Schistosomiasis: Discovery of New Molecules for Disease Treatment and Vaccine Development

Abstract

The parasite blood flukes belonging to the genus Schistosoma cause schistosomiasis. Among the Schistosoma species that infect humans, three stand out: Schistosoma japonicum (S. japonicum), which occurs in Asia, mainly in China and the Philippines; Schistosoma haematobium (S. haematobium), which occurs in Africa; and Schistosoma mansoni (S. mansoni), which occurs in Africa and South America and the center of Venezuela (Brazil). Research has shown that these species comprise strains that are resistant to Praziquantel (PZQ), the only drug of choice to fight the disease. Moreover, patients can be reinfected even after being treated with PZQ , and this drug does not act against young forms of the parasite. Therefore, several research groups have focused their studies on new molecules for disease treatment and vaccine development. This chapter will focus on (i) parasite resistance to PZQ , (ii) molecules that are currently being developed and tested as possible drugs against schistosomiasis, and (iii) candidates for vaccine development with a primary focus on clinical trials.

1. Introduction

From the public health and socioeconomic standpoints, schistosomiasis is a parasitic disease with significant prevalence in most developing countries, and it is the second-largest neglected disease in the world [1, 2, 3]. Schistosomiasis is caused by digenean trematodes belonging to the genus Schistosoma. S. mansoni, S. japonicum, S. haematobium, Schistosoma mekongi (S. mekongi), and Schistosoma intercalatum (S. intercalatum) are the main species underlying the disease in humans. The three former species are the main causative agents of schistosomiasis [4].

The number of people living in risk areas, which cover 78 countries in tropical and subtropical regions, is greater than 700 million [5, 6]. Transmission is high or moderate in 52 of these countries (World Health Organization, 2021). More specifically, S. mansoni occurs in Africa and Brazil; S. haematobium occurs in Africa; and S. japonicum occurs in China, the Philippines, and some places in Indonesia. According to the WHO, there were about 10.1 million deaths due to schistosomiasis in the world in 2016 [3]. However, controlling schistosomiasis depends on the diagnosis, sanitation, and disease treatment with praziquantel (PZQ), a drug recommended by the WHO and which has been used for over 30 years [3, 7]. In 2017, 46.3% of the population was treated with PZQ; 70.8% of the treated population corresponded to school-aged children [8, 9].

S. mansoni, S. japonicum, and S. haematobium have an intricate life cycle that involves different parasite forms (miracidia, sporocysts, cercariae, schistosomula, adult worms, and eggs), in which structural and metabolic changes occur. Their life cycle requires the presence of an invertebrate host and a vertebrate host (Figure 1). Snails belonging to the genera Biomphalaria, Bulinos, and Oncomelania (intermediate hosts) release cercariae into the water, which infects the vertebrate host through the skin. In the vertebrate host, cercariae lose their tail and develop into schistosomula, which reach the pulmonary artery. In the lung, schistosomula migrate to the heart through the venous circulation. Next, schistosomula migrate to the hepatic portal system, where they develop into adult worms, become sexually mature, and pair. At this time, adult S. japonicum and S. mansoni worms move to the intestine mesenteric veins and are lodged in different locations [10]—S. japonicum worms remain in the upper part of the vein, whereas S. mansoni worms remain in the lower part and close to the large intestine. Thereafter, eggs are laid, and some of them are released in the feces. In contrast, S. haematobium worms do not lodge in the intestine, but they inhabit the bladder pelvic venous plexus and the vesicular, where they lay eggs and cause urogenital schistosomiasis. Sometimes, S. haematobium worms can be found in the rectal venules. Part of the S. haematobium eggs is retained, whilst the other part is eliminated in the urine [11].

Figure 1.

S. mansoni, S. japonicum, and S. haematobium life cycle—(1) S. mansoni and S. japonicum worms move to the mesenteric veins of the intestine and are lodged in different locations. Eggs are laid, and part of the eggs is released in the feces. (2) Miracidia are released from eggs into the water and infect snails (intermediate hosts). (3) Within the snail, miracidia progress through two generations of sporocysts, to become cercariae. (3) Cercariae are released from the snail and penetrate the skin of the definitive vertebrate host, releasing enzymes from glands. During penetration, cercariae lose their forked tail and become schistosomula. (4) Schistosomula migrate to the dermis veins and, upon encountering the pulmonary artery, are carried to the lungs. From the lungs, they reach the venous circulation and travel to the hepatic portal system. (5) In the hepatic portal system, schistosomula develop into adult worms, where parasite sexual maturation and pairing take place. At this time, S. haematobium worms are not stored in the intestine, but they inhabit the bladder pelvic venous plexus and the vesicular. Sometimes, they can be found in the rectal venules. S. haematobium causes urogenital schistosomiasis.

Schistosomiasis is characterized by two phases: acute and chronic. Symptoms of acute illness include myalgia, abdominal pain, diarrhea, fatigue, fever, and, in the case of urogenital schistosomiasis, hematuria. Diarrhea occurs in patients with a greater parasite load; abdominal pain is diffuse. In chronic intestinal schistosomiasis, symptoms are more severe. Over time, patients have diarrhea with the presence of blood in stool, anemia, and retention of eggs in the anal region, not to mention hepatosplenomegaly due to egg deposition in the liver. Hepatosplenomegaly causes granuloma (Figure 2) and occurs in around 10% of patients, who present periportal fibrosis with portal hypertension, ascites, and gastrointestinal varices with bleeding [12, 13]. As for urogenital schistosomiasis, it affects the urogenital system so severely that it causes fibrosis in the bladder and ureter, calcification in the urinary tract, and kidney dysfunction. The greatest concern about this urogenital disease is that it causes bladder cancer and sterility, and, in the chronic phase, patients have bladder injury [2].

Figure 2.

Slides of (a) mouse liver infected with S. mansoni showing hepatic granuloma and fibrosis caused by parasite eggs; purple-colored cells are eosinophils, due to inflammation in the liver (b) mouse liver not infected with S. mansoni.

The differences in schistosomiasis pathology are due to parameters such as oviposition, granuloma size, and modeling of interleukins, which depend on the parasite load, host’s immunological profile (that is, the host’s ability to respond to the parasite, whether the parasite is in the form of schistosomula, adult worms, or eggs), and parasite virulence and infectivity [14]. Therefore, the parasite and host interact in a co-evolutionary and complex way (interplay) that interferes with disease transmission potential and pathology [2].

Worm maturation requires that host-derived signals be translated, to generate adaptive and innate immune responses. Much research is still needed to unravel the interrelationship of Schistosoma with the immune system during worm development, maturation, pairing, and oviposition [15]. After worm couples are paired and oviposition starts, the host responds strongly to the eggs. Immediately after deposition, the eggs are surrounded by cells and proteins from the host’s homeostatic system, including plasma proteins called egg-laying factors, von Willebrand factors, fibrin, and fibrinogen [15].

When it comes to schistosomiasis, egg antigens are the major problem: they are antigenic structures that secrete various toxic substances, the main one being SEA (Soluble Eggs Antigens). These toxic substances elicit the complex and multifactorial response in the mammalian host’s innate immune system [15]. The acute condition of the disease is characterized by the lesion around the eggs, with the release of interferon-ɣ and IL-10 by macrophages and IL-12 by dendritic cells [12]. Later, another eggshell protein, ɷ-1, is internalized in dendritic cells, directing the Th2 response and lowering IL-12 secretion [16]. However, this does not occur in infections caused by S. haematobium (urogenital schistosomiasis) because the female worm does not encode this eggshell protein [2].

Thus, these parasites activate the immune system and form the highly organized granuloma that is wrapped by the Th2 immune cells, namely macrophages, eosinophils, and cells that secrete cytokines of numerous types, including IL-2, IL-4, IL-13, and IL-5, which are surrounded by stromal cells and fibroblasts. In the case of S. haematobium, the main interleukin is IL-5, and the fibrous granuloma has a lot of collagen and eosinophils. Urinary eosinophilia occurs because these cells release toxic substances such as eosinophilic cationic protein, neurotoxin, and granulomatous protein [2, 12].

During infection with S. mansoni or S. haematobium, IL-13 production by the vertebrate host makes it susceptible to infection—antigens released by the eggs promote a polarized Th2-type response, with fibrosis developing around the eggs due to the release of transforming growth factor (TGF)-β [17].

In the infection period, there is a balance between the Th2 and Th1 responses. The Th2 anti-inflammatory effects control the immunopathology caused by the Th1 response [18]. In S. mansoni-infected mice, the Th2 response is epigenetically controlled and modulates dendritic cells and macrophages, as well as Th2 cells [2]. Recently, studies have been carried out to understand the molecular relationship between parasites and hosts. Genome integrity is essential for host cells, organisms, and species survival. Hence, errors in genome checkpoints trigger cellular apoptosis to eliminate the altered cell. However, pathogens can alter these pathways by manipulating both chromatin repair and cell signaling pathways. For this to happen, pathogens produce genotoxins and oncoproteins that modify the host’s epigenetic programs and influence metabolism. For this reason, they are called epigenators [19].

The Th2 response is crucial for granuloma maintenance and host survival. Proteins such as Cyclophilin A and lysophosphatidylserine (LPS), excreted from worms, can modulate the dendritic cell function, causing IL-10 to expand and activating regulatory T cells. The role of small fatty acid chains (SFACs) excreted by worms in regulating immune response is not yet known, but LPS and SCFA can modify the TLR2 signaling pathway in dendritic cells, altering maturation and regulatory T cell activation [15]. In children infected with S. haematobium in Uganda, regulatory B cells have been shown to induce T cells to secrete IL-10; however, in a study with Kenyan children, TNF-alpha has been correlated with inflammation and low IL-10 levels [2].

Several studies have reported that the host’s immune response plays a role in PZQ effectiveness. Studies using 0-, 1-, and 3-day S. japonicum schistosomula have shown that the percentage of surface-exposed antigens is 86.4%, 55.2%, and 3.9%, respectively. Resistance to PZQ (PZQ-R) in these stages has been related to the antigenic composition on the tegument surface, and in vitro research carried out with young S. mansoni and S. japonicum larvae (3, 7 and 14 days of maturity) has demonstrated that they are resistant to PZQ [20]. Currently, PZQ is the only drug of choice against schistosomiasis. Studies have indicated that some S. mansoni and S. japonicum strains are resistant to PZQ even in the adult worm stages, as seen from the many cases of reinfected patients following multiple PZQ administrations. This situation calls for the study of new therapies, such as drugs and vaccines [21].

2. Praziquantel and schistosomiasis

The WHO has planned strategies to control schistosomiasis through PZQ administration in endemic areas where the disease is highly prevalent, mainly in Africa, in regions such as the Nile Delta, Côte d’Ivoire, Mayuge District, and Uganda. These strategies have shown that the prevalence of morbidity due to S. mansoni and S. hematobium may originate from PZQ-R and parasite-host-drug interactions, especially in areas with longer history of PZQ treatment [2, 9].

Nevertheless, the greatest PZQ-R has been detected in parasite strains maintained in the laboratory. After the passage of S. mansoni in seven mouse generations treated with PZQ subdoses, resistance emerged. In infections with a single S. mansoni sex, resistance was greater than in infections with two sexes of the parasite. This fact was later analyzed in a group of human volunteers, infected with cercariae of a single sex and treated with PZQ at the usual dose of 40 mg/kg for 12 weeks. After treatment, parasites were detected in 43% of the volunteers [9, 22]. Despite the greater PZQ-R found in the laboratory, the fact that a single drug exists for treating a certain disease must be considered seriously because variations arise from both resistant strains of disease-causing microorganisms and resistant tumorigenic cells.

PZQ has a series of pharmacological and pharmaceutical limitations that are often disregarded because the effectiveness of oral, single-dose treatment has cure rates between 50 and 90%, whether for single- or mixed-species infections [13, 23, 24]. Regarding pharmacology, PZQ exhibits suboptimal pharmacokinetics with high intra- and inter-individual variability and extensive first-pass hepatic metabolism, which results in low oral bioavailability [25]. The PZQ mechanism of action is still poorly understood, but it seems to affect Ca²⁺ absorption through calcium channel opening, which interferes with muscle contraction and leads to antigens being present in the tegument [26]. Furthermore, PZQ is only effective against adult parasites; that is, it has no antiparasitic action against schistosomula. Thus, even during treatment, immature parasites develop into mature adult worms and continue to generate morbidity in reinfected patients [27, 28]. Schistosomiasis treatment with PZQ alone increases the possibility of resistance and hence treatment failure, especially in areas where infection occurs massively [13, 29]. Academically, resistance to any drug is defined as hereditary sensitivity acquired by a living organism; that is, it is transmitted between generations [9].

One of the consequences of PZQ-R is the increasing reproduction rate of parasites that survive treatment with PZQ. One of the possible explanations for this fact is that parasites have drug-resistant alleles, which are passed from generation to generation and are related to virulence [2].

Moreover, intergeneric, interspecies, and intraspecies interactions may occur because hosts are usually infected with more than one Schistosoma species; for example, S. mansoni and S. haematobium. In the case of mixed infections, patient’s morbidity due to hepatomegaly is lower because S. haematobium males recruit S. mansoni females from the portal vein to the vesicle plexus, where interspecies crossing takes place, with a greater number of eggs being laid in the urogenital tract [2]. Research has shown that patients infected with S. mansoni only have comorbidity in the bladder, which once again demonstrates an interaction between species and genetic variability and shows that the cure rate is lower in these patients than in patients infected with a single parasite species. This situation culminates in interspecies gene hybridization, with new genes being introduced. Consequently, new strains with greater transmission potential arise, making the disease difficult to control. This fact has been confirmed by genotypic analysis of S. mansoni schistosomula collected from patients in Senegal, who presented allelic variation at the locus L46951, where genes that transcribe mRNAs encoding proteins linked to egg production and fecundity are located. This allelic variation was increased in this lineage [2, 30].

Even the same parasite species have distinct lineages presenting greater or lesser infectivity and transmission in different endemic regions of Africa. This is due to genetic mutations caused by several factors, including environmental changes in both geographic regions and PZQ-R [30], which promote epigenetic changes in the parasite. Epigenetics is related to changes in gene expression while the DNA sequence remains unaltered. Epigenetics is one of the main regulatory systems of post-translational modifications (PTMs) in histones, which are proteins that form a unit called nucleosome [31].

In eukaryotes, chromatin is made up of genomic DNA (gDNA), RNA, and proteins. The main proteins are called histones, which are divided into isoforms. The main isoforms are H2A, H2B, H3, and H4, which form octamers around gDNA, consisting of two dimers, H3-H4 and H2A-H2B. At physiological pH, histones bear a positive charge and interact with the negative charge on gDNA, thereby constituting the basic unit called nucleosome, which closes the DNA structure. The nucleosome structure allows the terminal carbon and nitrogen tails (C-t and N-t, respectively) of these proteins to undergo PTMs [31]. The PTMs of these proteins include lysine acetylation and methylation (K), serine/threonine phosphorylation (Ser/Thr), and ubiquitination, among others. These PTMs are covalent modifications, and their set is called the “histone code” [32], with more than one PTM occurring in a histone molecule.

3. Parasite epigenetics interferes with the host’s immune response

Metabolic alterations and environmental changes (nutritional deprivation, temperature, and chemical agents) generate a stressful environment for living organisms. The stress mechanism is activated and causes activation of other regulatory mechanisms, including gene transcription, which generates an “epigenetic memory” in response to stress. This mechanism has been detected in S. cerevisiae and C. elegans [31, 32, 33, 34].

Studies have been carried out to understand how the molecular relationship between parasites and hosts works. Genome integrity is essential for host cells, organisms, and species survival. Thus, errors in genome checkpoints trigger cellular apoptosis, to eliminate the altered cell. However, pathogens can alter these pathways by manipulating both chromatin repair and cell signaling pathways. For this to happen, pathogens produce genotoxins and oncoproteins that modify the host’s epigenetic programs; that is, DNA expression, which consequently influences metabolism by altering the proteins that will be expressed. For this reason, pathogens are called epigenators. Some intracellular parasites such as Theileria parva and Theileria annulata encode proteins that manipulate the host’s intracellular pathways. For example, toxoplasma secretes the GRA16 protein, which is exported to the nucleus and interferes with the p53 protein pathway “checkpoint.” Studies have reported that Leishmania prevents or modulates the host’s immune response because it can methylate the macrophage DNA cytosine residue and silence immune response genes like calcium, JAK/STAT, MAPK, mTOR, and Notch [19, 35].

Some studies are being carried out on S. mansoni. As this parasite changes stages, metabolic and molecular alterations occur, naturally generating stress. In addition to environmental changes, there are nutritional differences between one environment and the other because the different S. mansoni forms pass through two hosts, a mammal and a snail of the genus Biomphalaria, not to mention water as an infection route. The parasite is suitable for diverse environments due to its hereditary phenotypic capacity [36]. Epigenetic regulation, which is linked to hereditary phenotypic capacity, occurs through “readers,” “erasers,” and “writers,” which respectively correspond to proteins with certain domains that recognize histone tagging; enzymes that remove histone labeling, such as histone deacetylases (HDACs); and histone demethylases, which promote both histone and gDNA labeling [37].

The S. mansoni genome comprises 363 megabases; 11,000 genes of these megabases encode proteins. Analysis of the parasite genome predicts methylation of 25 “readers,” 13 “erasers,” and 26 “writers.” All this machinery is regulated during parasite development, which makes it a target for the discovery of new drugs [36]. When the parasite changes stages, there are molecular changes and alterations in the signaling pathways, which are accompanied by “histone code” remodeling. A study using chromatin immunoprecipitation/sequencing (CHIP-Seq) and RNAseq from the cercaria to the adult worm stages has shown changes in H3K4 and H3K27 methylation. Besides that, when cercariae change to schistosomula, H3K27me3/H3K9me3 is demethylated, consequently activating gene replication and increasing H3K9 methylation and acetylation above and below the transcription site. H3K4me3 is a constant mark during the parasite life cycle; i.e., it does not vary. Bivalent H3K4 and H3K27 trimethylation at transcription initiation sites is a hallmark of cercariae, where these sites exist at a higher level. However, this feature is also present in primary sporocysts and adult worms and increases from miracidium to primary sporocysts, with H3K27 being a necessary hallmark in life cycle progression. H4K20me1 is also a strong marker in cercariae [38, 39].

Given that the parasite can act as an epigenator, to modify the host’s immune response to the disease, it is extremely important to know how chromatin epigenetic regulation occurs upon changes in temperature, pH, osmolarity, and physical and biochemical signals in Schistosoma species and upon alterations in the parasite/host relationship. The first question is about how the parasite manages to survive an average of 5 years in its definitive host; there are cases in which it can interact for up to 10 years [13]. The main barrier to parasite action is the host’s immune response to schistosomula, eggs, and adult worms.

With respect to S. mansoni schistosomula and adult worms, the parasite-host interface is still poorly studied. A large immunological barrier is known to exist when schistosomula infect the mammalian host, as previously mentioned. From the moment schistosomula leave the skin until they reach the host’s venous system and migrate to the lung, 3 days elapse. Eight days after infection, schistosomula reach the portal vein system, with few morphological changes. In this life cycle stage, the parasite tegument surface is modified through acquisition of molecules from the host. This stimulates receptors on inner membranes, aiding parasite development and escape from the host’s immune response. Upon reaching the portal system, parasite cell proliferation and cell biomass increase significantly, promoting morphological alterations [40].

New drugs or vaccines against schistosomiasis must be discovered—Schistosoma is a complex organism, which makes the discovery of new substances that can halt these parasites difficult. This is one of the reasons why PZQ has been used to treat schistosomiasis for 40 years. To overcome this issue, several investigations have been carried out to study PZQ-resistant Schistosoma species strains [9, 26, 41].

4. Developing vaccines and new drugs to treat schistosomiasis

Research aimed at discovering a vaccine against schistosomiasis involves selecting possible parasite antigens that are expressed in the intra-mammalian stages. These antigens activate the host’s immune system, forming memory cells. Reaction of immunoglobulins IgA, IgG, and IgM excreted by immune system cells is analyzed by the Enzyme-linked Immunosorbent Assay (ELISA) reaction with antigens from Schistosoma species adult worms, schistosomula, and eggs [42]. In immunology, ELISA is widely used for quantifying and detecting antigens and antibodies. The assay is performed in a high adhesion 96-well plate, and the antibody that binds to a specific antigen is added to the plate. Then, a chemiluminescent substrate is added, and the amount of antibody produced as a response to the studied antigen is detected through the color intensity of the sample in relation to a standard curve (Alice V. Lin).

As for the discovery of new drugs, it involves substances that act against tegumentary proteins or proteins that are linked to parasite metabolism. The initial tests on the investigated substances are called in vitro tests: these tests are carried out on parasites in culture medium containing the evaluated substance. Biochemical tests (Figure 3) are conducted to investigate the possible mechanism of action of the evaluated substance. Examples of such tests include proteins involved in autophagy and apoptosis as well as scanning and transmission electron microscopy for analysis of cellular structures and tegumentary injury and verification of changes in parasite biology that lead to parasite death [43, 44, 45, 46, 47, 48, 49, 50, 51].

Figure 3.

Scheme for discovering new drugs to treat schistosomiasis. The discovery of a new medication for schistosomiasis takes a long time, 10 years or more. The steps involve in vitro research when cultures of the parasites are treated with the study drug. If the parasite dies or if changes in the tegument and internal structures of the parasite occur, as detected by the methodologies mentioned in the diagram, tests on mammalian cells are performed to evaluate the toxicity of the substance. After this evaluation, in vivo tests are carried out. When the parasite load decreases by more than 80% and toxicity to liver cells is low, the substance is directed to clinical trials to determine whether there is a schistosomicidal effect, and if there are side effects in humans (Flavio C. Lombardo).

After the first in vitro phase for investigation of target proteins in the parasite (for vaccine development) or new molecules (for drug development) is performed, pre-clinical tests are accomplished in animals (mice, rabbits, or baboons). Later, clinical trials (divided into phases I, II, III, and IV) in humans are carried out. Phase I aims to generate safety and efficacy data for both vaccines and new drugs; phase II aims to establish vaccine immunogenicity; phase III demonstrates the effectiveness of the vaccine or drug and promotes their approval by regulatory bodies, such as the FDA; and phase IV is when the vaccine is made available to the population.

“In 2016, Science ranked the schistosomiasis vaccine as one of the 10 vaccines that urgently need to be developed to make a significant impact on reducing the global burden of diseases.” In 2013, a meeting with 70 experts from the Bill and Melinda Gates Foundation considered that an effective vaccine against schistosomiasis should reduce the parasite load and pathology caused by eggs by 75%; in other words, granulomas in the liver and urogenital tract should be reduced. In addition, an effective vaccine should elicit adaptive immune response and be effective against the three main Schistosoma species (S. mansoni, S. haematobium, and S. japonicum) or at least against the two former species, which cohabit the same regions of disease infestation [52].

4.1 Vaccines

New vaccines are developed by using recombinant proteins, and their effectiveness is tested by verifying whether they generate an immune response when applied to mammals. For the schistosomiasis vaccine, the recombinant protein system has proteins that are part of the surface of the parasite and that are secreted by it. These proteins have previously been selected by proteomics and transcriptome and analyzed in vitro. They are expressed in HEK-293 cells in in vitro culture. After evaluation of the humoral immune response by ELISA, preclinical and clinical assays are performed (Figure 4).

Figure 4.

Scheme for discovering schistosomiasis vaccine. Vaccine development involves several steps including in vitro, and clinical screening. The flowchart of how this process is carried out is outlined in the figure.

Developing a vaccine, which does not need to be 100% effective against schistosomiasis, will ensure that patients are not reinfected with the parasite, especially in endemic areas where morbidity is high. If this goal is reached, disease control is achieved [11]. However, discovering a vaccine is difficult because the parasite can escape the host’s immune system [53]. This escape can occur through epigenetic changes in the parasite genome [40, 54, 55]. Additionally, the parasite can act as an epigenitor, interfering with the expression of proteins linked to the vertebrate host’s immune system through genotoxins, also called bioactive molecules. Genotoxins can be enzymes or inhibitors that modify histone PTM, causing a balance between resisting reinfection and controlling the immune response (e.g., in relation to eggs retained in the liver) after treatment with PZQ [13].

The possibility that Schistosoma ssp act as an epigenitor comes from research showing that Schistosoma-specific proteins play an essential role in their hosts’ biological processes. In this context, 100 biomolecules of great importance for helminth survival have been researched and identified as vaccine targets [56, 57]. No vaccine against schistosomiasis has been approved, but these bioactive molecules have been identified by new techniques, including genomics, transcriptomics, microarrays, proteomics, and immunological profiling [57]. Immune response against Schistosoma species is multifactorial and complicated because it involves IgE and several cytokines of the immune system [13].

Vaccines that are being tested in humans include Sh28GST (Bilvax-Phase III), which offers 30–60% protection; Smp80 (phase I), which offers 30–70% protection; Smp14 (phase I), which offers 50–68% protection; and SmTPS1 and Sm-TSP-2 (phase I), which offer 65–69% protection [56, 57].

Techniques for producing vaccines with recombinant proteins are described below for further understanding of their tests. As an example, we will mention a vaccine that is in the test phase and which is based on the parasite protein p80, called calpain. The parasite protein p80 is present in the inner membrane of the tegument of adult worms and other S. mansoni, S. hamatobium, and S. japonicum stages. This protein accelerates the synthesis of proteins of signaling pathways, which in turn accelerates tegumentar membrane surface renewal and evasion of the host’s immune response [57].

Preclinical trials with many types of the Sm-p80-based vaccine, tested in mice infected with S. mansoni, have shown protection and reduced parasite load. The Deoxyribonucleic Acid-based vaccine (p80-pcDNA3 DNA) reduced oviposition by 84% and promoted humoral immune response (IgG2a and IgG2b Ab) in animals and mice vaccinated with either purified Sm-p80 or p80-pcDNA3 DNA; mononuclear macrophages secreted high IFN-γ and IL-2 levels (Th1 response). Then, the Sm-p80 DNA-based vaccine was tested in baboons, which had parasite-specific IgG activation. Protection was detected between 5 and 8 years after vaccination [42, 57, 58]. For this trial, recombinant Sm-p80 (S. mansoni) (Sm-p80 + GLA-SE) was encoded from the complete Sm-p80 sequence in pCold II vector for transformation into E. coli BL21 (DE3). Protein expression was confirmed by SDS-PAGE and immunoblotting. Initially, three different adjuvants were tested for vaccine formulation; the best adjuvant was the stable oil-in-water emulsion (GLA-SE), a TLR4 glucopyranosyl lipid A agonist. To verify the immunization strategy, 30 baboons were used: 15 controls were intramuscularly injected with 5 μg of GLA-SE, while 15 baboons were intramuscularly immunized with 25 μg of rSm-p80 in 5 μg of GLA-SE. After vaccination, the baboons were infected with cercariae. Baboons were used in this study because their immune system resembles the human immune system. The baboons were immunized with a higher dose of vaccine (250 μg), in a total of three doses (0, 4, and 8 weeks) from primary vaccination to the third boost, and they had their parasitological parameters monitored to verify disease progression. Then, each baboon was infected with 1000 S. mansoni cercariae 12 weeks post-vaccination [59].

After vaccination and infection with cercariae, the livers of the mice and baboons were removed, and transcriptome was performed by using RNAseq. This technique is used for sequencing and expression analysis of the mRNA set. In the case described here, this technique was used to analyze which host genes linked to the immune system would be active during the development of protection due to vaccination. RNAseq analysis of mouse liver showed high expression of genes linked to coding of innate immune response proteins; inflammatory cytokines such as IL-1, IL-15, IL-18, and the TNF superfamily; interferon; and complement factors. In addition, high IL-27 levels, related to IL-12 involved in CD4+ T cell proliferation and genes related to adaptive immune response, were detected. In baboon liver, expression of mRNA related to the Th1 immune response was identified. This was associated with differentiation and development of T cells, which are memory cells of paramount importance for immune response in the presence of parasite. CD8 and humoral responses with B cell differentiation were also detected [59].

When applied to mice, the vaccine mentioned above provided promising results with high IgM, IgA, and IgG levels and protection for up to 60 weeks after it was administered. At the end of the experiments, the recombinant vaccine showed between 30% and 70% protection. The next test, carried out on baboons, provided around 50% protection and 100% reduction in eggs in the liver and intestine, which should prevent disease transmission [56, 59]. The same vaccine was also administered to hamsters and baboons infected with S. haematobium, resulting in lower oviposition by female parasites and, consequently, reduced number of eggs in the tissues and a balance between the Th1 and Th2 responses [57].

For S. haematobium, a recombinant vaccine has been monovalently produced from the enzyme glutathione (S)-transferase (Sh28GST). The protein 28GST has been identified in the three main Schistosoma species, in the tegument, parenchyma, and genital organs of adult parasites and in schistosomula. As a vaccine, Sm28GST was also expressed as a recombinant protein in E. coli and purified (rSm28GST/BCG). In pre-clinical studies carried out in mice, the immunological response elicited by the vaccine encompassed the immunoglobulins IgG1/IgG2a and IgG2b. Sh28GS (S. haematobium) promoted cross-protection not only against S. haematobium but also against other Schistosoma species. Immunizations with rSh28GST in adult males (phase Ia) showed that the vaccine had no systemic toxicity or cross-reaction with human GST. The volunteers showed high levels of specific neutralizing antibodies against the parasite, especially after the third dose. In the screening phase Ib, performed with children, satisfactory results were obtained: IgG1, IgG2, IgG3, and Th2 cytokines (IL-5, IL-10, and IL-13) were produced [56].

Tetrapanin proteins (TSP) are transmembrane proteins of the tegument detected at all stages of the parasite life cycle. TSP is exposed to the host’s immune system. The main TSP is Sm-TSP-2, which has been used for testing vaccine development. In animal models, the recombinant Sm-TSP-2 vaccine protected the animals and decreased the parasite load and eggs in the liver. The neutralization response of the animals to the vaccine involved IgG1, IgG2 Abs., and IgG3. Later, a study was carried out with the recombinant vaccine, Recombinant Sm-TSP-2 vaccine formulated on aluminum hydroxide adjuvant (Sm-TSP-2/Al), in infected volunteers from non-endemic areas. The volunteers responded with increased IgG production. Projects encouraged by the Sabin Institute and in support of schistosomiasis vaccines have been launched, and a new recombinant vaccine, called Sm-TSP-2/Alhydrogel, is in phase 2 clinical trials in Brazil and the USA [57].

Another vaccine, still in pre-clinical testing for S. mansoni and S. japonicum, is based on a protein called Paramyosin (Pmy, 97 kDa). Pmy, which is present in the tegument and muscle of adult worms, tegument of schistosomula, and glands of cercariae, promotes the release of enzymes that help to penetrate the vertebrate host’s skin. Pmy is important for immune response evasion by the parasites given that it inhibits proteins C1 and C9 and binds to polymeric collagen and IgG, thereby inhibiting innate and acquired immune response activation through microorganism opsonization. Therefore, Pmy is linked to inhibition of host’s infection and reinfection. To test the vaccine based on Pmy, Swiss mice were immunized with three doses of purified Sm97. After immunization, the sera of these animals showed high levels of humoral immune response, linked to specific anti-Sm97 IgG1 and IgG2. Moreover, the parasite load, the number of eggs in the liver, and intestine of the animals decreased. However, this vaccine did not inhibit cercaria penetration in mice infected with S. mansoni, but it inhibited S. japonicum cercaria penetration through the host’s skin by 62–86%. These data led the vaccine to clinical screening phase I, carried out in 616 participants, to verify protection against S. japonicum reinfection. The participants’ protection profiles involved IgA, IgE, and IgG activation. Studies in the clinical phase are currently underway [57, 60].

The 14-kDa protein FABP is located in the basal part of the tegument and intestinal epithelium of all the stages of the parasite life cycle, including eggs. Because Schistosoma species do not have the machinery to produce fatty acids, FABP is responsible for the uptake, compartmentalization, and transport of fatty acids from the vertebrate host through the tegument of the parasite to its interior. The vaccine based on this protein has been produced by using the recombinant protein (rSml4/GLA-SE). When this vaccine was applied in rabbits and mice, it reduced the S. mansoni load by 89% and 67%, respectively. The clinical trial phase 1, which involved 20 male volunteers from endemic areas in Brazil, showed that the vaccine, applied in three doses, was highly immunogenic and safe. The humoral immune response increased, with high levels of specific total IgG1 and IgG2, IgG3, and IgG4, and low levels of IgE, IgA, and IgM being produced. An immune response dependent on IFNγ and TNFα took place. The next step will be phase II trials in endemic areas of Brazil and Africa [56, 61].

4.2 New drugs

Molecules that alter the parasite tegument structure must be considered as possible new drugs because the tegument is essential for parasite survival in mammalian hosts: indeed, the tegument plays an important role in evading immune response and acquiring nutrients from the host [53].

To date, no new molecule has reached the clinical screening phase, but several studies are in the preclinical and in vitro phases. Analyses of the transcriptome; that is, the Schistosoma mRNA set, have led to promising new targets against the parasite [13].

Some drugs are administered to treat schistosomiasis. One example is metrifonate, which has been used to treat urogenital schistosomiasis. Nevertheless, this drug requires that several doses be administered, and it has several side effects. Another drug is Oltipraz, which acts against S. mansoni and S. haematobium. However, 2 months are necessary for the patient to be cured, so patients usually give up the treatment. This drug reduces the levels of the parasite enzyme glutathione synthase, which facilitates worm elimination by the immune system. As for niridazole, it reduces the parasite glycogen levels and degenerates the female reproductive system. It is effective against S. haematobium, but protection against S. japonicum lies between 30% and 70%. However, today patients are no longer given the drug due to side effects on both the central nervous system and heart. Another problem is that this drug is no longer effective against S. japonicum males. The drug oxamniquine acts on S. mansoni by affecting the synthesis of nucleic acids, consequently impacting DNA, RNA, and protein translation. However, oxamniquine has been used for 20 years, so S. mansoni has become resistant to it [26].

The association of anthelmintic drugs with antimalarials is advantageous for research aimed at discovering combinations that eliminate not only the adult stage of the parasite but also schistosomula. Drug combinations are an alternative to treatment with PZQ monotherapy [62].

Concomitant administration of OXA and PZQ to treat S. mansoni-infected mice has been more successful than administration of individual drugs. OXA combined with PZQ has been orally administered in children aged between 8 and 20 years infected with S. mansoni or S. haematobium, and clinical screening has been carried out. Oral administration of 15–20 mg/kg PZQ associated with OXA (7.5–10 mg/kg) reduced egg shedding by between 93% and 99% in children infected with S. mansoni. In children infected with S. haematobium, this combination did not succeed in treating urogenital schistosomiasis [62].

Drugs used for malaria treatment have been tested in association with PZQ in pre-clinical trials. The combination that reduced the parasite load and the number of eggs went on to the clinical phase in endemic regions of Africa and Asia, where transmission and reinfection rates are high. Various combinations have been administered to patients infected with S. mansoni, S. japonicum, or S. haematobium. Among these combinations, Artesunate (AS) + PZQ and Sulfamethoxypyrazine/Pyrimethamine + AS can be mentioned, none of which has provided better results than monotherapy with PZQ [62].

Mefloquine (MFQ), an antimalarial drug, has been considered the best in vitro tests performed against Schistosoma. MFQ damaged the parasite tegument, muscles, and reproductive and digestive systems. Therefore, MFQ was tested in combination with PZQ and Artemisin, to see whether these combinations would be more effective than PZQ monotherapy. Clinical trials performed on children infected with S. mansoni and/or S. haematobium showed that the combinations did not outperform PZQ monotherapy even though association with antimalarial drugs was expected to increase the action of PZQ against schistosomula.

Despite the importance of PZQ monotherapy, this drug does not treat granulomas caused by eggs in the liver. Therefore, in addition to PZQ-resistant parasite strains, changes in liver histopathology are a problem in patients with chronic disease, especially in areas where reinfection occurs [62].

Recently, researchers have studied extracts of substances of plant origin, but most studies are in the in vitro phase. Among the studied extracts, we can mention (−)-6,6-dinitrohinokinin (DNK), which alters the tegument of S. mansoni couples, separating them and reducing the number of eggs and the rate of egg development. The in vivo test in mice infected with S. mansoni and treated with DNK revealed a smaller number of eggs per gram of tissue, which consequently reduced the size of the spleen and liver; the parasite load also decreased [63]. Cramoll-1,4-lectine, isolated from Cratylia mollis, has been shown to have antiparasitic activity in mice infected with S. mansoni and treated with 7 mg/kg for 7 days. Egg excretion, worm recovery, and granulomas decreased by 80%, 20%, and 73% in these animals [64].

Among medicinal plants with high schistosomicidal activity, Zingiber officinale can be cited. Zingiber has antifungal, antibacterial, and antioxidant effects. Moreover, it acts as anthelmintic, improving granulomatous inflammation. Due to their cost-effectiveness, nanoparticles (NPs) have been used against infectious agents. NPs can enter small capillaries, which allows better absorption, entry into target tissues, and lower toxicity. Zingiber extract is made up of nanoparticles (GNPs) that are easy to purify, which facilitates its use. In pre-clinical trials, GNPs (5 mg/kg or 2.5 mg/kg) were orally administered 3 days/week for 5 weeks. This treatment was started 4 weeks after mice were infected with S. mansoni. The number of eggs in the liver was evaluated: the egg load reduced by 65%, as verified by histopathology. Furthermore, worm recovery from infected mice decreased by 60%. Scanning electron microscopy images showed changes in tegument integrity, which occurred 10 weeks post-infection and was due to the antioxidant effect, as evaluated by enzymes linked to oxidative stress. Together, these results indicated that GNPs have hepatoprotective, schistosomicidal, and antioxidant functions [65].

Due to their anti- and pro-fibrotic function, small molecules, called microRNAs (miRNAs), have been researched for schistosomiasis treatment. miRNAs are small RNAs that are not translated into proteins. They contain around 70 nucleotides and are important for cellular homeostasis: they are involved in the post-transcriptional regulation of one-third of the protein-coding genes and hence participate in the activation or inhibition of cellular processes. miRNAs have been the target of research into the therapy of diseases such as cancer, diabetes, viral diseases, and other metabolic diseases. Through molecular biology techniques, they can be detected in tissues, plasma, serum, and biological fluids. These techniques include Polymerase Chain Reaction, Microarrays, and RNA Sequencing, which together amplify nucleotides and sequence them in order to discover their sequences [66]. Therefore, several miRNAs are being studied for the therapy of diseases such as solid tumors and hematopoietic diseases. Examples of such miRNAs include MiR-34 and MRX34 (the liposomal miR-34a mimic), which are in the phase I preclinical trials [67].

Along with the genotoxins produced by the parasites, which alter the host’s immune response [35], vertebrate host miRNAs play a role in the parasite-host relationship, so they have been studied as biomarkers for schistosomiasis detection and hepatic fibrosis gene therapy. Such studies are in the preclinical trial phase. Initial research has shown that miR-21 and miR-96 are involved in regulating the immune response and hence hepatic granuloma by regulation of the TGF β/SMAD pathway, linked to collagen formation. Therefore, they have a pro-fibrotic function, in contrast to miR-203-3p, which is anti-fibrotic. In the case of schistosomiasis, there are miRNAs that characterize liver changes and hepatosplenomegaly progression. Among these miRNAs, we can mention MiR-223: the serum of mice infected with S. japonicum showed high levels of this miRNA, which returned to normal levels after treatment with PZQ. Other miRNAs, such as miR-2c-3p, are related to fibrosis progression in mice infected with S. japonicum. Therapy through miRNA silencing, whose function is to decrease gene transcription of pro-fibrotic genes, is being tested in infected mice by using viral vectors such as lentiviruses and adenoviruses. The genome of these vectors has antisense sequences that can inhibit host profibrotic miRNAs. In independent experiments, the viral vectors lenti-let-7b and adeno-associated virus serotype 8 (rAAV8)-mediate (which inhibits miR-21 and miR-96) were injected into mice infected with S. japonicum, to slow down the collagen production activation pathway and to reduce hepatic granuloma. However, these studies are initial and further research is needed [68].

In view of what has been explained, the development of new vaccines for schistosomiasis is more advanced than the development of new drugs against this disease. As judged from the time that PZQ , the only drug of choice, has been used, developing new substances that are active against the parasite is difficult. When it comes to evading the host’s immune response, Schistosoma species have put together a strategy to follow their cycle from schistosomula to adult worm, so that they reach oviposition while balancing the host’s TH1 and Th2 immune responses. This strategy allows the parasite to survive for up to 10 years in the host. Therefore, the biological complexity of Schistosoma species prevents reinfections with these species from being avoided and causes the parasite to become resistant to PZQ. In conclusion, several biological molecules of the parasite (genotoxins and miRNAs, not mentioned in this chapter) interfere in the parasite-host relationship, not to mention the vertebrate host’s microRNAs that alter this relationship, generating a network of molecules that interacts with each other. Furthermore, the biological complexity of the parasite involves a cycle consisting of different phases (cercariae, miracidia, eggs, adult worms, schistosomula, and daughter sporocysts) during which the parasite structure and metabolism change according to the environment in which it is inserted (that is, whether the parasite is in the intermediate or definitive host or in water).