lactis prevented the onset of colitis in pigs [76].
Subsequent studies have shown that cystatins from other helminths, both nematodes, and
platyhelminths, can suppress inducible colitis [77–79].

Cytokine mimics and cytokine-binding proteins

There is a growing body of literature on helminth ESPs that possess cytokine-like functions
but not necessarily cytokine-like sequence homology or secondary structure. For example, H.
polygyrus secretes a protein called H. polygyrus TGF-β mimic (Hp-TGM) that binds to the
mammalian TGF-β complex and drives human and mouse Treg production but has no
sequence homology to mammalian TGF-β. Instead, Hp-TGM is a member of the complement
control protein (CCP) superfamily [80]. Recombinant Hp-TGM delayed allograft rejection in
mice and increased Treg numbers in draining lymph nodes at the site of graft transplant,
highlighting a potential use for this protein in a range of inflammatory settings. In like fashion,
N. americanus activation-associated secreted protein (Na-ASP-2) secreted by N. americanus

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 9 / 20

PLOS PATHOGENS

infective larvae is a member of the sperm-coating protein (SCP/TAPS) family and has struc-
tural- and charge-mimicking features of CXC-chemokines that recruit Tregs [81]. Na-ASP-2
was shown to bind to CD79A on human B cells whereupon it affected the expression of genes
involved in leukocyte transendothelial migration [82].
H. polygyrus secretes a cytokine-binding protein called H. polygyrus alarmin release inhibi-
tor (HpARI). This protein also belongs to the CCP family and inhibits the release of alarmins
[83]. HpARI binds directly to IL-33 and nuclear DNA, thereby tethering the alarmin within
necrotic cells and preventing its release. Recombinant HpARI administered to mice intrana-
sally suppressed ILC2s and eosinophil responses in the lungs of mice after administration of
Alternaria allergen, highlighting its utility for treating inflammatory diseases in the lungs in
particular.

Helminth molecules that accelerate wound healing

Helminths penetrate and migrate through skin and tissues without causing significant damage.
The mechanisms employed by helminths to concurrently reduce injury and stimulate healing in
the host are currently being explored [61, 84–86]. Most of the existing literature focuses on the
role of the TH2 response—type 2 macrophages in particular—in driving wound repair; however,
a handful of helminth-secreted molecules with wound healing properties have been described.
The liver fluke, Opisthorchis viverrini, secretes a granulin (GRN)-like growth factor, Ov-GRN-1,
which promotes wound healing and angiogenesis [87]. Recombinant Ov-GRN-1 is challenging to
express in scalable form, so a readily-synthesized bioactive peptide fragment of Ov-GRN-1 that
retained both in vitro and in vivo wound healing properties was identified and retained the in
vivo therapeutic properties of the parent protein [88]. Given the alarming incidence of T2D and
associated comorbidities such as nonhealing diabetic foot ulcers, topical growth factor-like pro-
teins and peptides from helminths address an area of great unmet need [89, 90].

Post-translational modifications
Many helminth immunomodulatory proteins are secreted and therefore undergo post-transla-
tional modifications (PTM). In some cases, the PTM are responsible for the addition of the
regulatory moiety of interest. For example, the dominant ESP of A. vitae is ES-62. This glyco-
protein that has therapeutic effects in a range of mouse models of inflammatory disorders such
as arthritis [91], asthma [92], and even systemic lupus erythematosus [93]. ES-62 is an amino-
peptidase that carries N-glycans decorated with phosphorylcholine (PC) [94, 95]. The fusion of
PC to an unrelated carrier protein demonstrated that it retained its therapeutic properties,
thus proving that PC is the bioactive moiety of ES-62 [96]. Small drug-like analogs of PC for
the treatment of arthritis and chronic lung fibrosis have overcome immunogenicity concerns
with the sizeable ES-62 protein [97, 98].
Chemical deglycosylation of ESPs from some helminths ablates protection against inflam-
matory diseases [99], highlighting the importance of glycans in driving regulatory responses.
One such glycan that decorates schistosome soluble egg antigens (SEAs) and secreted egg pro-
teins is the Lewis X-containing glycan found on dominant egg proteins such as omega-1 and
interleukin-4-inducing principle (IPSE)/alpha-1 [100]. Recombinant IPSE/alpha-1 expressed
in wild tobacco drives IL-10 production from Bregs [101] and suppresses inflammatory cyto-
kine responses by skewing inflammatory monocytes toward anti-inflammatory M2 macro-
phages [102]. Administration of SEA to mice induced TH2 immune responses characterized
by M2 macrophages and eosinophils in WAT and liver and reduced fat mass gain and lowered
insulin resistance and glucose intolerance [103, 104] (Fig 2). Protection against metabolic dis-
ease was dependent on the engagement of the mannose receptor CD206 and the release of IL-

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 10 / 20

PLOS PATHOGENS

33, inducing ILC2-dependent improvements in metabolic status [105]. The immunomodula-

tory Lewis X-containing glycan, lacto-N-fucopentaose III (LNFPIII) from the schistosome teg-
ument also improves glucose tolerance and insulin sensitivity in diet-induced obese mice, at
least in part via increased IL-10 production by macrophages and DCs, and resulted in reduced
WAT inflammation [106]. LNFPIII treatment also up-regulated the expression of the multi-
purpose farnesoid X nuclear receptor (FXR)-α to reduce lipogenesis in the liver, thereby pro-
tecting against hepatosteatosis [107]. Producing appropriately glycosylated recombinant
versions of schistosome glycoproteins has proven challenging. Nevertheless, recent efforts to
glycoengineer Lewis X-containing proteins in wild tobacco with coexpression of defined glyco-
syltransferases proved successful [105] and opened up new possibilities for generating appro-
priately glycosylated immunotherapeutics.

Helminth metabolites—an untapped resource for small-molecule

therapeutics
Although helminth proteins and their PTMs have been documented and discussed elsewhere
in this review, much less is known about parasitic helminth metabolomes. The metabolomics
revolution has begun to reveal small-molecule metabolites in parasitic helminth somatic
extracts and ESPs [108–111]. Helminthology has been slow to adopt cutting edge metabolo-
mics techniques [111], partly because of the difficulty in obtaining sufficient quantities of
ESPs, but also because of the diversity in physicochemical properties of metabolites results in it
being almost impossible for a single analytical method to provide the required full coverage of
the metabolome of a given biological sample. As such, particular analyses are biased toward
certain groups of metabolites. Only a handful of studies have characterized helminth metabo-
lomes, and even fewer have addressed the anti-inflammatory properties of these molecules. A.
caninum secretes metabolites that suppress both inducible colitis in mice and ex vivo produc-
tion of inflammatory cytokines from human PBMCs [112]. The identity of the protective moi-
eties is not yet known, but short-chain fatty acids secreted by the parasite (or the hookworm’s
resident microbiota) are prospective candidates. Another candidate is succinate, a metabolite
that is produced by the intestinal microbiota that is critical for inducing intestinal tuft cells to
initiate TH2 responses. Succinate was recently shown to be a major component of metabo-
lomes of gastrointestinal helminths [109, 112] and was significantly enriched in the ESP meta-
bolome of hookworms compared to the somatic tissue metabolites.
The lipidome of different developmental stages of S. mansoni was recently described, and
the egg stage was shown to be enriched in oxylipids with known immunoregulatory properties
such as prostaglandins [113]. Indeed, prostaglandin E2 is secreted in high amounts by T. suis
compared to other lipids and suppresses TNF and IL-12 secretion from lipopolysaccharide-
activated human DCs [114]. Of course, prostaglandins are not unique to helminths and are a
therapeutic target in their own right because of their up-regulation in some aggressive cancers
[115]. Helminth metabolites that are likely to be candidate small-molecule drugs are those that
are unique to parasites and have evolved to suppress defined immunopathological pathways
safely and are readily synthesizable. We anticipate substantial growth in this area in the coming
years given the increased access of researchers from diverse fields to metabolomics platforms.

Helminth-secreted extracellular vesicles in the treatment of inflammatory

disorders
The discovery that parasitic helminths secrete extracellular vesicles (EVs) has spurred a new
paradigm in the discovery of helminth-derived immunotherapeutics and antihelminth vac-
cines [116, 117]. EVs are a heterogeneous group of lipid-enclosed vesicles in the nano- to the

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 11 / 20

PLOS PATHOGENS

micrometer size range. There is increasing evidence that helminth EVs are essential players in
regulating host inflammation and immunity, and their application as anti-inflammatory thera-
peutics has been considered. However, to date, the exact mechanisms by which helminth EVs
polarize immune responses remain elusive.
In the Alternaria model of allergic asthma, administration of H. polygyrus EVs significantly
reduced lung immunopathology via ILC2-mediated suppression of innate immunity [118].
The anticolitic therapeutic potential of helminth EVs has also been demonstrated in several
recent reports. N. brasiliensis EV-treated mice were protected from T-cell-dependent acute
colitis, specifically by the suppression of inflammatory cytokines and increased expression of
IL-10 [119]. EVs from the liver fluke Fasciola hepatica can modulate T-cell-independent colitis
[120], characterized by reduced expression of intestinal proinflammatory cytokines that sup-
press both mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-
enhancer (NF-κB) signaling pathways.
Perhaps the most intriguing aspect of helminth EVs (from a drug development perspective)
is the abundance of vesicular microRNAs (miRNAs) that are predicted to target mammalian
host genes, particularly those involved in immune processes. Helminth EVs are actively inter-
nalized by host cells [119, 121], providing a mechanism by which the parasites transfer genetic
material to the host in a bid to actively manipulate host gene expression [116, 118]. There is a
growing body of literature demonstrating the presence of parasite-specific miRNAs secreted
by worms, but little is known about their specific interactions with host genes. H. polygyrus
miRNAs were shown to down-regulate the expression of the mouse phosphatase gene dusp1
(which corresponds to MKP-1 in humans), a key regulator of MAPK signaling and TH1
responses to toll-like receptor ligands using a luciferase reporter assay [118].
Looking forward, a deeper understanding of helminth miRNA immunobiology may lead to
advances in miRNA-based therapeutics for a whole host of immune dysfunction diseases,
including conditions like scleroderma, in which defined miRNAs (of host origin) are already
in preclinical and clinical development (reviewed in [122]). EV research has been critical for
demonstrating how miRNAs could be used to treat disease, but some practical challenges and
obstacles need to be overcome before this type of therapy enjoys mainstream application
[123].

Turning worm proteins into conventional drugs

The molecular diversity of known helminth ESPs with therapeutic properties is impressive,
and considering that we have only dipped our toes in the water, the potential breadth of moie-
ties is prodigious. Each family of molecules and even individual members within those families
present unique and shared challenges. Moreover, the pharmacokinetic (PK) properties of a
helminth-derived therapeutic protein and how best to deliver it to the target tissue/organ are
challenging. For example, an ideal IBD drug would be delivered orally, and although some hel-
minth ESPs have likely evolved to be stable in acidic environments, ensuring that a protein
enjoys safe passage through the stomach and delivery to the specific cell type in the gut is not
straightforward. Moreover, what does the optimal PK profile of a helminth protein look like?
Unlike most biologics (e.g., monoclonal antibodies) in which plasma half-life can be measured
in weeks, a helminth protein might only be present for hours. This longer half-life might, how-
ever, be sufficient for enduring functional activity via impact on defined cell types and mobili-
zation of those cells to inflamed tissues [51]. The foreignness of a helminth protein also poses
potential immunogenicity concerns, and the development of antidrug antibodies could be
problematic.

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 12 / 20

PLOS PATHOGENS

Furthermore, the recombinant expression platform is of paramount importance for the

production of helminth biologics. Many groups are expressing recombinant helminth proteins
in cell lines that are used for industrial-scale production of antibodies and other therapeutic
proteins. Levels of endotoxin and other host cell-derived contaminants need to be strictly con-
trolled for cGMP grade production of biologics, particularly for immunotherapeutics that aim
to wind down inflammatory responses (as opposed to promoting them with vaccines), so an
emphasis on eukaryotic cell expression platforms, such as HEK293, is recommended.
Helminth-derived miRNAs are predicted to target an impressive array of mammalian host
genes involved in inflammation, but how those miRNAs might be therapeutically delivered to
target cells, internalized, and then encounter their target gene is not clear [124, 125]. In the
natural setting, many miRNAs are delivered to target cells in the sheltered environment of
EVs, but recapitulating this process for a therapeutic purpose requires novel approaches, such
as the use of synthetic exosomes [126].
Despite these challenges, evolutionary selection pressure has tailored helminth ESPs to be
efficacious and safe, at least in the setting of active helminth infection. Of course, in a therapeu-
tic setting, the dose, the frequency, and route of administration are likely to be different. Fur-
thermore, it is important to note that numerous nature-inspired drugs, sourced from the
venom of various invertebrates and vertebrates, are commercially available for treating a range
of disorders [127]. It is timely that helminths now join this list and that drugs inspired by these
exquisitely adapted parasites get the attention they deserve. The scientific community implores
industry-based organizations to make long-term investments intended at deciphering and cap-
italizing on the extraordinary and diverse modes of action of these products to unearth the
next generation of novel therapeutics.

Supporting information
S1 Table. Completed and ongoing experimental helminth infection studies in healthy vol-
unteers.
(DOCX)

References
1. Hotez PJ, Alvarado M, Basanez MG, Bolliger I, Bourne R, Boussinesq M, et al. The global burden of
disease study 2010: interpretation and implications for the neglected tropical diseases. PLoS Negl
Trop Dis. 2014; 8(7):e2865. https://doi.org/10.1371/journal.pntd.0002865 PMID: 25058013; PubMed
Central PMCID: PMC4109880.
2. Chen F, Liu Z, Wu W, Rozo C, Bowdridge S, Millman A, et al. An essential role for TH2-type responses
in limiting acute tissue damage during experimental helminth infection. Nature medicine. 2012; 18
(2):260–266. https://doi.org/10.1038/nm.2628 PMID: 22245779; PubMed Central PMCID:
PMC3274634.
3. Bach JF. The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med.
2002; 347(12):911–920. https://doi.org/10.1056/NEJMra020100 PMID: 12239261.
4. Windsor JW, Kaplan GG. Evolving Epidemiology of IBD. Curr Gastroenterol Rep. 2019; 21(8):40.
https://doi.org/10.1007/s11894-019-0705-6 PMID: 31338613.
5. Biagioni B, Vitiello G, Bormioli S, Tarrini D, Lombardi C, Rossi O, et al. Migrants and allergy: a new
view of the atopic march. Eur Ann Allergy Clin Immunol. 2019; 51(3):100–114. https://doi.org/10.
23822/EurAnnACI.1764-1489.96 PMID: 30983310.
6. Fumagalli M, Pozzoli U, Cagliani R, Comi GP, Riva S, Clerici M, et al. Parasites represent a major
selective force for interleukin genes and shape the genetic predisposition to autoimmune conditions. J
Exp Med. 2009; 206(6):1395–1408. https://doi.org/10.1084/jem.20082779 PMID: 19468064; PubMed
Central PMCID: PMC2715056.
7. Yazdanbakhsh M, Kremsner PG, van Ree R. Allergy, parasites, and the hygiene hypothesis. Science.
2002; 296(5567):490–494. https://doi.org/10.1126/science.296.5567.490 PMID: 11964470.

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 13 / 20

PLOS PATHOGENS

8. Strachan DP. Hay fever, hygiene, and household size. BMJ. 1989; 299(6710):1259–1260. https://doi.
org/10.1136/bmj.299.6710.1259 PMID: 2513902; PubMed Central PMCID: PMC1838109.
9. Wiria AE, Sartono E, Supali T, Yazdanbakhsh M. Helminth infections, type-2 immune response, and
metabolic syndrome. PLoS Pathog. 2014; 10(7):e1004140. https://doi.org/10.1371/journal.ppat.
1004140 PMID: 24992724; PubMed Central PMCID: PMC4081794.
10. Hays R, Esterman A, Giacomin P, Loukas A, McDermott R. Does Strongyloides stercoralis infection
protect against type 2 diabetes in humans? Evidence from Australian Aboriginal adults. Diabetes Res
Clin Pract. 2015; 107(3):355–361. https://doi.org/10.1016/j.diabres.2015.01.012 PMID: 25656764.
11. van der Zande HJP, Zawistowska-Deniziak A, Guigas B. Immune Regulation of Metabolic Homeosta-
sis by Helminths and Their Molecules. Trends Parasitol. 2019; 35(10):795–808. https://doi.org/10.
1016/j.pt.2019.07.014 PMID: 31492623.
12. Harris NL, Loke P. Recent Advances in Type-2-Cell-Mediated Immunity: Insights from Helminth Infec-
tion. Immunity. 2017; 47(6):1024–1036. https://doi.org/10.1016/j.immuni.2017.11.015 PMID:
29262347.
13. Gazzinelli-Guimaraes PH, Nutman TB. Helminth parasites and immune regulation. F1000Res. 2018;7.
https://doi.org/10.12688/f1000research.13350.2 PMID: 29527296; PubMed Central PMCID:
PMC6206608.
14. Maizels RM. Regulation of Immunity and allergy by helminth parasites. Allergy. 2019; 75(3):524–534.
https://doi.org/10.1111/all.13944 PMID: 31187881.
15. Grencis RK, Humphreys NE, Bancroft AJ. Immunity to gastrointestinal nematodes: mechanisms and
myths. Immunol Rev. 2014; 260(1):183–205. https://doi.org/10.1111/imr.12188 PMID: 24942690;
PubMed Central PMCID: PMC4141702.
16. Lee SC, Tang MS, Lim YA, Choy SH, Kurtz ZD, Cox LM, et al. Helminth colonization is associated with
increased diversity of the gut microbiota. PLoS Negl Trop Dis. 2014; 8(5):e2880. https://doi.org/10.
1371/journal.pntd.0002880 PMID: 24851867; PubMed Central PMCID: PMC4031128.
17. Giacomin P, Croese J, Krause L, Loukas A, Cantacessi C. Suppression of inflammation by helminths:
a role for the gut microbiota? Philos Trans R Soc Lond B Biol Sci. 2015; 370(1675). https://doi.org/10.
1098/rstb.2014.0296 PMID: 26150662; PubMed Central PMCID: PMC4528494.
18. Giacomin P, Zakrzewski M, Croese J, Su X, Sotillo J, McCann L, et al. Experimental hookworm infec-
tion and escalating gluten challenges are associated with increased microbial richness in celiac sub-
jects. Sci Rep. 2015; 5:13797. https://doi.org/10.1038/srep13797 PMID: 26381211; PubMed Central
PMCID: PMC4585380.
19. Brosschot TP, Reynolds LA. The impact of a helminth-modified microbiome on host immunity. Muco-
sal Immunol. 2018; 11(4):1039–1046. https://doi.org/10.1038/s41385-018-0008-5 PMID: 29453411.
20. Zaiss MM, Rapin A, Lebon L, Dubey LK, Mosconi I, Sarter K, et al. The Intestinal Microbiota Contrib-
utes to the Ability of Helminths to Modulate Allergic Inflammation. Immunity. 2015; 43(5):998–1010.
https://doi.org/10.1016/j.immuni.2015.09.012 PMID: 26522986; PubMed Central PMCID:
PMC4658337.
21. Ramanan D, Bowcutt R, Lee SC, Tang MS, Kurtz ZD, Ding Y, et al. Helminth infection promotes colo-
nization resistance via type 2 immunity. Science. 2016; 352(6285):608–612. https://doi.org/10.1126/
science.aaf3229 PMID: 27080105; PubMed Central PMCID: PMC4905769.
22. Summers RW, Elliott DE, Urban JF Jr., Thompson RA, Weinstock JV. Trichuris suis therapy for active
ulcerative colitis: a randomized controlled trial. Gastroenterology. 2005; 128(4):825–832. https://doi.
org/10.1053/j.gastro.2005.01.005 PMID: 15825065.
23. Summers RW, Elliott DE, Urban JF Jr., Thompson R, Weinstock JV. Trichuris suis therapy in Crohn’s
disease. Gut. 2005; 54(1):87–90. https://doi.org/10.1136/gut.2004.041749 PMID: 15591509; PubMed
Central PMCID: PMC1774382.
24. Scholmerich J, Fellermann K, Seibold FW, Rogler G, Langhorst J, Howaldt S, et al. A Randomised,
Double-blind, Placebo-controlled Trial of Trichuris suis ova in Active Crohn’s Disease. J Crohns Colitis.
2017; 11(4):390–399. https://doi.org/10.1093/ecco-jcc/jjw184 PMID: 27707789; PubMed Central
PMCID: PMC5881737.
25. Voldsgaard A, Bager P, Garde E, Akeson P, Leffers AM, Madsen CG, et al. Trichuris suis ova therapy
in relapsing multiple sclerosis is safe but without signals of beneficial effect. Multiple sclerosis (Hound-
mills, Basingstoke, England). 2015; 21(13):1723–1729. Epub 2015/02/24. https://doi.org/10.1177/
1352458514568173 PMID: 25698173.
26. Fleming J, Hernandez G, Hartman L, Maksimovic J, Nace S, Lawler B, et al. Safety and efficacy of hel-
minth treatment in relapsing-remitting multiple sclerosis: Results of the HINT 2 clinical trial. Multiple
sclerosis (Houndmills, Basingstoke, England). 2017:1352458517736377. https://doi.org/10.1177/
1352458517736377 PMID: 29064315; PubMed Central PMCID: PMC5878983.

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 14 / 20

PLOS PATHOGENS

27. Loukas A, Hotez PJ, Diemert D, Yazdanbakhsh M, McCarthy JS, Correa-Oliveira R, et al. Hookworm
infection. Nat Rev Dis Primers. 2016; 2:16088. https://doi.org/10.1038/nrdp.2016.88 PMID: 27929101.
28. Feary JR, Venn AJ, Mortimer K, Brown AP, Hooi D, Falcone FH, et al. Experimental hookworm infec-
tion: a randomized placebo-controlled trial in asthma. Clin Exp Allergy. 2010; 40(2):299–306. https://
doi.org/10.1111/j.1365-2222.2009.03433.x PMID: 20030661; PubMed Central PMCID: PMC2814083.
29. Blount D, Hooi D, Feary J, Venn A, Telford G, Brown A, et al. Immunologic profiles of persons recruited
for a randomized, placebo-controlled clinical trial of hookworm infection. Am J Trop Med Hyg. 2009; 81
(5):911–916. https://doi.org/10.4269/ajtmh.2009.09-0237 PMID: 19861631.
30. Daveson AJ, Jones DM, Gaze S, McSorley H, Clouston A, Pascoe A, et al. Effect of hookworm infec-
tion on wheat challenge in celiac disease—a randomised double-blinded placebo controlled trial. PloS
ONE. 2011; 6(3):e17366. https://doi.org/10.1371/journal.pone.0017366 PMID: 21408161; PubMed
Central PMCID: PMC3050888.
31. Croese J, O’Neil J, Masson, Cooke S, Melrose W, Pritchard D, et al. A proof of concept study estab-
lishing Necator americanus in Crohn’s patients and reservoir donors. Gut. 2006; 55(1):136–137.
https://doi.org/10.1136/gut.2005.079129 PMID: 16344586; PubMed Central PMCID: PMC1856386.
32. Donath MY. Multiple benefits of targeting inflammation in the treatment of type 2 diabetes. Diabetolo-
gia. 2016; 59(4):679–682. https://doi.org/10.1007/s00125-016-3873-z PMID: 26868493.
33. Pierce D, Merone L, Lewis C, Rahman T, Croese J, Loukas A, et al. Safety and tolerability of experi-
mental hookworm infection in humans with metabolic disease: study protocol for a phase 1b rando-
mised controlled clinical trial. BMC Endocr Disord. 2019; 19(1):136. https://doi.org/10.1186/s12902-
019-0461-5 PMID: 31829172.
34. Diemert D, Campbell D, Brelsford J, Leasure C, Li G, Peng J, et al. Controlled Human Hookworm
Infection: Accelerating Human Hookworm Vaccine Development. Open forum infectious diseases.
2018; 5(5):ofy083. https://doi.org/10.1093/ofid/ofy083 PMID: 29780848; PubMed Central PMCID:
PMC5952933.
35. Donath MY, Dinarello CA, Mandrup-Poulsen T. Targeting innate immune mediators in type 1 and type
2 diabetes. Nat Rev Immunol. 2019; 19(12):734–746. https://doi.org/10.1038/s41577-019-0213-9
PMID: 31501536.
36. Hoogerwerf MA, Coffeng LE, Brienen EAT, Janse JJ, Langenberg MCC, Kruize YCM, et al. New
Insights Into the Kinetics and Variability of Egg Excretion in Controlled Human Hookworm Infections.
The Journal of infectious diseases. 2019; 220(6):1044–1048. https://doi.org/10.1093/infdis/jiz218
PMID: 31077279.
37. Maizels RM, Smits HH, McSorley HJ. Modulation of Host Immunity by Helminths: The Expanding Rep-
ertoire of Parasite Effector Molecules. Immunity. 2018; 49(5):801–818. https://doi.org/10.1016/j.
immuni.2018.10.016 PMID: 30462997; PubMed Central PMCID: PMC6269126.
38. Harnett MM, Harnett W. Can Parasitic Worms Cure the Modern World’s Ills? Trends Parasitol. 2017;
33(9):694–705. https://doi.org/10.1016/j.pt.2017.05.007 PMID: 28606411.
39. Ruyssers NE, De Winter BY, De Man JG, Loukas A, Pearson MS, Weinstock JV, et al. Therapeutic
potential of helminth soluble proteins in TNBS-induced colitis in mice. Inflamm Bowel Dis. 2009; 15
(4):491–500. https://doi.org/10.1002/ibd.20787 PMID: 19023900.
40. Cancado GG, Fiuza JA, de Paiva NC, Lemos Lde C, Ricci ND, Gazzinelli-Guimaraes PH, et al. Hook-
worm products ameliorate dextran sodium sulfate-induced colitis in BALB/c mice. Inflamm Bowel Dis.
2011; 17(11):2275–2286. https://doi.org/10.1002/ibd.21629 PMID: 21290484.
41. Ferreira I, Smyth D, Gaze S, Aziz A, Giacomin P, Ruyssers N, et al. Hookworm excretory/secretory
products induce interleukin-4 (IL-4)+ IL-10+ CD4+ T cell responses and suppress pathology in a
mouse model of colitis. Infect Immun. 2013; 81(6):2104–2111. https://doi.org/10.1128/IAI.00563-12
PMID: 23545299; PubMed Central PMCID: PMC3676036.
42. Wammes LJ, Mpairwe H, Elliott AM, Yazdanbakhsh M. Helminth therapy or elimination: epidemiologi-
cal, immunological, and clinical considerations. Lancet Infect Dis. 2014; 14(11):1150–1162. https://doi.
org/10.1016/S1473-3099(14)70771-6 PMID: 24981042.
43. McSorley HJ, O’Gorman MT, Blair N, Sutherland TE, Filbey KJ, Maizels RM. Suppression of type 2
immunity and allergic airway inflammation by secreted products of the helminth Heligmosomoides
polygyrus. Eur J Immunol. 2012; 42(10):2667–2682. https://doi.org/10.1002/eji.201142161 PMID:
22706967; PubMed Central PMCID: PMC4916998.
44. Shepherd C, Giacomin P, Navarro S, Miller C, Loukas A, Wangchuk P. A medicinal plant compound,
capnoidine, prevents the onset of inflammation in a mouse model of colitis. J Ethnopharmacol. 2018;
211:17–28. https://doi.org/10.1016/j.jep.2017.09.024 PMID: 28942135.
45. Maizels RM, Balic A, Gomez-Escobar N, Nair M, Taylor MD, Allen JE. Helminth parasites—masters of
regulation. Immunol Rev. 2004; 201:89–116. https://doi.org/10.1111/j.0105-2896.2004.00191.x
PMID: 15361235.

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 15 / 20

PLOS PATHOGENS

46. Hewitson JP, Grainger JR, Maizels RM. Helminth immunoregulation: the role of parasite secreted pro-
teins in modulating host immunity. Mol Biochem Parasitol. 2009; 167(1):1–11. https://doi.org/10.1016/
j.molbiopara.2009.04.008 PMID: 19406170; PubMed Central PMCID: PMC2706953.
47. Jolink H, de Boer R, Willems LN, van Dissel JT, Falkenburg JH, Heemskerk MH. T helper 2 response
in allergic bronchopulmonary aspergillosis is not driven by specific Aspergillus antigens. Allergy. 2015;
70(10):1336–1339. https://doi.org/10.1111/all.12688 PMID: 26179335.
48. Logan J, Navarro S, Loukas A, Giacomin P. Helminth-induced regulatory T cells and suppression of
allergic responses. Curr Opin Immunol. 2018; 54:1–6. https://doi.org/10.1016/j.coi.2018.05.007 PMID:
29852470.
49. Grainger JR, Smith KA, Hewitson JP, McSorley HJ, Harcus Y, Filbey KJ, et al. Helminth secretions
induce de novo T cell Foxp3 expression and regulatory function through the TGF-beta pathway. J Exp
Med. 2010; 207(11):2331–2341. https://doi.org/10.1084/jem.20101074 PMID: 20876311; PubMed
Central PMCID: PMC2964568.
50. Wensveen FM, Jelencic V, Valentic S, Sestan M, Wensveen TT, Theurich S, et al. NK cells link obe-
sity-induced adipose stress to inflammation and insulin resistance. Nat Immunol. 2015; 16(4):376–
385. https://doi.org/10.1038/ni.3120 PMID: 25729921.
51. Navarro S, Pickering DA, Ferreira IB, Jones L, Ryan S, Troy S, et al. Hookworm recombinant protein
promotes regulatory T cell responses that suppress experimental asthma. Sci Transl Med. 2016; 8
(362):362ra143. https://doi.org/10.1126/scitranslmed.aaf8807 PMID: 27797959.
52. Allen JE, Maizels RM. Diversity and dialogue in immunity to helminths. Nature reviews Immunology.
2011; 11(6):375–388. https://doi.org/10.1038/nri2992 PMID: 21610741.
53. Lewis G, Wang B, Shafiei Jahani P, Hurrell BP, Banie H, Aleman Muench GR, et al. Dietary Fiber-
Induced Microbial Short Chain Fatty Acids Suppress ILC2-Dependent Airway Inflammation. Frontiers
in immunology. 2019; 10:2051. https://doi.org/10.3389/fimmu.2019.02051 PMID: 31620118; PubMed
Central PMCID: PMC6760365.
54. von Moltke J, Ji M, Liang HE, Locksley RM. Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithe-
lial response circuit. Nature. 2016; 529(7585):221–225. https://doi.org/10.1038/nature16161 PMID:
26675736; PubMed Central PMCID: PMC4830391.
55. Ferreira LMR, Muller YD, Bluestone JA, Tang Q. Next-generation regulatory T cell therapy. Nat Rev
Drug Discov. 2019; 18(10):749–769. https://doi.org/10.1038/s41573-019-0041-4 PMID: 31541224.
56. Abdel Aziz N, Nono JK, Mpotje T, Brombacher F. The Foxp3+ regulatory T-cell population requires IL-
4Ralpha signaling to control inflammation during helminth infections. PLoS Biol. 2018; 16(10):
e2005850. https://doi.org/10.1371/journal.pbio.2005850 PMID: 30379806; PubMed Central PMCID:
PMC6231676.
57. Bluestone JA, Trotta E, Xu D. The therapeutic potential of regulatory T cells for the treatment of auto-
immune disease. Expert Opin Ther Targets. 2015; 19(8):1091–1103. https://doi.org/10.1517/
14728222.2015.1037282 PMID: 25881491.
58. Ferreira LMR, Muller YD, Bluestone JA, Tang Q. Next-generation regulatory T cell therapy. Nat Rev
Drug Discov. 2019; 18:749–769. https://doi.org/10.1038/s41573-019-0041-4 PMID: 31541224.
59. Hussaarts L, van der Vlugt LE, Yazdanbakhsh M, Smits HH. Regulatory B-cell induction by helminths:
implications for allergic disease. The Journal of allergy and clinical immunology. 2011; 128(4):733–
739. https://doi.org/10.1016/j.jaci.2011.05.012 PMID: 21684587.
60. Chen F, Wu W, Jin L, Millman A, Palma M, El-Naccache DW, et al. B Cells Produce the Tissue-Protec-
tive Protein RELMalpha during Helminth Infection, which Inhibits IL-17 Expression and Limits Emphy-
sema. Cell reports. 2018; 25(10):2775–83.e3. https://doi.org/10.1016/j.celrep.2018.11.038 PMID:
30517865.
61. Gao X, Ren X, Wang Q, Yang Z, Li Y, Su Z, et al. Critical roles of regulatory B and T cells in helminth
parasite-induced protection against allergic airway inflammation. Clinical and experimental immunol-
ogy. 2019; 198(3):390–402. https://doi.org/10.1111/cei.13362 PMID: 31397879; PubMed Central
PMCID: PMC6857085.
62. Nagashima H, Mahlakoiv T, Shih HY, Davis FP, Meylan F, Huang Y, et al. Neuropeptide CGRP Limits
Group 2 Innate Lymphoid Cell Responses and Constrains Type 2 Inflammation. Immunity. 2019; 51
(4):682-95.e6. https://doi.org/10.1016/j.immuni.2019.06.009 PMID: 31353223; PubMed Central
PMCID: PMC6801073.
63. Bouchery T, Le Gros G, Harris N. ILC2s-Trailblazers in the Host Response Against Intestinal Hel-
minths. Frontiers in immunology. 2019; 10:623. https://doi.org/10.3389/fimmu.2019.00623 PMID:
31019505; PubMed Central PMCID: PMC6458269.
64. Stoltzfus JD, Pilgrim AA, Herbert DR. Perusal of parasitic nematode ’omics in the post-genomic era.
Mol Biochem Parasitol. 2017; 215:11–22. https://doi.org/10.1016/j.molbiopara.2016.11.003 PMID:
27887974; PubMed Central PMCID: PMC5440216.

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 16 / 20

PLOS PATHOGENS

65. Holt DC, Fischer K, Allen GE, Wilson D, Wilson P, Slade R, et al. Mechanisms for a novel immune eva-
sion strategy in the scabies mite sarcoptes scabiei: a multigene family of inactivated serine proteases. J
Invest Dermatol. 2003; 121(6):1419–1424. https://doi.org/10.1046/j.1523-1747.2003.12621.x PMID:
14675192.
66. Zhu S. Did cathelicidins, a family of multifunctional host-defense peptides, arise from a cysteine prote-
ase inhibitor? Trends Microbiol. 2008; 16(8):353–360. https://doi.org/10.1016/j.tim.2008.05.007
PMID: 18632274.
67. Cantacessi C, Hofmann A, Pickering D, Navarro S, Mitreva M, Loukas A. TIMPs of parasitic helminths
—a large-scale analysis of high-throughput sequence datasets. Parasit Vectors. 2013; 6:156. https://
doi.org/10.1186/1756-3305-6-156 PMID: 23721526; PubMed Central PMCID: PMC3679795.
68. Zhan B, Badamchian M, Meihua B, Ashcom J, Feng J, Hawdon J, et al. Molecular cloning and purifica-
tion of Ac-TMP, a developmentally regulated putative tissue inhibitor of metalloprotease released in
relative abundance by adult Ancylostoma hookworms. Am J Trop Med Hyg. 2002; 66(3):238–244.
https://doi.org/10.4269/ajtmh.2002.66.238 PMID: 12139214.
69. Kucera K, Harrison LM, Cappello M, Modis Y. Ancylostoma ceylanicum excretory-secretory protein 2
adopts a netrin-like fold and defines a novel family of nematode proteins. J Mol Biol. 2011; 408(1):9–17.
https://doi.org/10.1016/j.jmb.2011.02.033 PMID: 21352830; PubMed Central PMCID: PMC3070796.
70. Cuellar C, Wu W, Mendez S. The hookworm tissue inhibitor of metalloproteases (Ac-TMP-1) modifies
dendritic cell function and induces generation of CD4 and CD8 suppressor T cells. PLoS Negl Trop
Dis. 2009; 3(5):e439. https://doi.org/10.1371/journal.pntd.0000439 PMID: 19468296; PubMed Central
PMCID: PMC2678263.
71. Ferreira IB, Pickering DA, Troy S, Croese J, Loukas A, Navarro S. Suppression of inflammation and tis-
sue damage by a hookworm recombinant protein in experimental colitis. Clin Transl Immun. 2017; 6(10):
e157. https://doi.org/10.1038/cti.2017.42 PMID: 29114386; PubMed Central PMCID: PMC5671989
72. Ochieng J, Chaudhuri G. Cystatin superfamily. J Health Care Poor Underserved. 2010; 21(1 Suppl):51–
70. https://doi.org/10.1353/hpu.0.0257 PMID: 20173285; PubMed Central PMCID: PMC2888135.
73. Murray J, Manoury B, Balic A, Watts C, Maizels RM. Bm-CPI-2, a cystatin from Brugia malayi nema-
tode parasites, differs from Caenorhabditis elegans cystatins in a specific site mediating inhibition of
the antigen-processing enzyme AEP. Mol Biochem Parasitol. 2005; 139(2):197–203. https://doi.org/
10.1016/j.molbiopara.2004.11.008 PMID: 15664654.
74. Hartmann S, Lucius R. Modulation of host immune responses by nematode cystatins. Int J Parasitol.
2003; 33(11):1291–1302. https://doi.org/10.1016/s0020-7519(03)00163-2 PMID: 13678644.
75. Gregory WF, Maizels RM. Cystatins from filarial parasites: evolution, adaptation and function in the
host-parasite relationship. Int J Biochem Cell Biol. 2008; 40(6–7):1389–1398. https://doi.org/10.1016/j.
biocel.2007.11.012 PMID: 18249028.
76. Danilowicz-Luebert E, Steinfelder S, Kuhl AA, Drozdenko G, Lucius R, Worm M, et al. A nematode
immunomodulator suppresses grass pollen-specific allergic responses by controlling excessive Th2
inflammation. Int J Parasitol. 2013; 43(3–4):201–210. https://doi.org/10.1016/j.ijpara.2012.10.014
PMID: 23174104.
77. Coronado S, Barrios L, Zakzuk J, Regino R, Ahumada V, Franco L, et al. A recombinant cystatin from
Ascaris lumbricoides attenuates inflammation of DSS-induced colitis. Parasite Immunol. 2017; 39(4).
https://doi.org/10.1111/pim.12425 PMID: 28295446.
78. Khatri V, Amdare N, Tarnekar A, Goswami K, Reddy MV. Brugia malayi cystatin therapeutically ame-
liorates dextran sulfate sodium-induced colitis in mice. J Dig Dis. 2015; 16(10):585–594. https://doi.
org/10.1111/1751-2980.12290 PMID: 26358507.
79. Wang S, Xie Y, Yang X, Wang X, Yan K, Zhong Z, et al. Therapeutic potential of recombinant cystatin
from Schistosoma japonicum in TNBS-induced experimental colitis of mice. Parasit Vectors. 2016;
9:6. https://doi.org/10.1186/s13071-015-1288-1 PMID: 26728323; PubMed Central PMCID:
PMC4700642.
80. Johnston CJC, Smyth DJ, Kodali RB, White MPJ, Harcus Y, Filbey KJ, et al. A structurally distinct
TGF-beta mimic from an intestinal helminth parasite potently induces regulatory T cells. Nat Commun.
2017; 8(1):1741. https://doi.org/10.1038/s41467-017-01886-6 PMID: 29170498; PubMed Central
PMCID: PMC5701006.
81. Ohue Y, Nishikawa H. Regulatory T (Treg) cells in cancer: Can Treg cells be a new therapeutic target?
Cancer Sci. 2019; 110(7):2080–2089. https://doi.org/10.1111/cas.14069 PMID: 31102428; PubMed
Central PMCID: PMC6609813.
82. Tribolet L, Cantacessi C, Pickering DA, Navarro S, Doolan DL, Trieu A, et al. Probing of a human pro-
teome microarray with a recombinant pathogen protein reveals a novel mechanism by which hook-
worms suppress B-cell receptor signaling. J Infect Dis. 2015; 211(3):416–425. https://doi.org/10.1093/
infdis/jiu451 PMID: 25139017.

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 17 / 20

PLOS PATHOGENS

83. Osbourn M, Soares DC, Vacca F, Cohen ES, Scott IC, Gregory WF, et al. HpARI Protein Secreted by
a Helminth Parasite Suppresses Interleukin-33. Immunity. 2017; 47(4):739–51.e5. https://doi.org/10.
1016/j.immuni.2017.09.015 PMID: 29045903; PubMed Central PMCID: PMC5655542.
84. Gause WC, Wynn TA, Allen JE. Type 2 immunity and wound healing: evolutionary refinement of adap-
tive immunity by helminths. Nat Rev Immunol. 2013; 13(8):607–614. https://doi.org/10.1038/nri3476
PMID: 23827958.
85. faz lopez B, Morales-Montor J, Terrazas L. Role of Macrophages in the Repair Process during the Tis-
sue Migrating and Resident Helminth Infections. BioMed Research International. 2016; 2016:1–11.
https://doi.org/10.1155/2016/8634603 PMID: 27648452
86. Ariyaratne A, Finney CAM. Eosinophils and Macrophages within the Th2-Induced Granuloma: Balanc-
ing Killing and Healing in a Tight Space. Infect Immun. 2019; 87(10). https://doi.org/10.1128/iai.00127-
19 PMID: 31285249; PubMed Central PMCID: PMC6759305.
87. Smout MJ, Sotillo J, Laha T, Papatpremsiri A, Rinaldi G, Pimenta RN, et al. Carcinogenic Parasite
Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia. PLoS
Pathog. 2015; 11(10):e1005209. https://doi.org/10.1371/journal.ppat.1005209 PMID: 26485648
88. Dastpeyman M, Bansal PS, Wilson D, Sotillo J, Brindley PJ, Loukas A, et al. Structural Variants of a
Liver Fluke Derived Granulin Peptide Potently Stimulate Wound Healing. Journal of medicinal chemis-
try. 2018; 61(19):8746–8753. https://doi.org/10.1021/acs.jmedchem.8b00898 PMID: 30183294.
89. Li J, Chen J, Kirsner R. Pathophysiology of acute wound healing. Clinics in Dermatology. 2007; 25
(1):9–18. https://doi.org/https://doi.org/10.1016/j.clindermatol.2006.09.007 PMID: 17276196
90. Ibrahim NI, Wong SK, Mohamed IN, Mohamed N, Chin K-Y, Ima-Nirwana S, et al. Wound Healing
Properties of Selected Natural Products. Int J Environ Res Public Health. 2018; 15(11):2360. https://
doi.org/10.3390/ijerph15112360 PMID: 30366427.
91. Pineda MA, Lumb F, Harnett MM, Harnett W. ES-62, a therapeutic anti-inflammatory agent evolved by
the filarial nematode Acanthocheilonema viteae. Mol Biochem Parasitol. 2014; 194(1–2):1–8. https://
doi.org/10.1016/j.molbiopara.2014.03.003 PMID: 24671112.
92. Rzepecka J, Siebeke I, Coltherd JC, Kean DE, Steiger CN, Al-Riyami L, et al. The helminth product,
ES-62, protects against airway inflammation by resetting the Th cell phenotype. Int J Parasitol. 2013;
43(3–4):211–223. https://doi.org/10.1016/j.ijpara.2012.12.001 PMID: 23291461; PubMed Central
PMCID: PMC3584281.
93. Aprahamian TR, Zhong X, Amir S, Binder CJ, Chiang LK, Al-Riyami L, et al. The immunomodulatory
parasitic worm product ES-62 reduces lupus-associated accelerated atherosclerosis in a mouse
model. Int J Parasitol. 2015; 45(4):203–207. https://doi.org/10.1016/j.ijpara.2014.12.006 PMID:
25666929; PubMed Central PMCID: PMC4355381.
94. Deehan MR, Harnett MM, Harnett W. A filarial nematode secreted product differentially modulates
expression and activation of protein kinase C isoforms in B lymphocytes. J Immun. 1997; 159
(12):6105–6111. PMID: 9550411.
95. Harnett W, Harnett MM. Immunomodulatory activity and therapeutic potential of the filarial nematode
secreted product, ES-62. Adv Exp Med Biol. 2009; 666:88–94. https://doi.org/10.1007/978-1-4419-
1601-3_7 PMID: 20054977.
96. Harnett W, Deehan MR, Houston KM, Harnett MM. Immunomodulatory properties of a phosphorylcho-
line-containing secreted filarial glycoprotein. Parasite Immunol. 1999; 21(12):601–608. https://doi.org/
10.1046/j.1365-3024.1999.00267.x PMID: 10583862.
97. Doonan J, Lumb FE, Pineda MA, Tarafdar A, Crowe J, Khan AM, et al. Protection Against Arthritis by
the Parasitic Worm Product ES-62, and Its Drug-Like Small Molecule Analogues, Is Associated With
Inhibition of Osteoclastogenesis. Front Immun. 2018; 9:1016. https://doi.org/10.3389/fimmu.2018.
01016 PMID: 29867986; PubMed Central PMCID: PMC5967578.
98. Suckling CJ, Mukherjee S, Khalaf AI, Narayan A, Scott FJ, Khare S, et al. Synthetic analogues of the
parasitic worm product ES-62 reduce disease development in in vivo models of lung fibrosis. Acta
Trop. 2018; 185:212–218. https://doi.org/10.1016/j.actatropica.2018.05.015 PMID: 29802846.
99. Matisz CE, Geuking MB, Lopes F, Petri B, Wang A, Sharkey KA, et al. Helminth Antigen-Conditioned
Dendritic Cells Generate Anti-Inflammatory Cd4 T Cells Independent of Antigen Presentation via
Major Histocompatibility Complex Class II. Am J Pathol. 2018; 188(11):2589–2604. https://doi.org/10.
1016/j.ajpath.2018.07.008 PMID: 30121255.
100. Wuhrer M, Balog CI, Catalina MI, Jones FM, Schramm G, Haas H, et al. IPSE/alpha-1, a major secre-
tory glycoprotein antigen from schistosome eggs, expresses the Lewis X motif on core-difucosylated
N-glycans. The FEBS journal. 2006; 273(10):2276–2292. https://doi.org/10.1111/j.1742-4658.2006.
05242.x PMID: 16650003.
101. Haeberlein S, Obieglo K, Ozir-Fazalalikhan A, Chaye MAM, Veninga H, van der Vlugt L, et al. Schisto-
some egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory B

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 18 / 20

PLOS PATHOGENS

cells. PLoS Pathog. 2017; 13(7):e1006539. https://doi.org/10.1371/journal.ppat.1006539 PMID:

28753651; PubMed Central PMCID: PMC5550006.
102. Knuhr K, Langhans K, Nyenhuis S, Viertmann K, Kildemoes AMO, Doenhoff MJ, et al. Schistosoma
mansoni Egg-Released IPSE/alpha-1 Dampens Inflammatory Cytokine Responses via Basophil Inter-
leukin (IL)-4 and IL-13. Frontiers in immunology. 2018; 9:2293. https://doi.org/10.3389/fimmu.2018.
02293 PMID: 30364177; PubMed Central PMCID: PMC6191518.
103. Hussaarts L, Garcia-Tardon N, van Beek L, Heemskerk MM, Haeberlein S, van der Zon GC, et al.
Chronic helminth infection and helminth-derived egg antigens promote adipose tissue M2 macro-
phages and improve insulin sensitivity in obese mice. Faseb j. 2015; 29(7):3027–39. Epub 2015/04/
09. https://doi.org/10.1096/fj.14-266239 PMID: 25852044.
104. Kahl J, Brattig N, Liebau E. The Untapped Pharmacopeic Potential of Helminths. Trends Parasitol.
2018; 34(10):828–842. https://doi.org/10.1016/j.pt.2018.05.011 PMID: 29954660.
105. Hams E, Bermingham R, Wurlod FA, Hogan AE, O’Shea D, Preston RJ, et al. The helminth T2 RNase
omega1 promotes metabolic homeostasis in an IL-33- and group 2 innate lymphoid cell-dependent
mechanism. Faseb j. 2016; 30(2):824–835. https://doi.org/10.1096/fj.15-277822 PMID: 26490658;
PubMed Central PMCID: PMC4973506.
106. Tang CL, Liu ZM, Gao YR, Xiong F. Schistosoma Infection and Schistosoma-Derived Products Modu-
late the Immune Responses Associated with Protection against Type 2 Diabetes. Frontiers in immu-
nology. 2017; 8:1990. https://doi.org/10.3389/fimmu.2017.01990 PMID: 29387059; PubMed Central
PMCID: PMC5776330.
107. Bhargava P, Li C, Stanya KJ, Jacobi D, Dai L, Liu S, et al. Immunomodulatory glycan LNFPIII allevi-
ates hepatosteatosis and insulin resistance through direct and indirect control of metabolic pathways.
Nature medicine. 2012; 18(11):1665–1672. https://doi.org/10.1038/nm.2962 PMID: 23104131;
PubMed Central PMCID: PMC3493877.
108. Becker AC, Willenberg I, Springer A, Schebb NH, Steinberg P, Strube C. Fatty acid composition of
free-living and parasitic stages of the bovine lungworm Dictyocaulus viviparus. Mol Biochem Parasitol.
2017; 216:39–44. https://doi.org/10.1016/j.molbiopara.2017.06.008 PMID: 28651962.
109. Wangchuk P, Kouremenos K, Eichenberger RM, Pearson M, Susianto A, Wishart DS, et al. Metabolo-
mic profiling of the excretory-secretory products of hookworm and whipworm. Metabolomics. 2019; 15
(7):101. https://doi.org/10.1007/s11306-019-1561-y PMID: 31254203.
110. Wangchuk P, Constantinoiu C, Eichenberger RM, Field M, Loukas A. Characterization of Tapeworm
Metabolites and Their Reported Biological Activities. Molecules. 2019; 24(8). https://doi.org/10.3390/
molecules24081480 PMID: 30991712; PubMed Central PMCID: PMC6514793.
111. Kokova D, Mayboroda OA. Twenty Years on: Metabolomics in Helminth Research. Trends Parasitol.
2019; 35(4):282–288. https://doi.org/10.1016/j.pt.2019.01.012 PMID: 30824203.
112. Wangchuk P, Shepherd C, Constantinoiu C, Ryan RYM, Kouremenos KA, Becker L, et al. Hookworm-
Derived Metabolites Suppress Pathology in a Mouse Model of Colitis and Inhibit Secretion of Key
Inflammatory Cytokines in Primary Human Leukocytes. Infection and immunity. 2019; 87(4). https://
doi.org/10.1128/iai.00851-18 PMID: 30670556; PubMed Central PMCID: PMC6434117.
113. Giera M, Kaisar MMM, Derks RJE, Steenvoorden E, Kruize YCM, Hokke CH, et al. The Schistosoma
mansoni lipidome: Leads for immunomodulation. Anal Chim Acta. 2018; 1037:107–118. https://doi.
org/10.1016/j.aca.2017.11.058 PMID: 30292284.
114. Laan LC, Williams AR, Stavenhagen K, Giera M, Kooij G, Vlasakov I, et al. The whipworm (Trichuris
suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells.
Faseb J. 2017; 31(2):719–731. https://doi.org/10.1096/fj.201600841R PMID: 27806992; PubMed
Central PMCID: PMC5240662.
115. Karpisheh V, Nikkhoo A, Hojjat-Farsangi M, Namdar A, Azizi G, Ghalamfarsa G, et al. Prostaglandin
E2 as a potent therapeutic target for treatment of colon cancer. Prostaglandins Other Lipid Mediat.
2019:106338. https://doi.org/10.1016/j.prostaglandins.2019.106338 PMID: 31100474.
116. Coakley G, Buck AH, Maizels RM. Host parasite communications-Messages from helminths for the
immune system: Parasite communication and cell-cell interactions. Mol Biochem Parasitol. 2016; 208
(1):33–40. https://doi.org/10.1016/j.molbiopara.2016.06.003 PMID: 27297184; PubMed Central
PMCID: PMC5008435.
117. Eichenberger RM, Sotillo J, Loukas A. Immunobiology of parasitic worm extracellular vesicles. Immu-
nol Cell Biol. 2018; 96(9)704–713. https://doi.org/10.1111/imcb.12171 PMID: 29808496.
118. Buck AH, Coakley G, Simbari F, McSorley HJ, Quintana JF, Le Bihan T, et al. Exosomes secreted by
nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity. Nat Com-
mun. 2014; 5:5488. https://doi.org/10.1038/ncomms6488 PMID: 25421927; PubMed Central PMCID:
PMC4263141.

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 19 / 20

PLOS PATHOGENS

119. Eichenberger RM, Ryan S, Jones L, Buitrago G, Polster R, Montes de Oca M, et al. Hookworm
Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice. Frontiers
in immunology. 2018; 9:850. https://doi.org/10.3389/fimmu.2018.00850 PMID: 29760697; PubMed
Central PMCID: PMC5936971.
120. Roig J, Saiz ML, Galiano A, Trelis M, Cantalapiedra F, Monteagudo C, et al. Extracellular Vesicles
From the Helminth Fasciola hepatica Prevent DSS-Induced Acute Ulcerative Colitis in a T-Lymphocyte
Independent Mode. Front Microbiol. 2018; 9:1036. https://doi.org/10.3389/fmicb.2018.01036 PMID:
29875750; PubMed Central PMCID: PMC5974114.
121. Marcilla A, Trelis M, Cortes A, Sotillo J, Cantalapiedra F, Minguez MT, et al. Extracellular vesicles from
parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host
cells. PloS ONE. 2012; 7(9):e45974. https://doi.org/10.1371/journal.pone.0045974 PMID: 23029346;
PubMed Central PMCID: PMC3454434.
122. Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a new era for the management of cancer
and other diseases. Nat Rev Drug Discov. 2017; 16(3):203–222. Epub 2017/02/18. https://doi.org/10.
1038/nrd.2016.246 PMID: 28209991.
123. Samanta S, Rajasingh S, Drosos N, Zhou Z, Dawn B, Rajasingh J. Exosomes: new molecular targets
of diseases. Acta Pharmacol Sin. 2018; 39(4):501–513. https://doi.org/10.1038/aps.2017.162 PMID:
29219950; PubMed Central PMCID: PMC5888687.
124. Pottoo FH, Barkat MA, Harshita, Ansari MA, Javed MN, Sajid Jamal QM, et al. Nanotechnological
based miRNA intervention in the therapeutic management of neuroblastoma. Semin Cancer Biol.
Forthcoming 2019. https://doi.org/10.1016/j.semcancer.2019.09.017 PMID: 31562954.
125. Sil S, Dagur RS, Liao K, Peeples ES, Hu G, Periyasamy P, et al. Strategies for the use of Extracellular
Vesicles for the Delivery of Therapeutics. J Neuroimmune Pharmacol. 2019. https://doi.org/10.1007/
s11481-019-09873-y PMID: 31456107.
126. Li SP, Lin ZX, Jiang XY, Yu XY. Exosomal cargo-loading and synthetic exosome-mimics as potential
therapeutic tools. Acta Pharmacol Sin. 2018; 39(4):542–551. https://doi.org/10.1038/aps.2017.178
PMID: 29417947; PubMed Central PMCID: PMC5888690.
127. Pennington MW, Czerwinski A, Norton RS. Peptide therapeutics from venom: Current status and potential.
Bioorg Med Chem. 2018; 26(10):2738–2758. https://doi.org/10.1016/j.bmc.2017.09.029 PMID: 28988749.
128. Summers RW, Elliott DE, Qadir K, Urban JF Jr., Thompson R, Weinstock JV. Trichuris suis seems to
be safe and possibly effective in the treatment of inflammatory bowel disease. Am J Gastroenterol.
2003; 98(9):2034–2041. https://doi.org/10.1111/j.1572-0241.2003.07660.x PMID: 14499784.
129. Fleming J, Hernandez G, Hartman L, Maksimovic J, Nace S, Lawler B, et al. Safety and efficacy of hel-
minth treatment in relapsing-remitting multiple sclerosis: Results of the HINT 2 clinical trial. Mult Scler.
2019; 25(1):81–91. https://doi.org/10.1177/1352458517736377 PMID: 29064315; PubMed Central
PMCID: PMC5878983.
130. Bager P, Arnved J, Ronborg S, Wohlfahrt J, Poulsen LK, Westergaard T, et al. Trichuris suis ova ther-
apy for allergic rhinitis: a randomized, double-blind, placebo-controlled clinical trial. J Allergy Clin
Immunol. 2010; 125(1):123-30.e1-3. https://doi.org/10.1016/j.jaci.2009.08.006 PMID: 19800680.
131. Rosche B, Wernecke KD, Ohlraun S, Dorr JM, Paul F. Trichuris suis ova in relapsing-remitting multiple
sclerosis and clinically isolated syndrome (TRIOMS): study protocol for a randomized controlled trial.
Trials. 2013; 14:112. https://doi.org/10.1186/1745-6215-14-112 PMID: 23782752; PubMed Central
PMCID: PMC3680966.
132. Sandborn WJ, Elliott DE, Weinstock J, Summers RW, Landry-Wheeler A, Silver N, et al. Randomised
clinical trial: the safety and tolerability of Trichuris suis ova in patients with Crohn’s disease. Aliment
Pharmacol Ther. 2013; 38(3):255–263. https://doi.org/10.1111/apt.12366 PMID: 23730956.
133. Feary J, Venn A, Brown A, Hooi D, Falcone FH, Mortimer K, et al. Safety of hookworm infection in indi-
viduals with measurable airway responsiveness: a randomized placebo-controlled feasibility study.
Clin Exp Allergy. 2009; 39(7):1060–1068. https://doi.org/10.1111/j.1365-2222.2009.03187.x PMID:
19400893; PubMed Central PMCID: PMC2728895.
134. Gaze S, Driguez P, Pearson MS, Mendes T, Doolan DL, Trieu A, et al. An immunomics approach to
schistosome antigen discovery: antibody signatures of naturally resistant and chronically infected indi-
viduals from endemic areas. PLoS Pathog. 2014; 10(3):e1004033. https://doi.org/10.1371/journal.
ppat.1004033 PMID: 24675823; PubMed Central PMCID: PMC3968167.
135. Croese J, Giacomin P, Navarro S, Clouston A, McCann L, Dougall A, et al. Experimental hookworm
infection and gluten microchallenge promote tolerance in celiac disease. J Allergy Clin Immunol. 2015;
135(2):508–516. https://doi.org/10.1016/j.jaci.2014.07.022 PMID: 25248819.
136. Langenberg MCC, Hoogerwerf MA, Koopman JPR, Janse JJ, Kos-van Oosterhoud J, Feijt C, et al. A
controlled human Schistosoma mansoni infection model to advance novel drugs, vaccines and diag-
nostics. Nat Med. 2020; 26:326–332. https://doi.org/10.1038/s41591-020-0759-x PMID: 32066978.

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008508 May 14, 2020 20 / 20