Clinical Cysticercosis

#### **Chapter 1**

## Cardiac Cysticercosis: Current Trends in Diagnostic and Therapeutic Approaches

*Ane Karoline Medina Néri, Danielli Oliveira da Costa Lino, Sara da Silva Veras, Ricardo Pereira Silva and Geraldo Bezerra da Silva Júnior*

#### **Abstract**

Cardiac cysticercosis is a rare infection and its diagnosis is usually incidental, as most patients are asymptomatic. Laboratory and imaging tests, such as echocardiogram and cardiac nuclear magnetic resonance, can also be used in the diagnostic approach. The clinical manifestations are broad and patients can present with symptoms that range from heart failure to arrhythmias. Treatment of this condition has been scarcely studied and no protocols have been well established to date. One can choose not to treat the asymptomatic cases or to use cestocides, in the case of symptomatic individuals. Patient monitoring through cardiac enzymes and electrocardiogram during treatment is recommended, as well as performing imaging tests after treatment. This chapter aims to discuss cardiac cysticercosis, divided into sessions that will cover everything from its epidemiology and clinical aspects to diagnostic methods, therapeutics and treatment monitoring, with emphasis on the most current aspects.

**Keywords:** Helminthiasis, Taenia Infections, Cysticercosis, Neglected Diseases, Cysticercus, Cardiovascular Infections, Cardiac Diseases

#### **1. Introduction**

Endemic in Asia, Africa and Latin America, cysticercosis is caused by the ingestion of *Taenia solium* eggs. Contamination is caused by autoinfection in individuals with taeniasis, due to poor hand hygiene, or by heteroinfection, from contaminated foods, especially raw vegetables and water [1, 2].

The incubation period after the ingestion of the egg lasts for 3–8 weeks, with symptom onset occurring in up to 3–5 years. It is believed that the cysticercus is able to survive for a period of 3 to 6 years, after which it begins to degenerate, causing fibrosis or necrosis in the affected tissues due to the triggered inflammatory process. Nonetheless, the infection usually remains asymptomatic. Morphologically, the cysticercus exhibits two forms: the cystic form that contains the scolex and the racemous form, which corresponds to a set of vesicles, without scolex and a configuration similar to grape clusters [1].

The most affected tissues in cysticercosis are muscle and eye tissues, and the most severe manifestations are, overall, those related to the central nervous system [1].

Cardiac cysticercosis is rare, although autopsy studies have shown a 20–27% prevalence of cardiac cysticercosis occurring concomitantly with neurocysticercosis [3, 4]. In cases of cardiac involvement, pericardial effusion, signs of edema and myocardial inflammation or even myocardial infarction can occur [5].

The diagnosis can be attained by conclusively demonstrating the presence of the cysticercus through histopathological techniques in biopsy material, by visualizing the scolex in computed tomography or magnetic resonance imaging tests or by fundus examination, in cases of intraocular cysticercosis. In the absence of direct demonstration of the parasite presence, serological tests allow the diagnosis of the disease, although these tests are not widely commercially available [6].

There is no consensus on the treatment of cysticercosis with cardiac involvement. Patients with extraneural cysticercosis should be evaluated and high-risk situations, such as disseminated infections, intraventricular cysts and ocular involvement, should be excluded. Cases of asymptomatic individuals may not require surgical or anthelmintic therapy [3, 7].

The role of anthelmintics, such as Albendazole and Praziquantel, in the treatment of cardiac cysticercosis has not been directly investigated. However, it seems that the use is valid due to their effectiveness in the treatment of cysticercosis in other sites, such as neurocysticercosis. The role of cardiac surgery in the treatment of this condition also remains unclear [2].

As it is a rare condition and, therefore, still little discussed, this chapter aims to discuss the existing evidence in the literature on the diagnosis and treatment of cysticercosis with cardiac involvement, emphasizing the most current trends.

#### **2. Epidemiology and clinical presentation of cysticercosis with cardiac involvement**

Cysticercosis with cardiac involvement, especially myocardial impairment, is considered rare, and has been scarcely studied, being more frequently asymptomatic, so its diagnosis is often incidental, usually attained during cardiac surgery or at autopsy [1, 2]. Retrospective studies with autopsies have shown a variable prevalence of cardiac involvement, between 22.6% and 26.8% [3, 4, 8, 9] of the cases identified with cysticercosis.

As for the presentation according to the age group, another autopsy study showed that, of patients with cysticercosis, 27.8% were elderly and 72.2% were non-elderly, and that among the first, 20% had cardiac involvement due to cysticercosis, whereas of the latter, 25% showed cardiac cysticercosis [10]. Research [8] has shown a higher prevalence of cysticercosis in male individuals in all age groups, except for those between 30 and 39 years old, in which there was a greater number of affected women.

Cysticerci appear as oval cystic structures with thin, semitransparent and serous walls, containing liquid and measuring up to 30 mm in diameter, which contain a characteristic scolex [6]. Cysticercal involvement and distribution are variable in cardiac tissues, including the pericardium, subendocardium and myocardium [1, 6]. Cardiac cysticerci are usually multiple and, rarely, a single cardiac cyst may be present [1].

The immune system of the infected individual may not recognize the cysts for many years. However, when cysticerci age, their cystic structures can rupture, which will result in an inflammatory response [9] with variable expression and the possibility of granulomas and also fibrosis [2]. Although most cases are asymptomatic, clinical manifestations may occur as a result of inflammation, precisely at

#### *Cardiac Cysticercosis: Current Trends in Diagnostic and Therapeutic Approaches DOI: http://dx.doi.org/10.5772/intechopen.97472*

the time of spontaneous cysticercus degeneration or during treatment, which may result in different degrees of cardiac involvement [1, 2].

One study [11] showed that non-elderly individuals had significantly more cardiac inflammation than the elderly and that the inflammatory infiltrate decreases with age and depends on the evolutionary stage of the cysticercosis. Moreover, the study showed there are gender differences regarding the intensity of the inflammatory response triggered by the presence of cysticerci in the heart, with women (elderly and non-elderly) showing a more intense response to the parasitosis than men.

Therefore, in cases of cardiac involvement, myocarditis with transient left ventricular dysfunction, pericardial effusion of variable extension, restrictive cardiomyopathy due to fibrosis formation [6], ischemic heart disease [12], in addition to valve pathologies [1, 2, 6] and conduction system defects, such as bradyarrhythmia and advanced atrioventricular block [13, 14] can occur. Dilated cardiomyopathy [6] and even severe ventricular dysfunction and cardiogenic shock have also been reported, in cases with severe cardiac or cardiopulmonary infestation [15].

The reasons why some patients have multiple cysticerci, while others have a single lesion remain uncertain. In a prospective follow-up study in India with 60 patients with disseminated cysticercosis, it was observed that changes in the Toll-like receptor-4 of *Asp299Gly* and *Thr399Ile* genes increased the risk (6.63 and 4.61-fold in the presence of polymorphisms, respectively) of disseminated cysticercosis [7].

The relationship between cysticercosis and immunosuppression remains uncertain, although post-chemotherapy cases of cysticercosis have been documented in Brazil and Mexico [15, 16]. Animal experiments using chemotherapeutic drugs have suggested that innate resistance contributes to the outcome of primary infection and there is a high degree of resistance to reinfection, both in the humoral and cellular mechanisms. This resistance to reinfection is altered by immunosuppression, probably due to the delay in antibody synthesis onset [17].

**Table 1** shows a summary of several cases of patients with cysticercosis and cardiac involvement reported in the literature, with patients' general characteristics, clinical manifestations and sites of disease presentation (cardiac and extracardiac).



**Table 1.**

*Clinical characteristics, sites of disease presentation (cardiac and extracardiac), and clinical manifestations in patients reported in the literature.*

#### **3. Current diagnostic and therapeutic approaches to cardiac cysticercosis**

#### **3.1 Diagnostic methods used in the analysis of cardiac involvement due to cysticercosis**

The diagnosis of cardiac cysticercosis can be attained by conclusively demonstrating the presence of the cysticercus through histopathological techniques in biopsy material or by visualizing the scolex, either by computed tomography or

#### *Cardiac Cysticercosis: Current Trends in Diagnostic and Therapeutic Approaches DOI: http://dx.doi.org/10.5772/intechopen.97472*

nuclear magnetic resonance imaging tests. Some authors consider these tests to be the gold standard in the diagnosis of cysticercosis, as they allow the visualization of the parasite and the host's reaction process [29].

The computed tomography shows greater sensitivity in the detection of calcified cysticerci, whereas the magnetic resonance imaging has greater resolution power, which may show the scolex with better accuracy [6, 30]. The echocardiogram may play a role in identifying cardiac cysts and occasionally identifies cysts consistent with cysticercosis during routine screening for other purposes [1, 6].

In the absence of direct visualization of the parasite, serological tests allow the disease diagnosis [1, 6, 30, 31]. The oldest tests, which used unfractionated antigens, including the enzyme-linked immunosorbent assay (ELISA), have been associated with high rates of false-positive and false-negative reactions [21, 30]. Currently, the Enzyme-linked immunoelectrotransfer blot (EITB) is considered one of the most reliable immunological tests for the diagnosis of cysticercosis and neurocysticercosis [29, 31]. The initial evaluation of this test indicates a 98% sensitivity and 100% specificity in serum and cerebrospinal fluid (CSF) samples. Other studies have reported an 86–100% variation in sensitivity in serum and 81–100% in CSF, while the variation in specificity ranged from 93–100% in both sample types [29]. Unfortunately, the availability of these tests is experimental, not being commercially available and rarely available at most medical centers, except for research centers working in this area [31].

Some laboratory alterations can be identified in cardiac cysticercosis, such as marked peripheral eosinophilia disclosed in the blood count, albeit only in the case of a ruptured cyst [1, 32]. Most individuals affected by this pathology do not have viable *Taenia solium* in the intestine, making the stool parasitological test ineffective [1, 31, 32].

**Figure 1** summarizes the main diagnostic methods that can be used for the diagnostic definition of cysticercosis with cardiac involvement.

#### **3.2 Treatment and monitoring of the patient with cysticercosis and cardiac involvement**

There is no consensus regarding the treatment of cardiac cysticercosis. In patients with extra-neural cysticercosis, the existence of high-risk conditions, such as disseminated infection, intraventricular cysts and ocular involvement, should be evaluated [1, 31]. Asymptomatic cases might not require more specific

#### **Figure 1.**

*Diagnostic methods that can be used for the diagnostic definition of cysticercosis with cardiac involvement.*



#### *Cardiac Cysticercosis: Current Trends in Diagnostic and Therapeutic Approaches DOI: http://dx.doi.org/10.5772/intechopen.97472*



**Table 2.**

#### *Cardiac Cysticercosis: Current Trends in Diagnostic and Therapeutic Approaches DOI: http://dx.doi.org/10.5772/intechopen.97472*

therapy, such as surgical procedures or anthelmintic pharmacological therapy [3, 7]. In cases of asymptomatic myocardial involvement, there is usually no justification for any type of intervention, given the benign prognosis associated with this condition [3, 6].

The role of anthelmintic drugs such as Albendazole and Praziquantel in the treatment of cardiac cysticercosis has not been directly investigated in large studies; however, it seems that their use is valid due to their efficacy in the treatment of cysticercosis in other sites, such as neurocysticercosis [6]. Therefore, these drugs are used at the same dose and duration utilized to treat neurocysticercosis, with Praziquantel at a dose of 50 mg/kg/day for 15 days and Albendazole at a dose of 15 mg/kg/day for 8–15 days [6, 30, 31]. In case of a solitary cyst or granuloma, monotherapy with Albendazole may be sufficient [31].

The role of the surgical removal of cysts through cardiac surgery for the treatment of cardiac cysticercosis is also not yet clear and may be indicated when some valvular apparatus is compromised, when there is left ventricular outflow tract obstruction or even when there is epicardial coronary artery compression, with subsequent myocardial blood supply reduction [6, 12, 25, 28].

Corticosteroids are used together with antiparasitic agents in the initial treatment of neurocysticercosis to decrease the pericystic inflammatory reaction that follows larval necrosis, but there is no definition regarding its use in patients with cardiac involvement, although it is theoretically possible [1, 2, 7, 17]. A randomized trial comparing 6 mg/day of Dexamethasone for 10 days with 8 mg/day for 28 days, followed by a gradual reduction over 2 weeks, suggested that increasing the dose of Dexamethasone results in fewer seizures during treatment for neurocysticercosis [30, 31]. However, in some cardiac conditions, such as pericarditis, steroids have been associated with increased relapse and recurrence. Due to the rapid response to anti-helminthic therapy in some cases, Albendazole can be used without steroids, but with adequate monitoring [18].

Cardiac monitoring is recommended, with cardiac enzymes and electrocardiogram, during the early stages of the treatment for cardiac impairment due to cysticercosis [6]. The cases reported in the literature show that the lesions on the MRI disappear 6 to 9 months after treatment [1, 16]. However, there is no recommendation on the most appropriate periodicity for the assessment of myocardial necrosis biomarkers, or imaging tests after treatment [6].

**Table 2** shows a summary of several cases reported in the literature regarding compromised cardiac structures, the several methods used for the diagnosis, the performed treatments and their monitoring, in addition to the disease evolution in each case of cysticercosis with reported cardiac impairment.

#### **4. Conclusion**

The clinical management of cysticercosis with cardiac involvement is complex, due to the rarity of the pathology and the broad spectrum of clinical presentation, ranging from asymptomatic cases to those with more severe manifestations, such as cardiogenic shock and advanced cardiac blocks. The lack of studies directly investigating the role of diagnostic methods for its detection, as well as drug therapy effectiveness with anthelmintic drugs and corticosteroids, and the role of the surgical approach are also factors that have an impact on the management of these cases. Therefore, we propose that individuals with cardiac cysticercosis should be evaluated individually by a multidisciplinary team, so that the best diagnostic and therapeutic conduct, as well as the best way of monitoring each specific case, can be implemented.

### **Acknowledgements**

We would like to thank the Brazilian Research Council (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq) for supporting our research (research grant to GBSJ # 310974/2020-8).

### **Conflicts of interest**

The authors declare no conflicts of interest.

#### **Author details**

Ane Karoline Medina Néri1,2,3\*, Danielli Oliveira da Costa Lino1 , Sara da Silva Veras4 , Ricardo Pereira Silva3,5 and Geraldo Bezerra da Silva Júnior1,2

1 Postgraduate Program in Collective Health, Health Sciences Center, University of Fortaleza, Fortaleza, Ceará, Brazil

2 School of Medicine, University of Fortaleza, Fortaleza, Ceará, Brazil

3 Cardiology Service, Walter Cantídio Teaching Hospital, School of Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil

4 Infectious Diseases Service, Walter Cantídio Teaching Hospital, School of Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil

5 Postgraduate Program in Cardiovascular Sciences, Department of Clinical Medicine, School of Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil

\*Address all correspondence to: karolinemedina@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Cardiac Cysticercosis: Current Trends in Diagnostic and Therapeutic Approaches DOI: http://dx.doi.org/10.5772/intechopen.97472*

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### Section 2

## Recent Advances in Cysticercosis Research: Vaccine Immune Response and Immunodiagnosis

#### **Chapter 2**

## Development of an Oral Vaccine for the Control of Cysticercosis

*Marisela Hernández, Anabel Ortiz Caltempa, Jacquelynne Cervantes, Nelly Villalobos, Cynthia Guzmán, Gladis Fragoso, Edda Sciutto and María Luisa Villareal*

#### **Abstract**

Parasitic diseases fecally transmitted, such taeniasis/cysticercosis *Taenia solium* binomial, represent a health problem whose incidence continues due to the prevalence of inadequate sanitary conditions, particularly in developing countries. When the larval stage of the parasite is established in the central nervous system causes neurocysticercosis a disease than can severely affect human health. It can also affect pigs causing cysticercosis causing economic losses. Since pigs are obligatory intermediate hosts, they have been considered as the targets for vaccination to interrupt the transmission of the parasitosis and eventually reduce the disease. Progress has been made in the development of vaccines for the prevention of porcine cysticercosis. In our research group, three peptides have been identified that, expressed synthetically (S3Pvac) or recombinantly (S3Pvac-phage), reduced the amount of cysticerci by 98.7% and 87%, respectively, in pigs exposed to natural conditions of infection. Considering that cysticercosis is orally acquired, it seems feasible to develop an edible vaccine, which could be administered by the pig farmers, simplifying the logistical difficulties of its application, reducing costs, and facilitating the implementation of vaccination programs. This chapter describes the most important advances towards the development of an oral vaccine against porcine cysticercosis.

**Keywords:** cysticercosis, *T. solium*, oral vaccine, transgenic plant, *Carica papaya*

#### **1. Introduction**

*Taenia solium* taeniasis/cysticercosis is a parasitic zoonosis that significantly affect economic and public health. Neurocysticercosis (NCC) is a most severe form of the disease caused by the establishment of the larval stage (cysticerci) of *Taenia solium* in the central nervous system (CNS). In 2010, the World Health Organization declared it one of the leading neglected diseases and aims to develop strategies for its eradication and prevention [1].

Between control measures it has been explored the improvement of health education, sanitary conditions, standards of meat inspection and the rearing of pigs. It has also been explored the impact of massive or individual treatment of taeniasis and the treatment and/or vaccination of pigs, all of them with promising results [2–4]. Vaccination of pigs would imply an unlikely immediate and potent effect

upon the number of tapeworm-carriers in rural communities, interrupting the parasite's life cycle and eventually reduce human neurocysticercosis. Developing an effective vaccine against *T. solium* pig cysticercosis is also being pursued by different research groups with promising results [5, 6].

In our group, an anti-cysticercosis vaccine named S3Pvac based on three peptides expressed was developed. The vaccine synthetically (S3Pvac) or recombinant (S3Pvac-phage) produced, reduced the number of cysticerci by 98.7% and 87% [7, 8] in randomized field trials, respectively. The recombinant vaccine was subsequently used in a control program applied in the State of Guerrero, confirming its usefulness. Indeed, S3Pvac-phage significantly reduce the prevalence of porcine cysticercosis from 7 to 0.5% and 3.6 to 0.3% estimated by tongue examination or ultrasound, respectively [3]. In the course of this control program, we were able to evaluate the difficulties involved in using an injectable vaccine. Pigs are produced free rurally reared, thus the application of an injectable vaccine requires their capture and subjection, a laborious procedure that increases the costs of vaccination and limits its massive application. Considering that cysticercosis is orally acquired, it seems feasible to develop an oral vaccine [9], which could be administered by the pig breeder, simplifying the logistical difficulties of its administration, reducing costs and facilitating the implementation of vaccination control programs.

For the design of an oral vaccine the use of plants is increasingly recognized as valuable platform. Plants offer the production of antigens at low costs, circumventing costly purification procedures. Plants also offer a natural way of antigen encapsulation preventing its degradation by the detrimental environment to which an oral intake vaccine is exposed such us antigen degradation by low pH, mucosal enzymes [10, 11] and the use of cell cultures will avoid non-desirable environmental effects due to the release of transgenic plants into the environment.

Moreover, plants also frequently include components with adjuvant properties like saponins that may increase the immunogenicity of the vaccine [12]. Considering this, the recombinant peptides KETc7, KETc1.6His, and KETc12.6His were expressed in transgenic clones of papaya embryogenic calli [13]. The three clones together constitute the oral S3Pvac-papaya vaccine candidate. Papaya was selected because the high efficiency of transformation and its own antiparasitic properties [14].

This third version of the vaccine has been shown to be immunogenic in mice and pigs and is being produced in suspended culture systems to massively produced this oral version of the vaccine that must be evaluated on the field against pig cysticercosis.

#### **2. Parasite and oral immunity**

Oral vaccination is an interest route to prevent infections caused by orally acquired pathogens overcoming the limitations of current injection-based vaccines in providing front-line protection against pathogen invasion and dissemination [15]. It offers a painless, safe and low-cost route that does not require trained personnel. Moreover, this route can elicit mucosal and systemic immunity. Vaccine antigen can be recognized and translocated by M cells, which act as sentinels and enter directly into the Peyer's patches. Then antigens can be transported to the intestinal mesenteric lymph nodes, stimulating the host's systemic and mucosal immune response resulting in the production of IgA and IgG antibodies with the ability to neutralize of invading pathogens before they are able to cause a widespread infection. Oral vaccination can also trigger an effective cellular immunity. However, the development of oral vaccines is a major challenge due to an inefficient


#### *Development of an Oral Vaccine for the Control of Cysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97227*

**Table 1.**

*Expression of antigens aimed at veterinary vaccine development.*

transport to reach M cells and the possibility to induce local and systemic immune tolerance. Considering that plant-based vaccines usually expressed low content of antigen it seems feasible to avoid oral tolerance using the proper dose and vaccine schedule. It remains to be elucidated if plants-derived vaccines could overcome mucosal tolerance when administered to human beings.

**Table 1** shows various plants that have been used to express antigens from different pathogens to be evaluated as edible vaccines. Tobacco has been used as an experimental model of transformation and expression. However, the use of other species such as tomatoes, lettuce, potatoes, corn, soybeans, alfalfa, Arabidopsis, papaya and carrots has been expanded [11, 28–33]. In some of these plants, the expressed recombinant antigen has shown efficacy when evaluated in experimental models or directly in the naturally affected host. Recombinant antigens have been reported to induce an immune response with the production of IgG, IgM or IgA antibodies, regardless of the route of administration [31].

#### **3. Transgenic plant platform**

Many different advantages of expression of recombinant proteins in transgenic plants for vaccine production can be mentioned over other commonly expression systems, such as bacteria, yeasts and baculoviruses. Plants can be constitutively or tissue-specific

expressed in single or multiple transgenes, antigens can be stable in seeds without the requirement of refrigeration, no purification nor cold chain for preservation.

Transgenic plants can also be used as bioreactors to produce high amounts of the recombinant protein of interest [34, 35]. They can also be produced as *in vitro* tissue culture, cell suspensions, hairy roots, moss protonema, microalgae and whole plants. There are many experimental plant-made veterinary vaccines produced in seeds, fruits, and leaves, that can be orally delivered as part of the animal feed, thus offering great convenience and economy in immunizing large populations of animals on farms [35]. The expression of antigens for the production of vaccines in transgenic plants is considered a safe and effective immunization system, which can avoid some of the difficulties associated with traditional vaccination methods, as well as a reduction in the costs of production, distribution and conservation.

One nice study of veterinary interest is the expression of the glycoprotein S of the porcine gastroenteritis virus in corn seeds for the production of an oral vaccine, which has also the ability to induce protection, through colostrum, in piglets [25, 26].

#### **3.1** *Carica papaya* L.

Classification of *Carica papaya L.* Family: *Caricaceae.* Gender: *Carica.* Species: *C. papaya L*. Morphological type: Arboreal. Climate: Equatorial tropical.

*Carica papaya* is a species of pantropical plant that grow in tropical regions of the Americas from Mexico to Argentina, Africa and Asia. The main importers are: United States, Japan, Hong Kong and the European Union. *Carica papaya* is known by different common names such as capaidso, naimi, nampucha, pucha, fruit bomb, milky, mamao, pawpaw. Papaya is an arborescent, semi-perennial plant that grows in areas with an average rainfall of 1800 mm per year and an average annual temperature of 20–22°C, a large number of varieties have been developed. Papaya fruiting occurs 10 to 12 months after transplantation, is maintained for ten years, and female, male or hermaphrodite [36] trees can be obtained. Papaya is a fruit known for its nutritional benefits and medicinal properties. Main papaya components and their reported properties are shown in **Table 2**.

#### **3.2** *Carica papaya* **as a cestode vaccine**

Papaya is an alternative system for the exploration of tropical tree genomes, containing a genome of 372 megabase (Mb), of diploid inheritance with 9 pairs of chromosomes and presents the smallest gene number, 24,746 genes [37]. Papaya exhibits some properties of possible advantages to be used as a platform to express *T. solium* vaccine antigens. Papaya components have antiparasitic properties per se [14]. Cells can be easily transformed by bioballistics and *in vitro* propagated and regenerated [38].

Among many papaya components, the papain contained in the latex has been widely evaluated in its ability to damage the cuticle of intestinal parasites by proteolytic digestion. **Table 3** shows some reports on the characterization and evaluation of antiparasitic activity of papaya against *Trichostrongylus colubrormis, Heligmosomoides polygyrus, Trichuris muris, Protospirura muricola* [39, 44–49] *Rodentolepis microstoma* [39], *Hymenolepis diminuta and microstoma* [40].

#### *Development of an Oral Vaccine for the Control of Cysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97227*


*included in papaya.*

#### **Table 2.**

*Papaya components and medicinal properties reported.*


#### **Table 3.**

*Antiparasitic activity of papaya against some cestodes.*

*Anoplocephala perfoliate* [41] *Hymenolepis diminuta* [42] *Hymenolepis microstoma* [43] without causing side effects to the host [50, 51].

#### **3.3 S3Pvac-papaya anticysticercosis vaccine**

For the development of the S3Pvac-papaya cysticercosis vaccine, three genetic constructions were used for the expression of recombinant peptides, KETc1.6His, KETc12.6His and KETc7 [13]. **Figure 1** summarize the methodology employed for the production of S3Pvac-papaya vaccine.

#### **3.4 Protective properties of S3Pvac papaya against cysticercosis**

The S3Pvac vaccine expressed in embryogenic papaya clones has demonstrated high protective capacity against experimental murine and *T. pisiformis* cysticercosis orally administered. **Table 4** shows the protective effect induced by oral immunization in mice at a dose range of 0.1 to 1 μg of soluble extract, whilst a higher dose lowered the percent of protection. In addition, different vaccine formulations also reduced the expected parasite load. On the other hand, the oral vaccine significantly reduced the number of infected rabbits and the percentage of cysticercus-free animals (83%), 21 days after the infection. The S3Pvac-papaya vaccine has not yet been evaluated in pigs, however, its immunogenic response in mice and pigs [27, 52], and its protective capacity in different models exhibit its potential to exert a protective response against naturally acquired porcine cysticercosis.

We previously reported non-specific protection that was induced by the wild type soluble extract [52, 54] has been attributed to the antiparasitic properties described to papaya itself mentioned above.

#### **Figure 1.**

*(A) Production of transgenic embryogenic papaya clones by a bioballistic method (B) Embryogenic papaya callus: a) Induction of embryogenic callus; b) Embryos in globular stage for transformation; c) Selection of transformed clones in selective medium.*



*† Mean ± standard deviation of the number of cysticerci recovered in each group.*

*§ Mice were fed with S3Pvac-papaya soluble extract (1* μ*g of total protein) into different vehicles. £*

*Rabbits received a suspension of 20 mg of each embryogenic transgenic papaya clone expressing KETc1, KETc12 and KETc7 in a gelatin capsule.*

*\*Protection statistically significant (P < 0.05).*

#### **Table 4.**

*Protective capacity induced by oral S3Pvac-papaya vaccine against experimental.*

#### **3.5 Biotechnological approach for the production of papaya anti-cysticercosis vaccine**

Plant biotechnology is a rapidly evolving area with major impact in the production of molecules with high pharmaceutical value. *In vitro* culture techniques offer central advantages in the manufacture of desired chemicals for human health. The benefits include a systematic supply of compounds under optimized controlled conditions, independence of weather, soil, disease, and socio-political problems; discovery of new compounds, bio-transformation systems and better adaptation to market changes. In an inclusive context, *in vitro* systems will give a better understanding of plant biochemistry and physiology, as well as some basic aspects of plant differentiation.

Plant biotechnology involves relevant procedures in the manufacture of oral vaccines enabling the production of higher amounts of active biomasses from transgenic plants, by means of massive propagation of cells, tissues and organs [12, 55]. Among others, these procedures include the growth of callus (aggregates of undifferentiated cells growing in solid media), suspension cultures (individualized undifferentiated cells growing in liquid media); as well as embryo cultures that could be grown in solid or in liquid nutrient media.

The three callus lines expressing KETc1, KETc7 and KETc12 peptides were generated, and further efforts to optimize the massive growth of the corresponding callus and suspension cultures, were conducted. These *in vitro* systems constitute adequate platforms for the massive production of papaya anti-cysticercosis vaccine in the near future.

#### *3.5.1 Callus cultures*

In the establishment and optimization of callus cell lines, *Carica papaya* L. (KETc7) embryogenic calli were used to obtain friable undifferentiated cells. Calli were placed in solid culture medium with 30 g/L sucrose, 3 g/L− polivinilpilorridone and 1.5 g/L− of phytagel. The nutrient media MS [56] and B5 [57] were evaluated; and the presence of the phytoregulators 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0, 2.0 and 3.0 mg/L− combined with kinetin (KN) at 1.0, 2.0 and 3.0 mg/L, was also tested. The cultures were maintained at 25°C and subjected to constant light as well as dark conditions. The best results were obtained for callus growing in B5 medium with 2 mgL−1 of 2,4-D combined with 2 mgL−1 KN in dark conditions (**Figure 2**). In these conditions after two subcultures non-phenolized calluses were developed, and after several subcultures the friable callus KETc7 cell line, was established.

#### *3.5.2 Cell suspension cultures*

Ten grams fresh weight (FW) of the friable callus line KETc7 were inoculated in 250 ml Erlenmeyer baffled flasks containing 100 ml nutrient medium without phytagel, and placed for 30 days on a rotary shaker at 100 rpm, 25°C and dark conditions. To disaggregate cell clusters and increase oxygen transfer, baffled flasks were used (**Figure 3**).

The cultures were sub-cultured in fresh medium every 15 days, and the best results were observed when using B5 nutrient medium, cultivated in darkness, and producing uniform suspended cultures without phenolization (**Figure 4**).

Cell viability was maintained at 95% until 45 days in culture, as confirmed by the fluorescein diacetate (FDA) method (**Figure 5**) [58].

#### **Figure 2.**

*Optimization of C. papaya KETc7 callus cell line under different growth conditions. (a) Photoperiod, (b) constant light, (c) darkness.*

#### **Figure 3.**

*Establishment of C. papaya KETc7 cell suspension line. (a) Culture in baffle flasks at 15 days (b) culture in Erlenmeyer flasks at 15 days (c) culture in Erlenmeyer flasks at 30 days.*

*Development of an Oral Vaccine for the Control of Cysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97227*

Once the friable uniform cell suspension cell line was established, it became possible to evaluate the growth kinetics of the culture during 45 days by collecting samples every 3 days, and determining the following growth parameters: fresh weight, dry weight, cell viability, pH and carbohydrate consumption [59].

The results showed that the KETc7 suspension cell line grew very well, reaching a doubling time of 6.9 days and a specific growth rate (μ) of 0.10 d−1. The maximum biomass value was 14.36 gPS L−1 obtained at 15 days in culture.

#### *3.5.3 Embryo suspension cultures*

The KETc7 embryos callus cell line generated in solid B5 medium without phytoregulators was used to establish embryo suspension cultures. An inoculum of 10% was added into 250 ml Erlenmeyer flasks containing 100 ml of liquid B5 medium (**Figure 6**). The flasks were kept on an orbital shaker at a stirring speed of 115 rpm, under constant light conditions (24 μmol.m−2. S−1) and 25°C. After 15 days, the biomass was sub cultivated in the same conditions described above, and the culture was propagated.

#### *3.5.4 Cell suspension cultures in bioreactors*

To scale-up the *C. papaya* suspension cultures 2 L airlift bioreactors were employed, with the following geometric design: height (52 cm), diameter (7 cm), draft tube height (27 cm), diameter of inner draft tube (2.7 cm), and bottom clearance (2.0 cm) [60]. The air was sprayed at the bottom of the draft, generating an internal loop in which the upcomer is in the draft, and the downcomer in the ring.

#### **Figure 4.**

*Growth of C. papaya KETc7 cell suspension line at 15 days in a rotary shaker at 100 rpm, 25°C, in the dark.*

#### **Figure 5.**

*Cell viability of C. papaya KETc7 cell suspension line by the fluorescein diacetate (FDA) method at day a) 0, b) 7, and c) 15, in an Epifluorescence Microscope Nikon Eclipse E400 (40X).*

The bioreactor was sterilized and then filled with autoclaved B5 medium (1.8 L) supplemented with 30 g/L sucrose, 2,4-D and KN (2 mg/L each). Fifteen-days-old *C. papaya* KETc7 cell suspension line was used to obtain an inoculum of 10% (v/v) FW. The culture in bioreactor was incubated at 25 ± 2°C under continuous light (white light flux density of 50 μmol/m2 /s) for 30 days. The bioreactor was operated in a batch mode at 0.1 vvm for 15 days and subsequently at 0.8 vvm, until the end of the culture period. Under these conditions, an adequate mixing of the cell suspension was obtained. Antifoam (Dow Corning FG-10) was applied as required, by injection of 0.5 mL (0.1% v/v). The culture was sampled every three days and the concentration of biomass, pH and sugars, was determined. Results show that the K ETc7 cell suspension culture was able to grow uniformly and that the exponential growth phase was reached from days 6 to 12, followed by a stationary phase. The maximum biomass was of 18.6 ± 0.7 g/L DW (**Figure 7**).

#### *3.5.5 Embryo suspension cultures in bioreactors*

Growth of *C. papaya* KETc7 embryo suspension line was scaled-up in the 2 L airlift bioreactors described before. Two weeks old embryo suspension line was used to obtain an inoculum of 10% (v/v) FW. The culture in bioreactor was incubated at 25 ± 2°C under continuous light (white light flux density of 50 μmol/m<sup>2</sup> /s)

#### **Figure 6***.*

*C. papaya KETc7 embryo suspension line grown in B5 medium at 100 rpm, 25°C, and constant light (24* μ*mol. m−2. S−1).*

**Figure 7***. C. papaya KETc7 cell suspension line growing in airlift bioreactor: (a) day 0, (b) day 15, (c) day 30.*

*Development of an Oral Vaccine for the Control of Cysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97227*

#### **Figure 8***.*

*C. papaya KETc7 embryo suspension line growing in airlift bioreactor (a) embryo culture in airlift bioreactor at 30 days, (b) harvested embryos after 30 days in culture, (c) embryos observed in stereoscopic microscope (10×).*

for 30 days. The bioreactor was operated in a batch mode at 0.1 vvm for 15 days and subsequently at 0.8 vvm until the end of the culture period. Embryo culture of the line KETc7in bioreactor batch type process, showed uniform growth. A maximum biomass of 30 g/L DW was obtained, which represents 4 times more respect to the initial inoculum and the number of generated embryos was of 279 (**Figure 8**).

#### **4. Conclusions**

This review addresses oral vaccination as a feasible approach to prevent parasitic diseases. Since most anti-parasitic vaccines currently available are parenterally administered, their use involves high production and logistic costs and become inaccessible for underdeveloped countries. To cope with this issue, the use of papaya transgenic clones is herein proposed to develop an anti-cysticercosis oral vaccine and to assay its effectiveness against other parasitic infections of veterinary and/or public health interest. The use of biotechnological tools by escalation of suspension cultures would allow us to produce a vaccine in sufficient, controlled amounts for its direct application, reducing the use of antibiotics, and therefore the risk of bacterial resistance.

#### **Acknowledgements**

Thanks to Georgina Díaz, B.Sc., and Gonzalo Acero, B.Sc., for their technical support. Special thanks to Juan Francisco Rodríguez, B.Sc., for his help in preparing the original manuscript. This work was supported by the Programa de Investigación para el Desarrollo y la Optimización de Vacunas, Inmunomoduladores y Métodos Diagnósticos, Institute of Biomedical Research, UNAM.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Marisela Hernández1†, Anabel Ortiz Caltempa2†, Jacquelynne Cervantes1 , Nelly Villalobos3 , Cynthia Guzmán<sup>2</sup> , Gladis Fragoso1 , Edda Sciutto1 \* and María Luisa Villareal2

1 Institute of Biomedical Research, National Autonomous University of Mexico, School Circuit, University City, Mexico City, Mexico

2 Biotechnology Research Center, Autonomous University of the State of Morelos, Cuernavaca, Mexico

3 Faculty of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico, School Circuit, University City, Mexico City, Mexico

\*Address all correspondence to: edda@unam.mx

† These authors contributed equally.

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Development of an Oral Vaccine for the Control of Cysticercosis DOI: http://dx.doi.org/10.5772/intechopen.97227*

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#### **Chapter 3**

## Regulation of the Immune Response in Cysticercosis: Lessons from an Old Acquainted Infection

*Jonadab E. Olguín and Luis Ignacio Terrazas*

#### **Abstract**

In the last decades, we have learned some critical lessons about the relationship between the human body and its interaction with many infectious diseases, where regularly, the immune system has a major role in protection. We learned to differentiate between the immune response occurring in either an intracellular or extracellular parasitic infection, between innate and adaptive immune response, between either inflammatory or anti-inflammatory responses, and finally, we learned to recognize very particular mechanisms, such as the inability of the immune system to respond during very particular scenarios, such as the inability of T cells to both proliferate and produce cytokines even after their exposure to mitogens or specificantigens. Along with our increase in the knowledge in immunology, we figured out that immunoregulation and immunosuppression are processes used by many parasites to reduce the capacity of the immune system to eliminate them, and to persist in the host favoring their transmission and also to complete their life cycles. Immunoregulation involves several mechanisms such as anergy, apoptosis, induction of both suppressive cytokines and membrane-bound molecules, as well as specialized cell populations of the immune system like regulatory T cells, Alternative Activated Macrophages, or Myeloid-derived Suppressor Cells, that together modify the outcome of the immune response. In this chapter we will review the general differences between the different types of immunoregulation, the kind of cellular populations of the immune system used by the helminths *Taenia solium* and *Taenia crassiceps* to induce immunoregulation and immunosuppression and also, the mechanisms used by these parasites such as mimicking molecules of the immune system to replace directly these mechanisms. Understanding and deciphering all these regulatory mechanisms could be useful to develop new tools to control this infection.

**Keywords:** Cysticercosis, Immunoregulation, Immunosuppression, *Taenia solium*, *Taenia crassiceps*, Regulatory T cells (Treg cells), Alternative Activated Macrophages (AAM), Myeloid-derived Suppressor Cells (MDSC)

#### **1. Introduction**

Taeniasis and cysticercosis, both neglected diseases, are two kinds of infections caused by the same parasite, *Taenia solium*. Taeniasis is the intestinal infection caused by the adult form of the tapeworm *T. solium*, while cysticercosis is the tissue infection caused by the larval stage, cyst or cysticercus of *T. solium* [1]. Whereas taeniasis only affects the human and it is restricted to the small intestine, cysticercosis affect two hosts, the human and the swine, besides this stage of the parasite can allocate at different anatomical sites including the brain, causing neurocysticercosis (NCC). The vast majority of medical findings by natural infection has been made during cysticercosis, given its clinical relevance when the parasite encroaches on the central nervous system including the brain and the eye [2]. In the past, NCC represented a major health problem mainly in developing countries [3], being highly prevalent in the general registration of autopsies [3]. The only way to find samples for the study of NCC, were in patients diagnosed by neuroimaging: magnetic resonance and computed tomography, and also by determination of specific antigens by ELISA and western blot from blood and cerebrospinal fluid samples [4, 5].

Because taeniasis remains asymptomatic, there are no symptoms directly associated with the disease, only general symptoms like abdominal bloating and abdominal pain [1, 5]. For this reason, a model to understand the immunological interactions between the host and the parasite and also, to understand the evolutionary capacity of *Taenia* to survive in the host was necessary. Animal models like hamsters, gerbils and chinchillas, were developed in the past to have a better understanding of the immunology in this field [6], but a limited information about it was published, having a focus in the inflammatory response in the intestinal mucosa of chinchillas receiving an immunosuppressant treatment with methyl-prednisolone [7]. Because of the nature to develop taeniasis and the necessity to have a better understanding of the immune response against *T. solium*, it was necessary to know the immunology and the mechanism of protection used by a "close familiar" to this parasite: *Taenia crassiceps*.

#### **2. General aspects of the immune response during NCC and Taeniasis**

*T. crassiceps* is a tapeworm that generates natural infections in some definitive canine hosts like dogs, red foxes, and wolves in the northern hemisphere of the world. It also has an extensive reproduction rate in the pleural and peritoneal cavities of their intermediate host like wild rodents [8]. For a better understanding of their life cycle, go to the reference [8]. *T. crassiceps* ORF strain was obtained by Dr. Reino Freeman in 1952. This strain has a deficient capacity to develop the scolex, therefore, cannot colonize the intestines of its definitive hosts [9]. Most of the research about the immunology of *T. crassiceps* has been developed with the ORF strain, injecting either 10 or 20 metacestodes of this parasite in the peritoneal cavity of syngenic female BALB/c mice [10, 11].

The immune response against *T. crassiceps* has been investigated in an extensive way. During the acute infection by this helminth a strong Th1 immune response characterized by high levels of IL-2 and IFN-γ is induced and has been associated with host protection, but as the infection becomes chronic, levels of both IL-2 and IFN-γ decrease as well as IL-12 produced by macrophages [12]. These reduced levels of Th1-type cytokines correlate with increased levels of IL-4 at chronic infection stages, suggesting a switch between inflammatory response in the acute infection to an anti-inflammatory response in chronic infection, favoring the adequate microenvironment for both, parasite development and their persistence in the host (**Figure 1**). Susceptibility to *T. crassiceps* infection is STAT6-mediated, characterized by strong IgG1, IgE, IL-4 and IL-13 production [13]. Therefore, unlike the case of the vast majority of helminth parasitic infections, protection during experimental cysticercosis is mediated by Th1-type immune responses, while parasite

*Regulation of the Immune Response in Cysticercosis: Lessons from an Old Acquainted Infection DOI: http://dx.doi.org/10.5772/intechopen.100137*

#### **Figure 1.**

*Hypothetical/integrative model of immune regulation triggered by* T. solium *and* T. crassiceps *infection and their released products on different types of cells. Products of both parasites can be recognized by innate cells through several pattern recognition receptors, including TLRs, CD205, among others, and induce tolerogenic DCs or modulate the activation of macrophages by inducing the expression of different inhibitory molecules in its membrane such as PDL1, PDL2, Galectin-9 (Gal-9) as well as soluble inhibitory factors such as PGE2, and IL-13, while favors the expression of genes associated with M2 polarization, such as arginase, FIZZ1 and YM1, leading to the inhibition of T cell proliferation. In contrast, the inflammatory properties of both DCs and macrophages are inhibited by these parasite molecules and the production of TNF-*α*, IL-12, iNOS and ROS are dramatically reduced. Additionally, TsES and TcES inhibit T cell responses through the induction of T regulatory cells as well as B regulatory cells which both are an important source of IL-10. These infections together with their secreted products can also induce a Th2-biased response and a reduced CD8 response.*

establishment is associated with Th2-type mediated immune responses [14]. One of the most relevant findings observed during experimental cysticercosis, was the reduced proliferative capacity of T lymphocytes obtained from infected mice to either nonspecific or specific antigens during chronic infection, suggesting the strong immunosuppressive capacity of *T. crassiceps* [12, 15].

On the other hand, evidence from subcutaneous, visceral, muscle, lung and cardiac tissue infected with *T. solium* in patients suggest that, out of the nervous central system (NCS), this infection causes no symptoms, reducing the possibility to understand and describe the occurring steps during early immune responses [1, 16]. Once inside of NCS, *T. solium* induces different levels of damage depending on the developing site. If the cysticercus is located in the brain parenchyma, it survives during different periods of time, from months to years, but eventually evolves in resolution [1, 2]. However, if the infection is located outside of the parenchyma's (subarachnoid) brain, it is associated with edema, inflammation and increased mortality rates around 20% in patients without a correct treatment [2, 17]. Besides its location on the NCS, also exists a relationship between the intensity of the symptomatology and the number and size of the larvae causing the infection. In fact, it was recently hypothesized that the gut-brain-axis has a major role in the manifestation of symptoms during NCC, mainly in patients with mental illness, depression and epilepsy [18], highlighting the importance for the microbiota in this field. Also, it was suggested that these interactions for the gut-brain-axis are dependent on galectin-7 (Gal-7) expression in brain endothelial cells during human *T. solium* cysticercosis [19]. Thus, the main actors for the development of cysticercosis are the host immune response, the microenvironment for the parasite development either the gut or NCS, the microbiota, and the host health status.

The immune response described in the *T. solium* rodent model has been helpful and relevant to understand the immunobiology during taeniasis and cysticercosis, being a main feature the suppression of the immune response. Next, we will try to describe both the immunoregulatory mechanisms and the direct effects of molecules secreted by *Taenia* parasites to induce immunoregulation.

#### **3. Immunoregulation during cysticercosis**

An inflammatory response during the initial infection process is necessary to induce immunity, to reduce parasite load and finally to have protection in cysticercosis. However, since basic science started to clarify the role of the immune response during cysticercosis, some special discoveries have been observed only in this helminth infection, suggesting a process of transformation from inflammation to an anti-inflammatory response, tipping the balance towards the parasite survival. Is necessary to pay attention in the fact that, during some helminth infections, the Th1 to Th2 switch is caused to keep the balance between immunity in the host with tissue repairing, and for the survival of the parasite o for its expulsion from the host, example of that are *Schistosoma mansoni*, *Nippostrongylus brasiliensis and Heligmosomoides polygyrus* infections [20, 21]. However, this Th1 to Th2 switching in cysticercosis appears to absolutely favor parasite survival. Also, this switch process is an initial step to induce an immunosuppressive process orchestrated by the parasite, or also, as a possibility, the microenvironment caused by the infection has a strong effect culminating in the incapacity to the immune response to react against the infection (**Figure 1**). Some evidence suggests that immunosuppression may be caused by T-cells, myeloid-derived cells or directly by parasite molecules like *Taenia crassiceps* excreted/secreted antigens (TcES) or *Taenia solium* excreted/ secreted antigens (TsES). Also, it was suggested that asymptomatic NCC is caused by a strong period of immunosuppression by live *T. solium* parasites, because brain inflammation is not observed during the development of the infection and while the parasite remains alive [22].

#### **3.1 Immunoregulation mediated by T-cells**

During some intracellular parasitic infections, like toxoplasmosis, trypanosomiasis and leishmaniasis, an incapacity of T lymphocytes to proliferate in response to antigen-specific or polyclonal mitogens has been described, mainly during acute infection [23–26]. Although the general observation is that the process of immunosuppression starts at the beginning of the chronic *T. crassiceps* infection, it was shown that during acute intraperitoneal (ip) infection in mice, there is a significant decreased percentage of T-CD4+ , T-CD8+ cells and B-CD19<sup>+</sup> cells at the infection site, starting at 3 days post infection (dpi) and culminating at 16 dpi [27]. These results correlate with increased levels of apoptosis, mainly in eosinophils. Perhaps, *T. crassiceps* begins to develop its suppressive microenvironment since the beginning of the development of the infection, such as reported for protozoan infections. During *T. crassiceps* chronic infection, a reduced proliferative capacity of T cells was described [12], especially in CD8-cytotoxic T cells [28]. Recent observations by our laboratory described that, this reduced capacity to induce cytotoxicity by CD8+ cells is caused by increased expression of Tim-3 and PD1 molecules in an IL4-Rα, STAT-1 and IFN-γ independent-pathway (**Figure 1**, Olguin JE et al., unpublished data). Interestingly, both expression of Tim-3 and PD1 is increased in adaptive regulatory CD4<sup>+</sup> Foxp3− T cells but not in natural Treg cells. In fact, during the chronic phase of experimental cysticercosis, we observed reduced percentages of Treg

#### *Regulation of the Immune Response in Cysticercosis: Lessons from an Old Acquainted Infection DOI: http://dx.doi.org/10.5772/intechopen.100137*

cells, which is contrasting with some published data. For example, a study reports that cocultured cysticerci of *T. solium* with human monocyte-derived DCs, induces Foxp3 expression in CD4+ naïve T cells *in vitro* and also, increased percentage of suppressive-related molecules (**Figure 1**) [29]. The same research group showed a descriptive study in patients with NCC, observing an increased expression of natural and adaptive Treg cells in blood [30], but the authors did not show, whether these induced and natural Treg cells had the capacity to suppress another population of immune cells, for example either T-CD8 or activated CD4 T cells. Maybe, the differences observed between NCC and experimental cysticercosis in Treg cells are explained by the site where the sample was obtained (NCS and blood in *T. solium*, peritoneum in *T. crassiceps*), and by the nature of the infection. However, it is clear that, whatever scenario is observed, T cell-mediated regulation is a critical mechanism involved during the development of experimental or natural cysticercosis. Also, observations made by our group in *T. crassiceps* infection are different to those done in other helminth infections. A recent study of hookworms like *Ancylostoma duodenale*, *Necator americanus*, *Ascaris lumbricoides* and others, showed by mass cytometry a clear profile of Th2 cells, favoring increased expression of CTLA-4 in Treg cells, and B cells producing IL-10 in infected Europeans and Indonesians patients [31]. Maybe, it is necessary to describe more specific surface markers to define the population of suppressive and/or regulatory T cells. For example, it has been described as a highly suppressive T regulatory Type 1 population (Tr1) during a helminth scenario by the co-expression of CD49 and LAG-3 [32]. Tr1 cells are different from natural regulatory (Treg) cells because they do not express constitutively the Foxp3 transcription factor [33].

As well as in *T. crassiceps* infection, during NCC a period of immunosuppression has been reported. One study extracting polymorphonuclear (PMN) cells from 11 patients diagnosed with NCC, showed a clear immunosuppression in response to TsES antigens from the scolex of *T. solium*, also NCC patients with calcified cysts displayed increased immunosuppression [34]. The same study showed the suppressor capacity of TsES, completely inhibiting the proliferative response to mitogens like phytohaemagglutinin (PHA) and concanavalin A (ConA) [34]. These results are different to those published by a different research group, under the same conditions, where NCC patients without treatment did not show differences in the percentages of CD3+ , CD4+ and CD8+ T cell populations. Moreover, blood cells from these NCC patients showed the same proliferative capacity to ConA or crude *T. solium* antigens compared with controls, and finally also PMN cells produced IL-2 [4]. Is not clear what is the correct scenario, maybe another process or data were not considered in the clinical history between these both studies, like age of the patient, sex, or any oncological or immunosuppressive constitutive status. In fact, more recently published data showed that if analyzed groups of patients with NCC are divided by the local site of the infection in either parenchymal (infection is resolved in general) or subarachnoid (increased symptomatology and pathology), the group of patients with subarachnoid infection has an increased immunoregulatory microenvironment characterized by IL-10, TGF-β and expanded Treg cell frequencies *ex-vivo* [35].

#### **3.2 Immune-regulatory myeloid-derived mechanisms**

The generation of an immune-regulatory environment could have an effect not exclusively in T cells, but in all immune cells, including all myeloid-derived lineages such as macrophages, dendritic cells (DCs) and PMNs. Thus, myeloid cells are key players in the immune response against cysticercosis. In fact, there is evidence suggesting an increased profile of alternative activated macrophages or

M2 macrophages involved in parasite expulsion and tissue repair during helminth infections, which induce protection to the host [36]. However, in cysticercosis, the scenario appears to be different.

During *T. crassiceps* infection it was suggested that recruited CD11b<sup>+</sup> Gr1+ myeloid-derived suppressor cells (MDSCs) impaired the T cell proliferation by secretion of high amounts of nitric oxide (NO) at early stages of an intraperitoneal infection. This classic inflammatory activation is switched at chronic stages, where CD11b+ Gr1+ cells express arginase, YM-1 and FIZZ1 genes, associated with an M2 phenotype (**Figure 1**). This immunosuppressive microenvironment favors IL-4 and IL-13 production, expanding the MDSCs population and also, inducing lipid mediators' activation like 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid, associated with the immunosuppression of T cell proliferation [37]. Also, alternative-activated macrophages (AAMs or M2) from chronic *T. crassiceps* infection have the ability to produce high levels of IL-6 and Prostaglandin E2 (see below), reducing the proliferative capacity of CD4+ T cells in a STAT-6 dependentpathway (**Figure 1**) [11], suggesting that AAMs are necessaries to induce the permissive microenvironment for the colonization of *T. crassiceps* infection. In fact, it was demonstrated that an early in vivo depletion of AAMs by using clodronate liposomes, increases the resistance against *T. crassiceps* [38], making stronger the hypothesis that experimental cysticercosis has an AAMs-dependence for a successful infection. Later, it was shown that these AAMs induce anergy on CD4<sup>+</sup> T cells during *T. crassiceps* infection, and that such an event depends on PDL1 and PDL2 expression in the surface membrane of AAMs [39].

On the other hand, monocyte-derived DCs co-cultured with CD4 naïve cells in presence of *T. solium* cysticerci promotes both, Treg and DCs cells differentiation towards a tolerogenic profile featured by a higher expression of Signaling Lymphocytic Activation Molecule 1 (SLAM1), B7-H1 and CD205 molecules (**Figure 1**). These results suggest that *T. solium* cysticerci has the ability to induce both Treg cells as well as suppressive or tolerogenic DCs [29].

#### **4. Regulatory mechanisms mediated by cytokines, antibodies, and soluble immune factors**

#### **4.1 IL-10**

IL-10 is produced by innate cells like myeloid and plasmacytoid DCs and macrophages, and is also produced by Breg cells, Th2, Th17 and Treg cells from the adaptive immune response. This capacity to be produced from several immune cell lineages, depends on the signal pathways activated like ERK, and also from transcription factors like STAT3, STAT4, STAT6 and cytokines like TGF-**β** [40]. One of the main features of IL-10 is its capacity to induce immunosuppression, reducing IL-12 and TNF-a levels. By transcriptomic array analyses, we observed that miR-125a-5p, miR-762, and miR-484 microRNAs, are associated with the targeting of inflammatory profiles of macrophages favoring the IL-10 signaling pathway, suggesting that *T. crassiceps* and its products induce post-transcriptional suppression mechanisms of the immune response [41]. Earlier studies performed in experimental cysticercosis caused by chronical *T. crassiceps* infection, indicated a strong Th2 biased immune response featured by high production of IL-4, IL-6 and IL-13 cytokines as well as increased IL-10 levels [12]. These findings were just recently confirmed by an independent group highlighting the importance of IL-10 cytokine [42].

*Regulation of the Immune Response in Cysticercosis: Lessons from an Old Acquainted Infection DOI: http://dx.doi.org/10.5772/intechopen.100137*

#### **4.2 Transforming growth factor beta (TGF-β)**

TGF-β is a cytokine involved in some situations during immune and not immune phenomena. It has a role in the control of cell proliferation and differentiation of some cell types like either Treg or Th17 cells [43, 44]. Also, by itself, it has the capacity to induce a suppressive environment in scenarios where required, like mucosal immune reactions. Dysregulation of TGF-β generates inflammatory disorders such as spontaneous colitis [45]. In the *T. solium* genome were found some genes homologs with the TGF-β receptor family, including some evolved in its down-stream transduction pathway. In fact, it was confirmed by RT-PCR and western blot assays that cysts of *T. solium* express the type I and type II receptor for TGF- β. Also, the addition of TGF- β to the culture media for both *T. crassiceps* and *T. solium* adequate conditions, promotes both the reproduction of *T. crassiceps* and the survival of *T. solium in vitro*. Finally, high levels of TGF-β were found in the cerebrospinal fluid from patients diagnosed with NCC [46]. All these results suggest a strong direct and indirect role for TGF-β in the process of immunosuppression during *T. solium* infection.

#### **4.3 Osteopontin**

Osteopontin (OPN) is a Th1 type cytokine upstream of IL-12 that has a role in the granuloma formation in inflamed tissues [47]. It was shown that blood cells co-cultured with TsES or viable cysticerci from *T. solium* led to decreased levels of OPN, IL-12 and IFN-γ. Injection of recombinant OPN into tissues surrounding implanted cysticerci enhances inflammatory responses, which suggests that TsES may have molecules that block OPN activity as a target for immunosuppression [48].

#### **4.4 Antibodies**

Humoral immune response has been described for its essential role against helminth infections, being IgE antibody isotype a cornerstone to induce protection [49]. However, during taeniasis and cysticercosis, little information about the role of B cells has been described. It was shown that an antibody called anti-GK-1 (IgG) obtained from the serum of pigs infected with *T. solium*, has an epitope shared by both *T. crassiceps* and *T. solium* [50] and also, has the capacity to inhibit the development of *T. solium* cysticerci into adult stage by recognition of the cyst protein KE7 [51]. This protective role for anti-GK1 antibodies is complement-mediated during *T. solium* infection [50, 52]. However, these results are the only data obtained for humoral immune response during taeniasis and cysticercosis. During experimental cysticercosis and during NCC, is clear that immunosuppression favors the establishment of the parasite, and this GK-1 antibody-mediated mechanism has naturally no success; maybe a molecule of the parasite has the capacity to inhibit this protective function, which in turn induces immunosuppression. Is necessary to clarify this point with specific and deeper experiments.

#### **4.5 Prostaglandin E2**

Some lipids from eicosanoid family derivatives from arachidonic acid, like prostaglandin E2 (PGE2), have been described as potent immunosuppressant molecules [53]. It has been described that administration *in vivo* of PGE2 in *T. crassiceps* infected mice favors both parasite growth and cytokine production of IL-10 and IL-6 by splenocytes and reduces the proliferative capacity of splenocytes stimulated with ConA. On the other hand, the administration of indomethacin, an inhibitor of PGE2 synthesis, induced the reduction of both the parasite growth and cytokine production of IL-10 and IL-6, increasing the ConA-proliferative response of splenocytes (**Figure 1**) [54]. These results suggest that *T. crassiceps* can induce the production of PGE2 indirectly from some cellular types, like almost all cells of the host, as a mechanism of immunoregulation [54]. Also, it is possible that some molecules from TcES could be a similar biomolecule like PGE2, mimicking their function and directly inducing immunosuppression, like the TGF-β phenomenon observed during *T. solium* infection.

#### **5. Immunoregulation mediated by Taenia-derived products**

#### **5.1 Paramyosin**

Paramyosin is an α-helical coiled coil 100 KDa protein that is present in muscle and tegument of the larval stage of *T. solium* [55]. This protein can bind to the protein C1q of the complement, therefore, inhibiting the complement cascade [56]. This was the first evasive mechanism described for this parasite. Vaccine strategies performed to block the activity of this protein resulted in almost 80% of protection [57].

#### **5.2 Glutathione transferase**

Glutathione transferase (GST) is an essential enzyme in the metabolism of cestodes, mainly for the detoxification of xenobiotics, it is localized on the cysticerci tegument of *T. solium* [58]. This molecule appears to have a immunomodulatory role given that its use as a putative vaccine was able to reduce parasite load on experimental cysticercosis, mainly by activating macrophages to produce proinflammatory cytokines [59]. These data indicate that *T. solium* and *T. crassiceps* may have pro and anti-inflammatory mixed molecules.

#### **5.3 TcES or TsES antigens**

Analysis of *T. solium* excreted/secreted antigens (TsES) showed a cysteine protease activity for these molecules, having the capacity to induce apoptosis specifically in CD4+ but no in CD8+ T cells, which is evidence of a direct mode of immunosuppression over a population of the immune response (**Figure 1**). Cocultured cysts of *T. solium* with lymphocytes *in vitro* have not the same effect to induce apoptosis like TsES [60]. Also, it was suggested that natural infection of pigs with *T. solium* cysticerci recruits CD3+ cells to the brain which are killed by apoptosis [61].

Studies in our laboratory demonstrated that TcES products have the capacity to block TLR4 and TLR9 initial signaling pathway in DCs, which has a negative effect over their maturation, their production of pro-inflammatory cytokines and also, to induce alloreactive T cell proliferation, but in an IL-10 independent pathway. All these regulatory effects were carbohydrate-dependent in the TcES, because the chemical alteration of glycans switch this tolerogenic environment to one favoring DCs maturation and secretion of pro-inflammatory cytokines (**Figure 1**) such as IL-12 and TNF-α [62]. Moreover, it was shown that the *in vivo* treatment with TcES, has the capacity to induce the differentiation of monocytes to AAMs expressing PDL1 and PDL2, which in turn down-modulate the activity of experimental autoimmune encephalomyelitis EAE [63]. Furthermore, it was shown that in the murine *Regulation of the Immune Response in Cysticercosis: Lessons from an Old Acquainted Infection DOI: http://dx.doi.org/10.5772/intechopen.100137*

model of NCC with the helminth *Mesocestoides corti*, the inhibition of TLR-initiated regulation of inflammatory cytokines exists. This effect is caused by an inhibition of acetylation and phosphorylation of both NF-kB and JNK which causes an accumulation of Ca2+ in the endoplasmic reticulum [22]. Probably, this phenomenon is similarly used by *T. solium* during initial establishment of the infection, however, deeper research is necessary in this field (**Figure 1**).

Also, it was shown that the nature of antigens of *T. solium* is essential to induce a proper immune response. Within *T. solium* crude lysate antigen, cyst wall antigen, and cyst fluid antigen, only low molecular weight fractions of cyst fluid are immunodominant, with the capacity to induce the production of inflammatory cytokines, but mainly higher levels of IL-10 and IL-4 by stimulated lymphocytes of patients with NCC [64]. Besides, it was suggested that the time of TcES obtention has a different impact over the kind of immunosuppression observed; TcES obtained early in infection, suppress the proliferative response of splenocytes stimulated with ConA than TcES obtained late in infection. Also, these early obtained TcES suppress the production of IFN-γ and IL-4 efficiently [65].

#### **6. Conclusions**

It has been largely known that helminthic infections induce strong Th2 mediated immune responses associated with regulation of inflammatory responses. Here, it has been described the different molecules and pathways altered by *T. solium* and *T. crassiceps* infection. Is noteworthy that some clinical studies point out that this immunomodulation favors both the parasite and host survival when the parasite is allocated in the brain, mainly because the inflammatory response is inhibited, avoiding the damage expected from a strong inflammatory response.

The anti-inflammatory activities and immunoregulatory properties found in both *T. solium* and *T. crassiceps* parasites and their products, can be useful beyond the host–parasite interactions. During some allergic diseases like asthma and rhinitis, the hygiene hypothesis has strengthened the idea of the historical necessity to down-modulate the immune response by mechanisms of natural coevolution, like the infection with helminth parasites [66]. In the same order of ideas, because of all these suppressive capacities of both *T. crassiceps* parasites and their TcES molecules, we hypothesized that it probably has the capacity to modulate chronic diseases associated with inflammation. We observed a clear role of both *T. crassiceps* and their TcES antigens in the modulation of experimental autoimmune encephalomyelitis (EAE) [67, 68], colitis-associated colon cancer (CAC) [69, 70], experimental colitis [71] and type 1 diabetes [72].

Lately, we have observed that the mechanisms used by the parasites that cause infectious diseases, such as taeniasis and cysticercosis, are very similar processes, and we dare to suggest that they are the same, to those occurring during the main oncological (solid) pathologies. The fact that a carcinogenic transformed cell induces an immunosuppressive process through immune-checkpoints such as PD1, CTLA-4 or Tim3, is a mechanism that had already been described in the past, during cysticercosis. So, immunoregulation and immunosuppression are natural selection mechanisms that pathogens take advantage of to be able to survive in a hostile environment and turn it to favor them, to face a variety of processes of continuous and varied attack of the immune response. Therefore, understanding and deciphering the why, how, and when these natural selection processes occur, we will be able to apply the lesson obtained during infectious diseases in processes affecting the current public health, like the main oncological pathologies.

### **Acknowledgements**

"This work was supported by CONACYT grant number A1-S-37879 and DGAPA-PAPIIT-UNAM grants numbers IA209720 and IN226519.

### **Conflict of interest**

"The authors declare no conflict of interest."

#### **Abbreviations**


*Regulation of the Immune Response in Cysticercosis: Lessons from an Old Acquainted Infection DOI: http://dx.doi.org/10.5772/intechopen.100137*


#### **Author details**

Jonadab E. Olguín1,2 and Luis Ignacio Terrazas1,2\*

1 Laboratorio Nacional en Salud: Diagnóstico Molecular y Efecto Ambiental en Enfermedades Crónico-degenerativas, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México (UNAM), Mexico

2 Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, UNAM, Mexico

\*Address all correspondence to: literrazas@unam.mx

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Regulation of the Immune Response in Cysticercosis: Lessons from an Old Acquainted Infection DOI: http://dx.doi.org/10.5772/intechopen.100137*

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#### **Chapter 4**

## The Long Road to the Immunodiagnosis of Neurocysticercosis: Controversies and Confusions

*Marcela Esquivel-Velázquez, Carlos Larralde, Pedro Ostoa-Saloma, Víctor Hugo Del Río Araiza and Jorge Morales-Montor*

#### **Abstract**

To date, even widely studied, there is not a standard diagnostic method to detect neurocysticercotic patients. The later due to the complex nature of cysticercosis disease and the simplicity of common immunological assumptions involved in explaining the low scores and reproducibility of immunotests in the diagnosis of neurocysticercosis. To begin with, the few studies dealing with the immune response during neurocysticercosis are not conclusive, which of course it is crucial to develop an immunodiagnostic test. Their full recognition should clear confusion and reduce controversy as well as provide avenues of research and technological design. In here, logical arguments add that even under common immunological assumptions, serology of neurocysticercosis will always include false negative and positive results. Thus, serology is no strong support for medical diagnosis of neurocysticercosis (NC). In contrast, immunotests performed in the cerebrospinal fluid (CSF) of neurological patients should have fewer false positive and fewer false negatives than in serum. To conclude, it is argued that high scores in serology for NC will not yield to usual approaches and that success needs of a concerted worldwide effort. A more punctilious strategy based on the design of panels of confirmed positive and negative sera needs to be construed, shared and tested by all interested groups to obtain comparable results. The identification of a set of specific and representative antigens of *Taenia solium* (*T. solium*) and a thorough compilation of the many forms of antibody response of humans to the many forms of *T. solium* disease are also to be considered as one of the most importants factors to the disease.

**Keywords:** Cysticercosis, Neglected Diseases, Neurocysticercosis, Immunodiagnosis

#### **1. Introduction**

Neurocysticercosis (NC) is a disease caused by the larvae (or cysticerci) of the intestinal parasite *Taenia solium* (*T. solium*) when the cysticerci lodges in the central nervous system (CNS). It is considered one of the most important parasitic disease of the CNS [1–3]. Cysticerci may infect humans and may also locate elsewhere of CNS, in skeletal muscles, heart, eyes, diaphragm, tongue and subcutaneous tissues, causing a condition simply referred to as cysticercosis. Cysticerci develop in humans and also in pigs from eggs produced by the adult tapeworm living in the intestine of humans and shed to the environment upon defecation, thus contaminating soil, waters and food.

The most serious condition of *T. solium* disease affecting human health is NC. An estimated 60% of NC cases are non-symptomatic [4], while the rest are symptomatic and exhibit a wide variety of neurological symptoms, being chronic epilepsy and headache the most noticeable [4–6]. Severe forms of NC develop meningitis, encephalitis, arteritis, areas of cerebral infarction and gliosis, as well as anatomical distortion and compression of intracranial structures causing blockade in the flow of cerebrospinal-fluid (CSF) [5–7], frequently leading to endo-cranial hypertension and requiring specialized medical attention and/or surgery to derive CSF and/or remove the parasite. The severe forms of NC seriously impair the patients´ health and may lead to death. Medical diagnosis of NC is impossible on clinical data alone as it presents a variety of nonspecific symptoms [8], while confirmatory diagnosis is established by biopsy, cranial CAT-scans and/or cranial NMR images showing nodular lesions of the brain usually suffice in most cases.

Immunodiagnosis of NC (IDxNC) has long been sought because of the disease's prolonged silent or ambiguous clinical pictures and also because of the low accessibility and impossibly high costs of CAT-scans and NMR-images in endemic countries [1, 9–11]. Not only an effective IDxNC would be a most practical way to facilitate medical diagnosis for millions of poor people in endemic countries, it would also supply sero-epidemiological studies with a low-cost indicator of prevalence of infection. In addition, a positive immune-test would rise the clinical suspicion of early non-symptomatic NC which, if confirmed, would allow to offer early treatment before the parasite does much irreversible CNS damage. Further, simplification of copro-parasitological studies in stools by an immune-test would help to identify carriers of live tapeworms and treat them in order to interrupt transmission in the explosive stage of massive egg production.

Many immunological methods have been tried to detect antibodies and/or antigens of *T. solium* in serum or CSF and feces, and even in urine and saliva [12–16], with variable levels of success in detecting NC cases and tapeworm carriers [17, 18]. The gallery includes *in vitro* tests using complement fixation, precipitation, agglutination, radioimmunoassay and enzyme-based detection systems (ELISA and Western Blots) [8, 19, 20]. Antigens used in diagnosis also vary from whole antigen extracts [14, 21, 22], secreted antigens [23–27], semi-purified fractions and purified natural proteins [6, 12, 16, 28] to recombinant proteins [4, 6, 8–11, 29, 30], and synthetic peptides [31–33], either from *T. solium* or from homologous parasites as *Taenia crassiceps* [2, 13, 19, 21, 32, 34], *Taenia saginata* [22, 35] or *Taenia taeniaeformis* [36]. Most reports initially claim very high specificity/sensitivity scores, sometimes even as high as 100/100%. Enthusiasm soon calms as the methods are applied by different laboratories, in larger numbers of cases and in various epidemiological scenarios of the disease [14, 28, 37, 38]. A sober statement about the state of the art at present times would claim a sensitivity that ranges from 50 to 85% (15–50% false positives) and a specificity of about 80–90% (10--20% false negatives), with large variations within and between tests and low reproducibility between laboratories [14, 28, 38].

#### **2. Generalities of immune response to** *Taenia solium* **cysticercosis**

In recent times it has been found that cysticercosis is importantly driven by the hosts neuroendocrine system function, particularly sex steroid hormones (Morales-Montor and Larralde c, 2005). *Taenia* parasites have developed elaborate

#### *The Long Road to the Immunodiagnosis of Neurocysticercosis: Controversies and Confusions DOI: http://dx.doi.org/10.5772/intechopen.98723*

mechanisms of interacting with their intermediate hosts. The oncospheres which invade the intermediate host are susceptible to antibody and complement. However, by the time the host has generated an antibody response, the parasites have begun to transform to the more resistant metacestode. The metacestodes have elaborate means of evading complement-mediated destruction, including paramyosin which inhibits C1q, taeniaestatin which inhibits both classical and alternate pathways, and sulfated polysaccharides which activate complement away from the parasite. Similarly, antibody does not seem to be able to kill the mature metacestode. In fact, the parasites may even stimulate the host to produce antibody, which could be bound via Fc receptors and used as a source of protein. Finally, taeniaestatin and other poorly defined factors may interfere with lymphocyte proliferation and macrophage function, thus paralyzing the cellular immune response. Since the symptoms of NC are typically associated with a brisk inflammatory response, we hypothesize that disease is primarily caused by injured or dying parasites. This hypothesis raises important questions in assessing the role of chemotherapy in the management of NC, as well as in the evaluation of clinical trials, most of which were uncontrolled (Morales-Montor et al., 2006).

The generation of protective T cell responses to cysticercosis is a complex process in which cytokines and costimulatory molecules provide signals that direct the development of adaptative immunity (13). The characterization of T cell responses as belonging to either Th1-type responses (dominated by the production of IFN-γ and associated with cell-mediated immunity) or Th2-type responses (characterized by production of IL-4 and IL-5, and associated with humoral immunity) was important because it provided a basis for understanding how T cells contribute to resistance, or susceptibility to cysticercosis (14). Subsequent studies distinguished the role of IL-12 and IL-4 in the development of Th1 and Th2 responses, respectively, but there are other cytokines involved in this process (13). Succintly, it can be sustained that immune response to the worm (adult stage of *T. solium*) is limited to Th2-type mechanisms, while the line of defense against the cysticercus is a mixed

#### **Figure 1.**

*Cysticerci or adult parasite and associated host immune cells. Mi, microglia/dendritic cell; Mo, macrophage; No, neutrophil; Eos, eosinophil; Th, T helper lymphocyte; Th1, T helper lymphocyte type 1; Th2, T helper lymphocyte type 2; BL, B lymphocyte; PC, plasmatic cell.*

Th1-Th2-type immune response, with dominance of Th1-type immune response mechanisms involved in limiting parasite growth and expansion (**Figure 1**).

#### **3. Sources and effects of controversy**

Low sensitivity and specificity of IDxNC, as well as variability of results within each method and irregular reproducibility between different laboratories, are cause of discussion and confusion. More than 50 years of insufficiently planned and disaggregated individualistic research using different materials, reagents, techniques and conditions of endemial are involved. Policy of publication favoring alleged breakthroughs tells the luminous half of the stories, creating the false impression that similar results are to be expected by all. The surging of commercial kits and their accompanying propagandistic fanfare has fueled dispute and nurtured distrust because of suspected conflicts of interest without much improvement in diagnostic capacity. The serious problem caused by all this is that the jingle of controversy and confusion has reached medical practice and introduced doubts on the significance of serology in medical diagnosis and epidemiological study surveys. This has in turn retarded the recognition of *T. solium* disease as the great threat it is to human health and the high costs it incurs to public health in endemic countries. It might be of help to clarify the major causes behind the low performance of IDxNC as a preliminary step to reach a consensual agreement on the meaning of its results, its limitations and the ways for improvement.

Low performance and variability are usually thought to rise from the technical virtues or pitfalls of the different available immunological tools and reagents used. There is some reason for argument here but there is much more than that to fully explain the incoherent results and to incorporate in the design of a strategy with a chance of worldwide solid success. Rarely is it recognized faulty results may rise from over-simplified immunological assumptions about this particular host–parasite relationship, incomplete knowledge of the *T. solium* antigen repertoire and/or the immunological complexities derived from the many forms *T solium* has of affecting humans. Because the pleomorphism in *T. solium* disease sets the levels of difficulty for immunological discrimination and is the least recognized cause of controversial results, here we shall describe in somewhat fastidious detail its many different faces.

The exercise illustrates how hard it is the task of immunotests when put to effectively discriminate from the multiple faces of *T solium* disease the one and only of NC. It will also suggest ways of clustering the significant from the insignificant discriminations, for medical as distinguished from epidemiological purposes, as well as point to what is possible and what impossible. Inevitably, some of the major immunological assumptions behind the presence or absence of antibodies and/or antigen in an individual must be dealt-with to some extent since they interact with the disease polymorphism to increment the difficulties of immunodiagnosis for NC. The exercise also explains many of the discrepant findings and should clear some of the controversy as well as point to ways of improvement.

#### **4. The many faces of** *T. solium* **disease**

Any human population under consideration may be divided in two sets according to their having had come in contact with *T. solium* (*I*) or not (*0*) (**Figure 2**).

*The Long Road to the Immunodiagnosis of Neurocysticercosis: Controversies and Confusions DOI: http://dx.doi.org/10.5772/intechopen.98723*

#### **Figure 2.**

*Schematic representation of the different possible subsets of the contact and infection of human population with* Taenia solium*.*

The set *I* includes at least 48 different subsets depending on whether the contact occurred a long (*l*) or short (*s*) time before sampling; the parasite was rejected (*r*) or it victoriously established in the host (*v*); the parasite is in the stage of a tapeworm (*t*) or as a cysticerci (*c*); if the cysticerci is located in the nervous system (*n*) or elsewhere (*e*) or in both (*ne*); if the cysticerci are few (*f*) or multiple (*m*); and if they are dead (*d*) or alive (*a*) or degenerating (*x*) (**Figure 2**).

The projection of positive serology upon the I set involve a number of immunological assumptions listed in **Table 1**.

Assuming the minimal, it may be concluded that:


In consequence:


If additional assumptions are added, then:


#### **Table 1.**

*Immunological assumptions involved upon positive serology within the I group (persons who has or had contact with* Taenia solium*).*

3.Antibodies and antigens would be more likely (but not exclusively) to be found in all the *r* subsets of *I* that combine with *m* and *a* (that is, in all cysticercosis cases, acquired shortly or long before sampling, with many and live cysticerci located in the brain or elsewhere) and in tapeworm carriers. But more likely they would be found in the CSF in the neurocysticercosis (*n*) subsets combining with *m* and *a*, in the SERUM for the *e* (elsewhere cysticercosis) subsets also combining with *m* and *a*, and in the feces of *t* (carriers of live tapeworms). The precise magnitude of each likelihood is to be assessed in perhaps each endemic situation.

#### **5. Clearing some discrepancy**

From the above description of the variety in *T. solium* disease of humans and the usual and rather liberal assumptions about the quality of the immunological reactants and the nature of the immune response to this particular parasite many of the discrepancies in the performance of different immunotests in different trials may be explained. The most important being the variation in the composition of the set of control not-NC individuals (i.e., some containing more members of the *e* or *t* subsets of *I* would thwart specificity due to many false positive results) and/or in the control NC individuals (in which an undue number of the *d* subset would lower sensitivity). Likewise, the control NC individuals are frequently a mixed lot of NC patients, differing in time of evolution, number and location of cysticerci, form and time of medical and surgical treatment, general health and nutritional status, age, gender, race, etcetera, that can possibly affect their immune reactivity [39–46] (**Figure 3**).

The use of domestic and probably differently composed sets of presumed control *I* and *0* individuals and of NC and not-NC individuals accompanying each immunotest trial is widespread and thus suspect of being a major cause of incoherent results between trials.

Variation between different trials would also follow from differences in the probability distribution of immunologically positive and negative individuals in different situations of endemia (i.e., high and low endemia, urban and rural transmission) and in the simplification of the forms of disease by way of binomial variables (i.e., long or short time of exposure before sampling, single or multiple cysticerci,

*The Long Road to the Immunodiagnosis of Neurocysticercosis: Controversies and Confusions DOI: http://dx.doi.org/10.5772/intechopen.98723*

#### **Figure 3.**

*Factors involved in neurocysticercosis. The development of neurocysticercosis depends on many factors from either the host or the parasite. The factors affecting the immune response of the host are particularly important for the immunodiagnosis of NC as they may affect the results between individuals.Differences in representativity of the whole of the parasite antigens and in cross-reactivity with other antigens in the geographic and endemic background, as well as differences in relative concentrations of reactants and conditions of reaction are additional suspect sources of variation.*

dead or alive (not dying) when they are not really so (i.e., individual may be carrying 1, 2, ..., n cysticerci) and some are continuous (i.e., time of exposure before sampling) and even non-disjunctive (i.e. dead, degenerating and live cysticerci may coexist in an individual).

The selection of the *T. solium* antigens to be used as reactants in the immunotests also vary widely among the different immunotests and also within the same immunotest applied to different endemic conditions and geographic locations.

#### **6. Immunological assumptions**

Of all the immunological assumptions necessary to interpret the results of immuno tests in diagnosis of NC, the less tenable are those implying there are no cross-reactions with other parasites endemic in the area, that all humans react equally to infection and that the set of antigens selected for the immunotest are shared by all individual cysticerci and tapeworm in the species (**Figure 4**).

The question of antigen cross-reactivity is usually dealt-with by selection of the set of *T. solium* antigens most reactive with positive control samples (confirmed NC cases) and less reactive with negative control samples (presumably without NC), all gathered from donors residing in the endemic areas [47, 48]: a sensible procedure in principle but usually lacking in proof of the statistical representation of the population affected by the other pathogens and in the certainty of negative control samples with respect to clinically silent NC and cysticercosis located elsewhere. Failure to control antigen cross-reactivity results in false positive tests. That not all humans react equally to infection is an additional source of false negative immunotests. Heterogeneous immune response of humans to pathogens is well known in a number of infections, possibly all, and although not thoroughly explored in *T. solium* disease it follows from differences in levels of antibodies of control and problem samples as well as differences in the published images of WB [47–49]. Besides, NC cases donating samples to use as positive controls usually differ in some

**Figure 4.**

*Failure in immunodiagnosis. Cross-reactivity occurs due to some, but not all, of the secretion and excretion antigens of Taenia solium that are shared, not only during the different stages of its biological cycle, but also with some other endemic parasites (eg: Taenia saginata).*

or various characteristics of the disease likely to be of immunological consequence (i.e., form and duration of treatment, natural history of the disease, site of residence, age [39], gender [40, 43–45, 50], race [28, 51–54]).

Thus, IDxNC is placed between the wall of false positives and the sword of false negatives and forced to negotiate selecting the antigen(s) most frequently found to react with control NC samples in order to decrease false positives but conceding some false negatives with the consequent loss in both sensitivity and specificity scores.

The antigen repertoire of *T. solium* is known to be numerous and varied [47, 48, 55, 56] but the distribution of the antigens in the members of the species, in the different developmental stages of the parasite and in different geographic locations is perhaps the most neglected possibly crucial need of information for the design of successful immunodiagnosis of *T. solium* disease.

### **7. Proposals for improvement**


#### *The Long Road to the Immunodiagnosis of Neurocysticercosis: Controversies and Confusions DOI: http://dx.doi.org/10.5772/intechopen.98723*

proposed [55, 57, 58] they are lacking in satisfactorily meeting with either one or both of these conditions. A way of avoiding the high costs and demanding technical skills involved in the purification of natural antigens is the use of those present in phage display peptide libraries [59–61]. Antigens present in only *T. solium* but not in all specimens of the species would constitute the candidate antigen preparation (CAP).


Three are the classes of *T. solium* disease that matter the most and perhaps require different strategies: the contact case (members of the *I* set), the NC case (all *n* and *ne* subsets) and the tapeworm carrier (*t* subset). For this purpose, it is indispensable to construct representative and certified negative and positive control panels of the samples CSF, serum and feces from each geographic area upon their reaction with DAP. Certification of the members of the *e* subset and *0* set is complicated by its need of whole-body scans in search of cysticerci located elsewhere of CNS. Additional negative control samples from a culturally and historically certified community or geographic area to be rid of *T. solium* disease and low in infectious disease in general would be useful to estimate blank readings of immunotesting with DAP.


### **Acknowledgements**

This work was supported by Project Grants IN-209719 from Programa de Apoyo a Proyectos de Innovación Tecnológica (PAPIIT), Dirección General de Asuntos del Personal Académico (DGAPA), Universidad Nacional Autónoma de México (UNAM) and grant FC 2016-2125 from Fronteras en la Ciencia, Consejo Nacional de Ciencia y Tecnología (CONACYT), both granted to Jorge Morales-Montor. Project IA- 202919 from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT), Dirección General de Asuntos del Personal Académico (DGAPA), Universidad Nacional Autónoma de México (UNAM) to Karen Elizabeth Nava-Castro. Also project IA- 206220 from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT), Dirección General de Asuntos del Personal Académico (DGAPA), Universidad Nacional Autónoma de México (UNAM) to Víctor Hugo Del Río Araiza.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Marcela Esquivel-Velázquez1 , Carlos Larralde1†, Pedro Ostoa-Saloma1 , Víctor Hugo Del Río Araiza<sup>2</sup> and Jorge Morales-Montor1 \*

1 Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Ciudad de México, Mexico

2 Departamento de Parasitología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México (UNAM), Ciudad de México, Mexico

\*Address all correspondence to: jmontor66@biomedicas.unam.mx

† Passed away

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Long Road to the Immunodiagnosis of Neurocysticercosis: Controversies and Confusions DOI: http://dx.doi.org/10.5772/intechopen.98723*

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### Section 3
