New Insight into the Immunity during Echinococcus granulosus Infection

Abstract

Echinococcus granulosus is a causative agent of cystic echinococcosis disease which represents a real challenge of health and economic sectors by threatening human and animal life. In E. granulosus-infected intermediate hosts, the local immune responses represent by balance between T helper-1 (Th1)/Th2 responses and involving of IL-10-secreting CD8+ T cells, as well as induction of antigen presentation and production of antibodies were suggested. Spill out of hydatid cyst fluid from ruptured cysts can induce deadly anaphylactic reactions. Although the host promotes effective immune responses against E. granulosus infection, the parasite can be survived, suggesting exist of mechanisms of immune evasion that help E. granulosus to grow and develop. Several mechanisms of immune evasion have been suggested during E. granulosus infection including; antigenic variation that lead to produce useless antibodies, alteration of Th1/Th2 cytokine profile, anti-apoptotic process, molecular mimicry and interfering with Antigen presentation, as well as fibrosis of hydatid cysts can be occurred in chronic cases. Furthermore, as an efficient drug against E. granulosus infection still not available, immunization of hosts could be necessary. Interestingly, combination of multiple EG95 proteins of oncospheres from the different isomers could possibly maximize the EG95 vaccine efficacy.

1. Introduction

Cystic echinococcosis is a neglected zoonotic disease as recognized by world Health organization. The disease is caused by a parasite tapeworm of the Echinococcus granulosus genus in intermediate hosts such as human and other warm-blooded animals. The parasite is transmitted by fecal–oral route. The parasite exploits bile acid and salt in the host duodenum to hatch its eggs into oncospheres. The oncospheres transfer to internal organs in particular liver through portal vein to establish cystic echinococcosis disease. The disease is described by a slow growing of fluid-filled cysts called hydatid cysts (metacestode stage) and this may be accompanied with serious chronic complications including; destruction of liver tissues and anaphylactic reactions [1].

The early stage of E. granulosus infection is asymptomatic and with progression in growth of hydatid cysts lead to interaction between host immunity and parasite. Hydatid cyst fluid of E. granulosus contains a complex mosaic group of antigens such as Ag5, a 67-kDa glycoprotein, and antigen B (AgB), a 160-kDa lipoprotein, which represent immune modulator antigens responsible for parasite survival in the intermediate hosts. Additionally, protoscolex tegumental surface antigens (PSTSA) and oncosphere surface antigen can also stimulate protective immune responses in immunized hosts [2]. Based on that, this chapter will cover three related sections of host–parasite interactions including; host anti-parasite immune responses, immune evasion mechanisms of parasite and host immunization.

2. Host immune responses in E. granulosus infection

2.1 Innate immune responses

The innate immunity is a part of immune system responsible for generating the early immune responses against non-specific pathogens. Host–parasite interaction complex is the baseline of immune response regulation, where cross-stimulation that mediated by a package of immune cells, receptors and sensors can work together to induce effective immune response against pathogen [3]. The level of Toll-like receptor2 (TLR2) and 4 (TLR4) can increase in the early stage of cystic echinococcosis suggesting a trigger role of them in innate immune responses by activation of myeloid differentiation factor 88 (MyD88) and important transcription factors, such as nuclear factor-κB (NF-κB), interferon regulatory factors) IRFs) and mitogen-activated protein kinase (MAPKs(leading to promote secretion of pro-inflammatory cytokines [4].

Furthermore, during establishment stage of hydatid cysts, induction of the innate immune responses are generated by an increase in the number of monocytes and macrophages with infiltration of neutrophils and macrophages into the site of damaged tissues. Involvement of the innate susceptibility/resistance (s/r) factors in host–parasite interaction include; activation of complement, nonspecific phagocytosis and cytolytic leukocytes. Moreover, feature of leukocytosis as a result of increase in the number of circulating neutrophils, eosinophils, lymphocytes, and macrophages are common indicators during E. granulosus infection [5]. The circulating monocytes can migrate and permanently settle in the particular tissues (i.e., the Kupffer cells of the liver and alveolar macrophages), for detection and elimination of invading pathogens or their products and host-belong injured materials [6].

2.2 Adaptive immune responses

Adaptive immune response is another type of immune responses generated against specific pathogen. There are two types of adaptive immunity including; cellular and humoral immunity. Understanding the local liver immune responses may contribute in designing new therapies and blocking immune evasion mechanisms during establishment stage of parasite infection. The second challenge of host by oncospheral antigen after 21 days from primary infection provided high level of protection. This likely due to the role of antibody-dependent cell-mediated cytotoxicity (ADCC) reactions [7]. In CD4 T cells- deficient mice that immunized with protoscoleces (E4+) antigen, production of CD4+ T cell-independent antibodies was determined in the early stage of infection [8]. The term of humoral immunity refers to antibody-mediated immune response that occurs when antigen presenting cells such as macrophages and DCs engulf antigens and expose them on their surfaces to amplify immune responses against specific pathogen [9]. In early stage of experimental infection of E. granulosus, although the activation of B cells has been detected that resulting in production of IgM and IgG2b, the E. granulosus can still grow and develop into hydatid cysts [10].

3. Mechanisms of immune evasion of E. granulosus

The real challenge for any pathogen is been survival as unwanted visitor in inconvenient environment. Therefore, to avoid effective immune responses of the host, E. granulosus developed several techniques called immune evasion strategies. The two main mechanisms are used by E. granulosus to equivocate the immune system of host; first is a passive escape by avoiding damaging effects of immune system components in the early stage of infection, and the second is by modulating of immune responses [11].

3.1 Protective role of hydatid cyst wall

In intermediate hosts, the first protective barrier following hydatid cyst (metacestode) formation is cyst wall. The hydatid cyst wall consist of outer membrane called laminated layer (antigenic variant layer) and inner layer called germinal layer responsible for production of protoscoleces. Although the hydatid cyst wall consist of different proteins which they can motivate immune responses against parasite, but it also provides physical protection from immune components [12]. Additionally, some of the host molecules that involve in immune response and protective action can be sequestrated in hydatid cyst fluid suggesting that the host molecules may absorbed by laminated germinal layers during host–parasite interaction [13].

3.2 Control of host complement system

Complement is a part of immune system represented by a serial of soluble proteins, membrane expressed receptors and regulators, where blocking of complement can cause perturbation in defense system of host against pathogens [14]. In the early stage of infection, deactivation of host complement can be occurred during several diseases including cystic echinococcosis. This complement deactivation lead to inhibition of acute inflammatory responses which include; boost of vascular permeability, infiltration of leukocytes, and chemotaxis of immune cells into infection site [15].

In alternative way of complement activation, triggering of complement cascade occurred by cleavage C3 into C3b and iC3b which is essential for producing membrane attack complex (MAC) of complement. The complement activation is regulated by host inhibitor factor H (FH), where FH binds with C3b component of complement leading to complement inhibition to protect host cells from self-attack by complement [16]. Preventing accumulation of complement cascade on the pathogen surface and perturbation in phagocytosis has been described by interaction between complement inhibitor FH and FH-binding proteins of many pathogens including E. granulosus, which causes down-regulation of opsonization process and increase phagocytic resistance of pathogens [17]. Furthermore, Sequestration of host complement inhibitor FH by high charged components on the hydatid cyst wall is suggested as a mechanism of complement evasion by E. granulosus [18].

3.3 Interference with infiltration of immune cells

Migration of immune cells from circulatory system into the various organs and tissues during normal and inflammatory conditions are common events in host body. In experimental cystic echinococcosis following by ingestion of mice with ovalbumin (OVA) after 3 months post infection to induce asthma model, the histopathological data indicated the ability of E. granulosus to reduce eosinophil infiltration and mucus secretion [19]. Furthermore, hydatid cyst fluid has ability to modulate infiltrating monocytes from differentiating into dendritic cells [20]. Although the T lymphocytes are the most frequent infiltrating cells in mice organs infected with of E. granulosus, but it is not correlated with the fertility of hydatid cysts [21, 22].

3.4 Manipulation of dendritic cells

Dendritic cells (DCs) are the most efficient antigen presenting cells that uptake the pathogen antigens and exposed them to the Th2 cells to trigger production of antibodies by B lymphocyte cells. It has been found that hydatid cyst fluid can directly modulate predifferentiated DCs and impairing their ability to release interleukin 12 (IL-12), IL-6 and prostaglandin E2 (PGE2) [20]. The effect of purified AgB and sheep hydatid fluid (SHF) of E. granulosus were evaluated to their ability in host monocyte maturation and differentiation into DCs, as well as interleukin secretion. The outcome data indicated the ability of AgB and SHF to impair maturation of monocytes to DCs, alternation of DCs differentiation towards nonprotective Th2 cell responses and inhibition of DCs-mediated proinflamatory responses [23] Interfering of Toll like receptors (TLRs) in DCs-mediated immune responses against E. granulosus was investigated, where the role of TLRs in trigger of effector DCs that promote MyD88-dependent negative signal for Th2 cell development was suggested [24].

3.5 Molecular mimicry

Molecular mimicry refers to the similarity in sequencing between specific pathogen antigens with some host’s self-antigens, where many pathogens have ability to share molecules has sequencing similar to the host antigens. According to the molecular mimicry strategy, the pathogen antigens can be recognized as a “Self” antigen, which helps to protect parasite from host immune responses [25]. The metacestodes of E. granulosus and E. multilocularis avoid the prospective immune responses in intermediate hosts by molecular mimicry, immunomodulation and alternation of leukocyte functions [26].

3.6 Interference with secretion of cytokines and chemokines

The interaction between E. granulosus and host immune system can be occurred including avoid host effective immune responses. E. granulosus has ability to induce T-helper 2 (Th2) responses leading to limitation of anti-parasitic immune responses which involves in metacestode parasite survival, rather than T-helper 1 (Th1) responses, that effectively suppress growth and development of hydatid cysts [10]. Induction of immune suppressor cells such as CD8+ T suppressor cells has been suggested as a mechanism of evasion by E. multilocularis, where CD8+ T suppressor cells detected in spleen of infected mice [27]. Depletion of CD4+ T cells can promote growth and development of protoscoleces to hydatid cysts in E. granulosus infected mice, indicating the role of CD4+ T cells in suppression and elimination of hydatid cysts in experimental model. However, the balance between CD4+ T cells-mediated cellular immune responses and IL-10-produced CD8+ T cells has a critical role in growth and development of hydatid cysts of E. granulosus [28].

In another study, interfering with immune response against protoscoleces in the early stage of infection has been suggested as a mechanism of evasion, where co-culture of protoscoleces with peripheral blood mononuclear cells in vitro indicated an increase in protoscoleces survival. This indicator was supported by using IL-10 and IL-4 antibodies which caused reduction of kill percentage of protoscoleces with inhibition of NO production that released by peripheral blood mononuclear cells. Additionally, IL-10 and IL-4 can reduce Th1 responses causing an increase in the probability of parasite survival [29]. AgB of E. granulosus metacestode stage that contributes in lipid metabolism in the parasite, also has a key role in host immune modulation by inhibiting inflammation response, where down-regulation of IL-1β and TNF-α secreted from LPS-induced macrophages and monocytes in IL-10-dependent manner were determined [30].

Although, the glycan antigens of E. granulosus including; glycoprotein and glycolipid molecules that are expressed in the protoscoleces, hydatid cyst fluid and laminated layer have a high antigenicity features, but parasite can reduce their effects by inhibition the expression of glycan antigens on the surface of laminated layer leading to generate limited immune responses against parasite [31]. Two main antigens including; tegumentary antigen and Antigen B in protoscoleces and metacestode, respectively, has shown a key role in host immune evasion, where tegumentary antigen can inhibit chemotaxis process, induction of IL-4-producing lymphocytes, production of non-complement fixing antibodies like IgG4 and stimulating a nonprotective Th2 cell response [32].

Further in vivo investigation was obtained by Rigano et al. [33] for evaluating the rule of parasite AgB in escape form the early immune responses mediated by specific immunoglobulin E (IgE) and IgG4 antibodies, where the data showed inhibition of chemotaxis process of polymorphonuclear neutrophils (PMN) and high level production of IL-4 and IL-13, but not IL-12 with significant lower concentration of IFN-γ in AgB-induced peripheral blood mononuclear cells.

3.7 Anti-apoptosis strategy

Apoptosis, a first type of programmed cell death is controlled by a serial of proteins and enzymes responsible for cascade of events in unicellular and multicellular organisms during infection and normal conditions. Apoptosis is a route of cell transformation during normal growth, as well as it serves as a protective mechanism by eliminating of damaged cells, infectious agents and malignant cells. There are two ways to promote apoptosis including; extrinsic and intrinsic routes. The protoscoleces of E. granulosus can undergo to both ways of apoptotic destruction that mediated by caspase-3 enzyme (Figure 1) [34].

Figure 1.

Host innate immune response (inflammasome and apoptosis) against E. granulosus—the figure modified from [34]—SEM image of protoscoleces from [35].

Furthermore, it has been shown that some of hydatid cysts can be free of protoscoleces called infertile hydatid cysts and this may be attributed to high expression of apoptotic components. In related to that, high level of DNA fragmentation and caspase-3 were detected in infertile hydrated cysts as compared to fertile cysts suggesting that apoptosis can be involved in hydatid cyst infertility [36]. The study obtained by Amirmajdi et al. [37] suggested that apoptosis can serves as a mechanism of survival by help E. granulosus to overwhelms host immune defends, where they detected that the expression ratio of pro-apoptotic (Bax)/anti-apoptotic markers (Bcl-2) and activity of caspase-3 are higher in lymphocytes treated with hydatid cyst fluid after 6 hrs exposure in comparison with control group. Additionally, two anti-apoptotic proteins of E. granulosus including; apoptotic protein inhibitor (Eg-IAP) and Eg-BIRP can reduce apoptosis in protoscoleces during establishment stage of the E. granulosus in intermediated hosts [38].

3.8 Camouflage strategy

According to the previous literatures, using camouflage strategy by invading pathogens allow them to stay survival in their hosts, where pathogens can exploit host components and secretions such as cells, proteins and enzymes to avoid effect of immune responses against them. The outermost layer around hydatid cysts of E. granulosus is produced by host tissues such as liver and lung consist of host cells which playing an important role in parasite survival [39]. It has been shown that high level of arginase-1 has an immune suppressive effect during infections. In cystic echinococcosis disease, high level of arginase-1but not arginase-2 that is produced by peritoneal cells helps parasite to be survive in intermediate hosts, where protective effects of arginase-1 by impeding the removal process of E. granulosus and cancer cells has been shown in BALB/c mice through down-regulation of T-cell receptor expression [40].

3.9 Antigenic variation

Antigenic variation or antigenic alteration is the ability of pathogens such as parasites, bacteria and viruses to alter the exposed proteins and carbohydrates on their body surfaces regularly to avoid been recognized by host immune cells. This strategy can help pathogen to dispose the generated effective immune response [41]. It has been shown the ability of E. granulosus to avoid host immune responses by modification of its exposed antigens which lead to produce useless antibodies [42].

3.10 Interfering with antigen presentation capability

Antigen presentation refers to expose components of pathogen on the surface of antigen presenting cells including; macrophages, DCs and B cells through the major histocompatibility complex type two (MHC-II) receptors to the specific immune cells. Antigen presentation is a major process of adaptive immune response that includes; recognition, phagocytosis and exposure of pathogen antigens by antigen presenting cells following by recognition of antigens through Th lymphocyte cells [43]. In vitro study suggested the role of hydatid cyst fluid of E. granulosus in modulation of immune responses by interfering with the function of antigen presenting cells to ensure its continued survival, where, impairment of antigen presentation process by down-regulation of MHC-2 has been determined in Balb/c mice experimentally infected with E. granulosus [44]. Excretory–secretory products (ES) and adult worm antigens of E. granulosus were caused impairing the development of Th1 cells and inhibition of DCs function which may reduce antigen presentation process and cytokine secretion from DCs [45].

4. Vaccination against E. granulosus parasites

At the early stage of E. granulosus life cycle, the oncosphere components can be a potent source of host protective antigens in sheep. The new EG95 vaccine was originally described by Lightowlers et al. [46] which consists of a single recombinant antigen of E. granulosus oncosphere and the Quil A adjuvant has ability to reduce the burden of hydatid cysts and providing protective immune responses by inducing complement-fixing antibodies. Vaccination trails using EG95 were applied in sheep, goats and cattle in New Zealand, Australia, Argentina, Chile and China [47]. Interestingly, combination of multiple EG95 proteins of oncospheres from the different isomers could possibly maximize the EG95 vaccine efficacy [48]. The protection program of sheep from E. granulosus infection using EG95 vaccine has been applied as follow; the first dose been offered at 2 months of mice age, following by a booster 1 month later, and yearly vaccination. In experimental study to evaluate the protective immune efficacy of recombinant E. granulosus (Chinese strain) glutathione S-transferase (rEgGST) as a new vaccine against protoscoleces development in mice indicated potential reduction of hydatid cyst formation, as well as elevated levels of IgG1, IgG2a, IgG3, IL-2, IL-4, IL-10 and IFN-γ which reflecting an increase in the activity of Th cells [49]. Moreover, antigen B in hydatid cyst fluid is another candidate vaccine that used in vaccination of intermediated hosts against E. granulosus infection. DNA vaccines encoding 8-kDa subunit of E. granulosus antigen B (HydI) with murine interleukin 12 (IL-12) as a genetic adjuvant was used in BALB/c mice vaccination. The HydI/MIL12-vaccinated group showed high significant levels of IFN-γ and IgG2a antibodies comparing with only HydI-vaccinated and control groups [50].

Another candidate vaccine that has been used to immunize intermediate hosts is protoscolex tegumental surface antigens (PSTSA). The data from PSTSA-immunized sheep showed an increase in the titer of antibodies after single and double immunization [51]. Furthermore, three recombinant proteins of egM gene family of E. granulosus named egM4, egM9 and egM123 were used to immunize dogs against E. granulosus infection. The egM gene subcloned in E. coli bacteria to express glutathione S-transferase (GST) fusion proteins, where 3 doses of 80 mg/protein/dog provided 97–100% of protection in terms of elimination of worm and reduction of egg production. This can breakdown the parasite life cycle in the dogs which is important for preventing the parasite transmission in intermediated hosts [52].

More recently, it has been shown that multi-epitope combination vaccine of E. granulosus tegumental protein (EgTeg) that consist of two CD4 T-cell epitopes, three CD8 T-cell epitopes and four B-cell epitopes has high level of antigenicity and immunogenicity in both in vivo and in vitro studies. In vivo and in vitro experiments of effectiveness evaluation of EgTeg vaccine indicated significant increase in the level of IFN-gamma, perforin and granzyme-B that produced by activated CD4+ T cells and CD8+ T cells, respectively. Additionally, the titer of antibodies was higher in immunized group in comparison with control group [53].

5. Conclusions

Overall, the outcome data from previous literatures about E. granulosus infection discovered the ability of intermediate hosts to generate effective immune responses against parasite, but growth and development of E. granulosus in these hosts with existence effective immune responses suggesting present of immune evasion strategies that enhance the parasite survival and causes successful infection. The suggested mechanisms of immune evasion of E. granulosus in intermediate hosts include; antigenic variation, interfering with Th1/Th2 cytokine profile, anti-apoptotic process, molecular mimicry and perturbation of antigen presentation. The vaccination against E. granulosus can reduce the destructive effects of parasite in intermediate and definitive hosts. The most common candidate vaccines that can provide protective immune responses against E. granulosus infection are EG95 and antigen B with Freund’s and Quil A adjuvants.

Acknowledgments

I would like to thank the Ministry of higher Education and Scientific Research of Iraq, University of Mosul, College of Education for Pure Science, Department of Biology for their unlimited support and encourage their academic staff to participate in international Publishing and collaboration with international organizations.