There is a rapidly growing body of evidence that production of extracellular vesicles (EVs) is a universal feature of cellular life. More recently, EVs have been identified in a broad range of both unicellular and multicellular parasites where they play roles in parasite–parasite intercommunication as well as parasite–host interactions. Parasitic helminth-derived EVs traverse host target cell membranes whereupon they offload their molecular cargo — proteins, lipids, and genetic information such as mRNAs and miRNAs — which are thought to hijack the target cell and modulate its gene expression to promote parasite survival. As such, EVs represent a novel mechanism of intercellular communication that could be targeted for vaccine-mediated interruption, given the abundance of surface antigens expressed on helminth EVs, and the ability of antibodies to block their uptake by target cells. In this Perspective article, we review recent developments in the field of helminth-derived EVs and highlight their roles in helminth vaccine discovery and development.

Introduction

Extracellular vesicles (EVs) are heterogeneous vesicles of membranous origin released by different types of cells. EVs comprise a complex mixture of genetic information, proteins, lipids, and glycans. EVs can broadly be categorized into three classes based on their cell of origin, molecular contents, function, physical characteristics, specific protein markers, and isolation techniques. These EV classes include (i) exosomes, (ii) microvesicles (MVs) or ectosomes, and (iii) apoptotic bodies (ABs). A shared characteristic of all three EV types is the surrounding lipid bilayer membrane with specific molecular cargo. However, their specific densities and sizes vary considerably, and are diagnostic features [1], although both measurement ranges for the different EV classes have been heterogeneously documented [2]. Nevertheless, exosomes, first recognized in the 1980s [3], are thought to range from 30 to 150 nm in diameter and with a specific density of 1.10–1.14 g/ml [3]. Exosomes originate from inward budding of membranes of multivesicular bodies, followed by their fusion with the cell plasma membrane and release into the extracellular space [3,4]. On the contrary, MVs, first discovered in the late 1960s [5], are relatively larger vesicles ranging from 100 to 1000 nm in diameter [3], are ubiquitously packaged and originate from the plasma membrane by outward budding or protrusion [3]. ABs are released from both normal and cancerous cells undergoing apoptotic cell clearance or programmed cell death [6], and are considered to range from 50 to 5000 nm in diameter [1,7].

Parasitic helminth exosome-like vesicles were first described in the rodent model intestinal fluke Echinostoma caproni and the livestock liver fluke Fasciola hepatica [8]. Since this initial publication, helminth EVs have received increasing attention, particularly given the role of EVs in the transfer of RNAs and other signaling information to target cells [912], and as novel diagnostic biomarkers of disease [13]. Subsequent studies reported a more detailed molecular analysis of F. hepatica EV proteins [14]. Exosome-like EVs with immunoregulatory properties were identified in the excretory/secretory (ES) products of the gastrointestinal parasitic nematode of mice Heligmosomoides polygyrus, whereupon they modulate innate immune responses in intestinal epithelial cells via transfer of miRNAs [9]. These findings prompted others to speculate that suppression of inflammation was a characteristic of parasitic helminth EVs [15]. The Asian blood fluke Schistosoma japonicum secretes EVs that influence immune pathways, notably modulation of macrophage activation pathways [10,16]. Larvae [17] and adults [18] of the African, South American and Middle East schistosome, Schistosoma mansoni, also secrete exosome-like EVs, and the carcinogenic human liver fluke, Opisthorchis viverrini, secretes EVs into the bile ducts of its human host, whereupon they are internalized by cholangiocytes and promote inflammation that may contribute to bile duct cancer [19]. To date, most of the literature on helminth EVs is focused on molecular documentation of vesicle cargo, with less emphasis on the roles of EVs in host–parasite communication. Table 1 summarizes the current state of play.

Table 1
Helminth-derived extracellular vesicles and their roles
Taxonomic classification Helminth Type of vesicle EV origin Cargo composition characterized EVs target Applied Roles References 
Trematodes F. hepatica Exosome-like vesicle Adult worms Proteins Uptake by intestinal cells In vitro Not reported [8,20
Exosome-like vesicles Adult worms Proteins, miRNAs Not reported NA Not reported [14
 EVs Adult worms miRNAs Not reported NA Not reported [21
Dicrocoelium dendriticum Exosomes Adult worms Proteins and miRNAs Not reported NA Not reported [22
S. japonicum Exosome-like vesicles Adult worms Proteins Macrophage In vitro Polarization of host macrophage [16
 Exosome-like vesicles Adult worms Proteins, miRNA Uptake by mouse liver cell In vitro Not reported [10
S. mansoni Exosome-like vesicles Adult worms Proteins Not reported NA Not reported [18
 EVs Schistosomules Proteins, miRNAs Not reported NA Not reported [17
O. viverrini EVs Adult worms Proteins Uptake by human cholangiocytes In vitro Immune regulation; antibodies to EVs prevent EV uptake [19
E. caproni Exosomes Adult worms Proteins Uptake by intestinal cells In vitro Not reported [8,23
  Exosomes Adult worm Not reported Systemic blood In vivo Immune-modulation [24
Cestodes Echinococcus multilocularis Vesicles derived from metacestodes Metacestodes Not reported Mononuclear cells/dendritic cells In vitro Immune-regulation [2527
Echinococcus granulosus Exosomes Hydatid cyst Proteins Not reported NA Not reported [28
 Taenia crassiceps
Mesocestoides corti
Echinococcus multilocularis 
EVs Larvae Protein and miRNAs Not reported NA Not reported [29
Nematodes H. polygyrus Exosomes Intestinal tract of adult nematode Proteins, mRNAs, small RNAs and Y RNAs Intestinal epithelial cells of the host In vivo and in vitro Immune-modulation in favor of parasite survival [9
 EVs Adult/larval worms Not reported Uptake by macrophage In vivo and in vitro Activates macrophages; Protection immunity [30
B. malayi Exosome-like vesicles Larval stage Protein and miRNA Internalization by macrophage In vitro Classical activation of macrophages [11
Trichuris suis EVs Larvae miRNA NA NA Not reported [13
Teladorsagia circumcincta Exosome-like vesicles Larvae Proteins Immunoglobulins In vitro Recognized by IgA and IgG [31
Trichuris muris Exosome-like vesicles Adult worms Proteins, mRNAs and miRNAs Uptake by murine colonic organoids In vitro Not reported [12
Taxonomic classification Helminth Type of vesicle EV origin Cargo composition characterized EVs target Applied Roles References 
Trematodes F. hepatica Exosome-like vesicle Adult worms Proteins Uptake by intestinal cells In vitro Not reported [8,20
Exosome-like vesicles Adult worms Proteins, miRNAs Not reported NA Not reported [14
 EVs Adult worms miRNAs Not reported NA Not reported [21
Dicrocoelium dendriticum Exosomes Adult worms Proteins and miRNAs Not reported NA Not reported [22
S. japonicum Exosome-like vesicles Adult worms Proteins Macrophage In vitro Polarization of host macrophage [16
 Exosome-like vesicles Adult worms Proteins, miRNA Uptake by mouse liver cell In vitro Not reported [10
S. mansoni Exosome-like vesicles Adult worms Proteins Not reported NA Not reported [18
 EVs Schistosomules Proteins, miRNAs Not reported NA Not reported [17
O. viverrini EVs Adult worms Proteins Uptake by human cholangiocytes In vitro Immune regulation; antibodies to EVs prevent EV uptake [19
E. caproni Exosomes Adult worms Proteins Uptake by intestinal cells In vitro Not reported [8,23
  Exosomes Adult worm Not reported Systemic blood In vivo Immune-modulation [24
Cestodes Echinococcus multilocularis Vesicles derived from metacestodes Metacestodes Not reported Mononuclear cells/dendritic cells In vitro Immune-regulation [2527
Echinococcus granulosus Exosomes Hydatid cyst Proteins Not reported NA Not reported [28
 Taenia crassiceps
Mesocestoides corti
Echinococcus multilocularis 
EVs Larvae Protein and miRNAs Not reported NA Not reported [29
Nematodes H. polygyrus Exosomes Intestinal tract of adult nematode Proteins, mRNAs, small RNAs and Y RNAs Intestinal epithelial cells of the host In vivo and in vitro Immune-modulation in favor of parasite survival [9
 EVs Adult/larval worms Not reported Uptake by macrophage In vivo and in vitro Activates macrophages; Protection immunity [30
B. malayi Exosome-like vesicles Larval stage Protein and miRNA Internalization by macrophage In vitro Classical activation of macrophages [11
Trichuris suis EVs Larvae miRNA NA NA Not reported [13
Teladorsagia circumcincta Exosome-like vesicles Larvae Proteins Immunoglobulins In vitro Recognized by IgA and IgG [31
Trichuris muris Exosome-like vesicles Adult worms Proteins, mRNAs and miRNAs Uptake by murine colonic organoids In vitro Not reported [12

NA, not applicable.

Most current studies in the field of EVs focus on the smaller sized exosomes and MVs and, because the methods used to isolate and purify membrane vesicles differ significantly between published studies, we will not strictly distinguish exosomes from MVs throughout, and instead will refer to them collectively as EVs, unless specifically mentioned as it appears in the sourced literature. In this Perspective article, we focus on presenting the available information based on the recent literature about roles of helminth EVs at the host–parasite interface, further highlighting their importance as targets for anti-helminth vaccines.

Helminth EVs are internalized by, and manipulate gene expression in, host target cells

A role for EVs in cell–cell communication in various mammalian microenvironments is well established [3234]. The observation that helminth EVs are endocytosed by mammalian host cells, however, implies a role in host–parasite communication. Marcilla et al. reported the release of exosomes from the liver fluke F. hepatica and the intestinal fluke E. caproni, and their subsequent internalization by a rat intestinal epithelial cancer cell line [8]. EVs of F. hepatica contain peroxiredoxins and cathepsin cysteine proteases [8], and HDM-1/MF6p has been identified from D. dendriticum exosomes [22]. These proteins have been shown to possess immunomodulatory activity in recombinant form as components of ES products prior to the discovery of helminth EVs [35]. Zhu et al. demonstrated that the murine macrophage-like cell line RAW264.7 endocytosed S. japonicum EVs and produced increased levels of nitric oxide and other markers of classical activation [10]. The L3 stage of the human filarial parasite Brugia malayi secretes exosome-like vesicles which were endocytosed by a murine macrophage cell line in which they drove the classical activation pathway [11]. EVs from H. polygyrus are internalized by murine epithelial cells whereupon they suppress expression of genes involved in innate immunity including IL-33 [22]. More recently, a study by Coakley et al. also showed that H. polygyrus-derived EVs are internalized by macrophages whereupon they induce suppression of type 1 and type 2 immune response-associated molecules — TNF, IL-6, RELMα, and Ym1 — and down-regulate expression of the IL-33 receptor subunit ST2 [30].

EVs from helminth parasites are also involved in the pathogenic progression of some helminth infections. Chaiyadet et al. showed that O. viverrini secretes EVs that are endocytosed by human cholangiocytes in vitro and elicit a cascade of inflammatory and pro-tumorigenic changes within the cell [19], thereby providing a plausible mechanism by which ES products are internalized by biliary cells of infected hosts [36] and contribute to the progress of cholangiocarcinoma (CCA) [37]. Moreover, O. viverrini EVs elicit production of IL-6 from recipient human cholangiocytes, showing a role for these EVs in liver disease progression [19]. Elevated IL-6 levels have been linked to chronic periductal fibrosis and CCA in O. viverrini-infected individuals [38] and to the maintenance of chronic inflammation that could progress to tumor formation [39]. O. viverrini EVs were also involved in driving proliferation of cholangiocytes [17], a condition that has been reported in both infected human subjects and the hamster infection model [40]. This persistent cell proliferation, together with other carcinogenic factors such as chronic immunopathology and high intake of dietary nitrosamines [41], triggers the establishment of malignant changes. Recent characterization of the miRNA content of exosome-like vesicles from S. japonicum revealed the presence of the Bantam miRNA, and its putative transfer to liver cells [10]. In Drosophila, Bantam miRNA has been reported to target a tumor-suppressor pathway (Hippo signaling), resulting in cellular growth and the suppression of cell death [42]. Consequently, schistosome-specific miRNAs, such as Bantam, may have a role in liver pathology of schistosomiasis. In support of this concept, Zhu et al. assessed the mRNA expression of three potential target genes (Utp3, Gins4, and Tysnd1) of schistosome Bantam miRNA in mice. Both in vitro cell culture (liver cells treated with S. japonicum EVs) and in vivo animal studies (in the livers of S. japonicum-infected mice) clearly showed conserved suppression of the same mRNAs in liver cells [10]. In another study which focused on F. hepatica EVs, the cargo molecules were shown to be developmentally regulated, and are most likely to assist the parasite's migration through host tissue and to evade attack by host immune cells [14].

EVs as anti-helminth vaccines

There are emerging indications from murine model studies that EVs from parasites have a potential role as vaccine candidates. A schistosome tetraspanin (TSP), Sm-TSP-2, belonging to a family of widely conserved exosome markers (CD63) is abundant in the S. mansoni EV membrane, and is currently in clinical development as a vaccine for S. mansoni infection [18,43,44]. While not yet proven, we hypothesize that the Sm-TSP-2 vaccine exerts its efficacy by generating antibodies that block internalization of schistosome EVs by host target cells and interrupt the parasite's ability to hijack host gene expression for its own survival. In support of this hypothesis, antibodies raised against recombinant Ov-TSP-1 from O. viverrini blocked EV uptake by cholangiocytes and suppressed cholangiocyte proliferation and IL-6 production [19]. TSPs are efficacious vaccine antigens in numerous helminth infections [43,4547] and these data suggest that the mechanism of vaccine efficacy is associated with blockade of parasite EV internalization by host cells in vivo and subsequent interruption of key physiological and pathological processes.

Sotillo et al. demonstrated the existence of numerous vaccine candidates on EVs secreted by S. mansoni [18]. Adult S. mansoni release exosome-like vesicles which are 50–130 nm in size, with over 80 identifiable proteins, 5 of which are TSPs (Figure 1). Some of these EV proteins had been previously identified as schistosomiasis vaccine candidates prior to the discovery of schistosome EVs, and other abundant S. mansoni EV proteins shared sequence identity with vaccine candidate proteins from other parasitic trematodes [18]. If parasite EV internalization by host cells plays a significant role in establishing parasitemia, the disruption of this process via neutralizing antibodies may explain why vaccines directed against EV membrane proteins show efficacy.

Schematic representation of a schistosome EV showing selective proteins of interest that were identified from larval [17] and adult [18] flukes.

Figure 1.
Schematic representation of a schistosome EV showing selective proteins of interest that were identified from larval [17] and adult [18] flukes.

Surface proteins presenting large extracellular loops are hypothesized to be accessible to antibody binding and possible blockade of EV uptake by target host cells.

Figure 1.
Schematic representation of a schistosome EV showing selective proteins of interest that were identified from larval [17] and adult [18] flukes.

Surface proteins presenting large extracellular loops are hypothesized to be accessible to antibody binding and possible blockade of EV uptake by target host cells.

Similarly, studies have specifically addressed the role of targeting entire EVs as an anti-parasite strategy [10,24,48,49]. The abundance of proteasome components in S. japonicum EVs [10] implicated the ubiquitin–proteasome system in a regulatory role during schistosome infection. Because of the vital role of the proteasome in parasite invasion [50], targeting proteasome molecules could be a candidate strategy for anti-schistosome therapy. In another study, the administration of E. caproni EVs to mice primed an immune response and reduced the severity of clinical signs of E. caproni infection [24]. More recently, H. polygyrus-derived EVs formulated with alum induced protective immunity in mice against larval challenge [30]; however, the target antigens of the protective immune response were not identified.

Challenges and perspectives of using EVs as anti-helminth vaccines

Recently, it has become evident that EVs are used by both parasite and host to influence the outcome of an infection. Continued studies in this relatively new arena of science should lead to a greater understanding of the roles of EVs in cell–cell communication at the host–parasite interface. The full extent of the roles of EVs in helminth infections, including the molecular mechanisms underlying EV formation and the packaging of selective cargo, as well as the docking and fusion of EVs with recipient cells, are not fully known. Dissecting these roles will require a better knowledge of the cell types that produce EVs and their content, as well as an understanding of the mechanisms involved in vesicle trafficking, packaging of their cargo, subsequent release and their uptake. Some of the fundamental challenges involved in fully understanding the roles of EVs in host–parasite interactions include discovery of new comprehensive methods for the isolation and characterization of EVs, identifying specific recipient host cells for parasite EVs, and identifying whether specific host cell receptors are required for EV fusion. Moreover, the mechanisms used by EVs in targeting specific cell types, the cellular responses to helminth EVs, and the interaction between EV-mediated responses and other components of the host immune response remain to be fully investigated. To be effective, however, we require the development of new methods of investigation, including the ability to specifically block the uptake of helminth EVs and/or their production, together with approaches for the evaluation of disease outcome. Furthermore, understanding the mechanisms by which helminth EVs communicate with target cells is an essential step for informed antigen discovery as it pertains to EVs. These approaches will help us understand whether helminth-derived EVs benefit the host (for example by suppressing inflammation), or whether they are mostly functioning as virulence factors. It is likely that both of these mechanisms exist (possibly in tandem) and that the ultimate fate is based on a balance of the contribution of EVs toward stimulation of immune responses and immune evasion. Understanding the full impact of helminth EVs on the infective process is required to understand physiological functions and may offer the perspective for applying EVs in vaccination and other therapeutic approaches.

Summary
  • Extracellular vesicles (EVs) are heterogeneous vesicles of membranous origin released by different types of cells.

  • Helminth EVs are internalized by, and manipulate gene expression in, host target cells.

  • There are emerging indications from murine model studies that EVs from parasites have a potential role as vaccine candidates.

Abbreviations

     
  • ABs

    apoptotic bodies

  •  
  • CCA

    cholangiocarcinoma

  •  
  • ES

    excretory/secretory

  •  
  • EVs

    extracellular vesicles

  •  
  • MVs

    microvesicles

  •  
  • TSP

    tetraspanin

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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