**4. Genomic and proteomic profile of** *T. spiralis*

There are 12 species and genotypes of *Trichinella* which are distributed worldwide and cause serious disease *Trichinellosis* in humans which leads to morbidity and mortality [1, 2, 12]. Based on larvae appearance in the muscle cells of the host only encapsulated and non-encapsulated clades (morphological distinct) *Trichinella* is recognized. Based on molecular studies, nine species and three genotypes of *Trichinella* show a wide biological diversity. Based on genetic data, only *Trichinella* encapsulated clade infects mammals includes *Trichinella spiralis* (T1), *Trichinella nativa* (T2), *Trichinella britovi* (T3), *Trichinella murrelli* (T5), *Trichinella nelson* (T7), and *Trichinella patagoniensis*(T12). The three *Trichinella* genotypes includes T6, T8, and

*Perspective Chapter: Advances in the Development of Anti-*Trichinella spiralis *Vaccine… DOI: http://dx.doi.org/10.5772/intechopen.103027*

T9. The *Trichinella* non-encapsulated clade includes *Trichinella pseudospiralis* (T4), which infects birds and mammals only, *Trichinella papuae* (T10), and *Trichinella zimbabwensis* (T11), they infect reptiles and mammals [1].

Proteomics (because of bioinformatics and mass spectrometry) is an effective technique to examine the modifications after the translation of genes such as proteolysis or glycosylation. These are powerful techniques to examine the samples obtained from pathogens to find the possible proteins involved in the pathogenesis of the disease [12]. *Trichinella* is substantially different in molecular and biological characteristics from other crown groups. The assembly of *Trichinella* is 64 million bp in length and about 15,808 proteins are encoded by this genome assembly. In *T. spiralis* genome, the estimation of repeat content is about 18% having low GC content (about 27%) relative to the overall genome (34%) and protein-coding region (43%) of *Trichinella spiralis* [14]. Microsatellites are present in the entire genome and many are distributed in the non-coding sequence of the genome. It leads to genetic diversity due to mutation [28]. During the early stage of *Trichinella* infection, *Trichinella spiralis* 14-3-3 protein is a strong immunogenic antigen [29]. Ts14-3-3 is an immunodominant antigen and this protein is also used to detect the whole period of infection with *Trichinella.* During the early phase of *Trichinella* infection, HSP70, cysteine protease, and Ts14-3-3 play a crucial role in balancing the host–parasite relationship. Therefore, these proteins are a good target for the development of vaccines and early immunodiagnostic measures [15].

#### **4.1 DNA based vaccine**

DNA vaccines got a glare in the early 1990s and evoked both humoral and cellular responses, when tested and identified, particularly induced cytotoxic T cell response, and abolished the safety concerns associated with the live vaccine [17]. Such vaccines tend to sustain host immune system stimulation in comparison to the Recombinant protein-based vaccines [6]. DNA vaccines emerged as a strong way of eliciting a humoral and cellular immune response against many parasitological antigens in small animal models. Moreover, DNA vaccines produce a concurrent Th1 and Th2 immune response against *T. spiralis* [30, 31].

The TspE1gene encoding a 31 kDa antigen of *T. spiralis* has been cloned to an expression vector pcDNA3 and administered in a mouse as a DNA vaccine [31]. Naturally, *T. spiralis* challenge suppresses the type 2 immune system response which inhibits them [17]. The mice immunized with the TspE1-pcDNA3 presented a significant larval reduction rate and an increased serum anti-*Trichinella* antibody level, hence this DNA vaccine proved to be partially protective against *T. spiralis* challenges [31]. Spleen cells after stimulation with the TspE1 recombinant protein exhibited a lymphoproliferative response, which is an indication of cellular response elicited by the DNA vaccine. Sequence of a serine protease (Ts-NBLsp) cDNA from newborn larvae of *T. spiralis*, cDNA sequence of recombinant TsNd (*Trichinella spiralis* nudix hydrolases) has been cloned to the plasmid pcDNA3.1 [17, 31, 32]. The antibody response against the serine protease of *T. spiralis* inhibits the protease activity thus hindering invasion of the parasite. The DNA vaccines Ts-NBLsp-pcDNA3.1 and pcDNA3.1-TsNd presented a balanced systemic Th1\Th2 immune response. The immunization with recombinant TsNd DNA vaccine resulted in an increased intestinal IgA and total IgG response with an exalted IgG1 than that of IgG2a [31]. To compare the recombinant nudix hydrolase DNA vaccine, the Ts-NBLsp-pcDNA3.1 vaccine showed a dominant IgG2a anti *trichinella* antibody and a predominantly Th1 immune response [17]. DNA vaccines elevated IFN gamma, IL-2, IL-4, and IL-10 levels [31]. Secretory IgA causes a significant

reduction in the female worm fecundity and this response is enhanced by cytokine IL-10 specifically. The intestinal mucosa of the infected animals produces a specific antibody response against *T. spiralis*. Ts-NBLsp-pcDNA3.1 and reduces the muscle larvae burden (77.93%) greater than that of the TsNd vaccine (53.9%).

In another study of the TsDNase II, the complementary DNA sequence of *T. spiralis* serine protease 2.1 has been cloned to the eukaryotic expression vector pcDNA3.1 and administered as a DNA vaccine through an attenuated *Salmonella typhimurium* to avoid degradation [30]. To elicit a persistent systemic and mucosal immune response against *T. spiralis*, attenuated salmonella is an effective live carrier that gives an efficient mode of vaccination. *T. spiralis* DNase II is an excretory-secretory product associated with adult worms and IIL which is expressed in the cuticle of IIL. *T. spiralis* serine protease appeared to be present in the spliceosome and cuticle of adult worms and intestinal infective larvae. Both of these vaccine candidates against *T. spiralis* resulted in the significant rise of specific IgG responses. IgG1 titer after the first dose of vaccination and then an increased level of IgG2a after the second dose of vaccination, furthermore they produced mixed Th1\Th2 response which can be described through elicited cytokines response as Th1(IFN gamma) and Th2 cytokines (IL-4, IL10) [30]. TsSP 1.2-pcDNA3.1 vaccine resulted in a 71.84% reduction in the muscle larvae in comparison to the TsDNase II DNA vaccine which caused a 59.26% reduction in the muscle larvae [30].

*T. spiralis* adult-specific DNase II-1 (TsDNase II-1) and DNase II-7 recognized in the excretory-secretory proteins of the AW [30] has been analyzed for their immune response against the worm. Antibody-dependent cell-mediated cytotoxicity assay (ADCC) revealed that both recombinant anti-TsDNase II-1 and anti-TsDNase II-7 sera mediated the attachment of mouse peritoneal exudate cells (PECs) to NBL and finally killing of the NBL. Paramyosin is a thick myofibrillar protein [6, 30, 33], which is an immunomodulatory protein that evades host immune response by inhibiting complement C1q and C8\C9. TsPmy and Ts87 both are efficient vaccine candidates against *T. spiralis*. The DNA encoding TsPmy and Ts87 have been cloned in a eukaryotic vector pVAX1 and the recombinant DNA was transformed in the *S. typhimurium* strain SL7207. The resulting DNA vaccines produced protective immunity against *T. spiralis* when administered in mice, both resulted in mucosal sIgA response in the intestine and systemic anti TsPmyIgG response. The antibody-secreting cells from the spleen and mesenteric lymph nodes of the mice immunized with TsPmy vaccine expressed the intestinal homing receptors CCR9 and CCR10. [30] determined that SL7207\ pVAX1-TsPmy vaccine came out with a 44.8% reduction in muscle larvae and a 46.6% reduction in adult worms. While SL7207\ pVAX1-Ts87 caused a 34.2% reduction in muscle larvae and a 29.8% reduction in adult worm burden. By using B and T cell epitopes from TsPmy a novel multi-epitope vaccine has been designed which elicits an immune response more efficiently as compared to traditional vaccines, TsPmy MEP vaccine reduced the muscle larvae up to 55.4% [33].

DNA vaccines have many advantages as they are inexpensive, focused immune response against the antigen of interest, heat stable and a broad-spectrum vaccine can be developed by mixing plasmids.

#### **4.2 Protein-based vaccine**

In recent studies, it is reported that specific protein molecules from numerous *T. spiralis* life cycle stages have been considered and expressed properly, so that their immune protection was also estimated, such as paramyosin (Ts-Pmy) obtained from

### *Perspective Chapter: Advances in the Development of Anti-*Trichinella spiralis *Vaccine… DOI: http://dx.doi.org/10.5772/intechopen.103027*

an adult cDNA library [21], TspGST and fructose-1,6-bisphosphate aldolase (Ts-FBPA) taken from the *T. spiralis* draft genome utilizing high expression at the ML stage, Ts31 from the ML ES proteins, serine protease (TsSP) from IIL (intestinal offensive stage) and ML surface proteins and cathepsin B (TsCB) from the *T. spiralis* draft genome [8]. On the other hand, when these recombinant proteins were used for vaccinating mice, they showed only 36.2–53.50% ML reduction following the *T. spiralis* challenge. In the current study, we determine the protective immunity persuaded by vaccination through a novel TsE protein. TsE is highly expressed and acts as a secretory protein at the *T. spiralis* intestinal offensive stage (IIL), TsE shows potency to be exposed first to the host's intestinal mucosa and then produce the local immune response through its working. It is observed that vaccination with rTsE persuaded significantly high levels of TsE-specific sIgA, which can simplify adult worm removal from the intestine. TE immune protection having 64.06% ML reduction, with this novel TsE vaccination was considered superior to those of the above-mentioned other *T. spiralis* proteins act as candidate vaccine target molecules. This study also recognized a foundation to develop polyvalent anti-*T. spiralis* vaccines in the upcoming period.

The immune response stimulated by a vaccine based on an exclusive antigen and multi-epitope (that work more efficiently than the large protein molecules) vaccines against *T. spiralis* has now been proposed. Therefore, the amalgamation of three selected epitopes from Ts-Pmy and Ts87 from *T. spiralis* adult produced in immunized mice IgG and IgG1 production and higher protection of about 35% in contrast to the parasite challenge in comparison to that encouraged by individual epitope peptides [8]. To achieve higher shielding immune responses counter to *T. spiralis*, it will be essential to propose a vaccine with multi-epitopes from different parasite stages focusing on NBL and adult stages (**Table 1**).



#### **Table 1.**

*Immunoregulatory kinetics of different* T. spiralis *based protein after binding with host immune cells.*

### **5. Role of progesterone receptor in** *trichinella spiralis*

Progesterone (P4) is a sex steroid hormone that plays roles in the physiology of the reproductive system such as corpus luteum of the ovary and placenta in females, while testes and adrenal cortex in males also participate in many other functions such as brain activity, immune modulation, metabolism of bones heart and lungs physiology. P4 is also responsible for the maintenance of pregnancy and shows an immunosuppressive effect [35]. when a high level of progesterone is present during the luteal phase of the estrus/menstrual cycle in females. Recent studies showed that these hormones also influence the course of parasites infections and also restrict the invasion of parasites when a high level of P4 in female animals is produced. Restricts the invasion of parasites [11]. P4 has an immunomodulatory effect on fetal antigens during pregnancy by suppressing the maternal immune response. However, progesterone can be either an inhibitory or stimulatory effect on the immune response mechanism depending upon cell type, concentration, and exposure time to steroids. It has nematotoxicity against newborn larvae of *T. spiralis*. Progesterone is responsible for decreased parasite load during pregnancy [11].

Sex steroids are known as immune response modulators and play a major role in *T. spiralis* susceptibility at two levels viz. (1) protective immune response and (2) direct effect on the development of worms. Besides, P4 up-regulates many molecules expressions from major histocompatibility complex class I and it also participates in

*Perspective Chapter: Advances in the Development of Anti-*Trichinella spiralis *Vaccine… DOI: http://dx.doi.org/10.5772/intechopen.103027*

the down-regulation of genes that are responsible for the fecundity and oviposition of the worms and inhibits the nuclear factor kappa B (NFƙB) activation in innate immunity [11].

### **6. Role of progesterone and mifepristone against** *T. spiralis*

Progesterone is a gonadal hormone primarily involved in the preparation of the endometrium for implantation of an embryo and necessary for the maintenance of pregnancy, while mifepristone is a drug that works as an antagonist of progesterone and glucocorticoid. It has an abortifacient effect and terminates early pregnancy by binding to intracellular progesterone receptors. Mifepristone has an antagonistic effect on the *T. spiralis* (Ts) membrane-associated progesterone receptor component-2 (Ts-MAPRC2). It also down-regulates the expression of the Ts-MAPRC2 gene and results in the abortion of the pregnant adult female worms [11].

Mifepristone (RU486) can be taken as an example that works as an antagonist in contrast to the progesterone receptor (PR) and glucocorticoid receptor (GR) with some lethal properties such as aborting agent and anticancer activities in the body. In the case of helminths, several research studies are concentrated on PGRMC receptors. Similarly, RU486 was one of the first medications accepted for surgical abortion and is frequently used to terminate an early or midterm pregnancy. Hereafter, PR and binding of P4 molecules (agonist) and RU486 (antagonist) can be helpful to elaborate *T. spiralis* species regarding differentiation and reproductive development as well as creating potential pharmacological targets that might be used as a drug therapy against *Trichinellosis*.

Progesterone is known for its immune-modulatory effects, which happen during pregnancy that is done by suppressing the response from the mother toward paternal antigens expressed in the fetus [11]. Taking into the description, we can conclude that progesterone is an adequate inducing activation of the effector cell populations responsible for cell death in an antibody-independent cytotoxic mechanism. This cytotoxicity should also be activated by soluble antigens released by the parasite because at constant self-aggression of tissues by these activated cells 0% NBL mortality 10 10 100 Progesterone (ng/ml) cells [35] .

### **7. Challenges to developing an efficient vaccine against** *T. spiralis*

The control of helminths in animals is usually through anthelmintics. Vaccine development against *T. spiralis* infection in pigs is an alternate method for the prevention of parasite *T. spiralis* from zoonosis. Effective vaccine development against *Trichinellosis* is conducted in mice instead of pigs. Effective development of a vaccine, is not only due to high price of experimental pigs but also due to poorly satisfied antigens detected from the mice. Moreover, the immune response induced by the same antigen in swine and mice is extremely different. So, [2]. concluded that in mice, poor immunogenic vaccine candidates are not capable to induce a strong protective immune response against *T. spiralis* infection in pigs.

TsT was a *T. spiralis* somatic antigen and at adult-stage with specific surface antigen it had a good antigenicity. If vaccination of mice is done with TsT, it will induce a systemic mixed Th1/Th2 response and an intestinal local sIgA response, which can produce partial protection against *T. spiralis* larval challenge. Then these results suggested that TsT plays a role in *T. spiralis* growth and survival in the host, and it might be deliberated as a potential target antigen for anti-*T. spiralis* vaccines. However [9], revealed that oral anti-Trichinella vaccines comprised of multiple antigenic epitopes of various *T. spiralis* life cycle phases should be recognized.

#### **7.1 Diversity within** *T. spiralis* **parasites**

*T. spiralis* is a nematode parasite that is prevalent throughout the world and translocated by humans and their animals. They occupy well-defined geographic ranges [36]. There is a big diversity among the *T. spiralis* parasites present in different geographic locations [24]. *T. spiralis* nematode belongs to the clade that diverged early in the phylum Nematoda evolution [14]. *T. britovi* parasitizes many sylvatic mammals such as Felidae, Canidae, Ursidae, Mustelidae, Suidae, Viverridae and is endemic to Northern-western Africa and Eurasia while *T. murrelli* is the only present in wild animals in North America. Millions of years ago, *Trichinella* could infect human beings evidenced by the ingestion of other parasites in meat [36].

The nematode *T. spiralis* is involved in the most common cause of human *trichinellosis*, which is considered a zoonotic disease worldwide. The heredity of *T. spiralis* giving rise to the genus *Trichinella* and reported that the last shared common ancestor was approximately 275 million years ago (Lower Permian Period) identified, however the modification of extant *Trichinella* species happened about 16–20 million years ago [14].

We compare the molecular physiognomies of nematodes and former metazoans by using the *T. spiralis* genome as standard. This comparative approach by using the *T. spiralis* permitted us to categorize conserved protein and gene sequences through the superficial model, particularly for the phylum Nematoda. We bring an approach that intrachromosomal modifications were common all over the phylum. However, this was in divergence to other features such as births and deaths of a protein family, which exhibited clear discrimination among the parasitic and non-parasitic nematodes. The identification of well-maintained physiognomies predicated based on this work will advance the more accurate research on pathogens from a phylum embracing thousands of pathogens that are mainly to infect humans, animals, and plants and behaves like infectious agent. The advances possibly will one day be responsible for complete strategies to prevent and control diseases that are caused by pathogens from across the Nematoda family around the globe [14, 36, 37].

Commencing from the time of the discovery of *Trichinella* which is in 1835 in anticipation of the middle of the next century. During the last decade, the use of molecular and biochemical methods in combination with experimental studies on biology, have resulted in the identification of seven *Trichinella* species that have different epidemiological and topographical distributions. Even though these species are very difficult to differentiate morphologically, this can be done with the molecular and positive biological characters for further identification [16].

#### **7.2 Genetic diversity related to multiple hosts**

A total of 30 species of *T. spiralis* having mtDNA genomes has 20 unique haplotypes that were observed containing 86 isolating sites. So, with four out of five shared haplotypes taking place in European and North American samples. Samples from North America had one haplotype, which is present in each geographic

*Perspective Chapter: Advances in the Development of Anti-*Trichinella spiralis *Vaccine… DOI: http://dx.doi.org/10.5772/intechopen.103027*

sampling site [38]. Out of the total, mostly the variations were limited to the Asian *T. spiralis* samples. There are about 7 Asian samples, and from these 8 haplotypes were identified; these differed on an ordinary by 24.9 nucleotides. In comparison to this, western samples are averaged with only 3.2 nucleotide differences per haplotype with only 13 haplotypes in 23 samples; the most different pair of western haplotypes differed by only 6 nucleotide differences between any two isolates. Similarly, nucleotide diversity (pi) was 0.00016 in western samples while Asian nucleotide diversity was 10-fold greater (0.00179). As a result, we can say that all Asian samples are different from the western samples by at least 45 bp and averaged 49 bp differences [24].

The most noticeable properties of this parasite's epidemiology are its requisite transmission by mode of meat ingestion in consumers. There is another important feature, which is present in two normally isolated ecological systems, which are sylvatic and domestic. In certain situations, the two biotopes are connected from end to end man's activities, which results in the revelation of humans to *Trichinella* species [38]. Usually, it is restricted to sylvatic animals. The species furthermost often associated with human infection is *T spiralis*, which is the reported species that is usually found in the meat of domestic pigs. The life cycle of *T spiralis* includes a multipart set of possible routes. Farm transmission can be the result of predation on or hunting other animals for food purposes (rodents), hog anthropophagy, and the feeding of uncooked meat leftovers [16].

Most outbreaks resulting from ingesting of *T spiralis* infected pigs can lead to its outbreak through local single-source but, progressively, the mass marketing of meat can distribute the disease-causing parasite in the entire population. There has been a great increase in the reported cases due to Trichinella species, just because of having so many species that are involved in the food chain. The reason for having genetic diversity is also stated that we are lacking in vaccines to eradicate it. The main source is considered as the meat from the game and domestic animals. From recent reports, we can conclude that it also specifies that infected herbivores including horses, sheep, goats, and cattle have been the source of the outbreak [14, 16].

#### **7.3 Multiple stage complexity of** *T. spiralis*

*T. spiralis*also has several stages of the complex life cycle that completes in two niches viz. intra-multicellular niche occurs in the intestine epithelium of host where adult male and female worms are involved with the help of (proteolytic digestive enzymes and become mature adult worms). Whereas intracellular niche occurs in the striated muscles of the organism where muscle larvae participate in the development of nurse cells [16], *T. spiralis* life cycle represents different antigens specific for a particular stage, where these antigens elicit immune responses and facilitate the developmental cycle of the parasites by modifying the host immune responses. To complete their life cycle, they skip the defensive mechanism of the host against invading the foreign body.

Once newborn larvae invade the lymphatic or circulatory system, they can drive anywhere in the organism and survive only in the skeletal muscles of the host. Humans can acquire *T. spiralis* infection only if they consume undercooked or raw meat containing muscle larvae [36]. Several genes are differentially expressed among the life cycle stages and up-regulated genes in the newborn larvae. The genome of *T. spiralis* is regulated in the developmental stages [34].

### **8. Future perspective**

Trichinella infection is an emerging zoonosis in many countries and where it become the reason for *trichinellosis* disease. Due to its widespread prevalence and high amount of pork meat consumption more clinical awareness is required. The acute infection is characterized by two phases viz. enteral which disrupt intestinal functions and parenteral phases are associated with the inflammatory and allergic reaction. The diagnosis of this disease contains new specific serological tests such as immunoblot or ELISA. Anthelmintics and anti-inflammatory drugs are the drug of choice for *Trichinella* infection [16]. Vaccines formulated for veterinary purposes have made a great impact not only on animal welfare, production, and health but also on human health. Vaccines are considered reliable, efficient, and sustainable for the control and prevention of parasitic infection.

In *Thrichinellosis*, induction of protective and therapeutic responses should evoke both innate and adaptive immune systems to prevent the establishment of parasites in the organism. The life cycle of *T. spiralis* is complex, and the immune response is not strong that induced by a vaccine containing specific antigen to overcome the challenging infection. Therefore, a vaccine containing multiple epitopes against *T. spiralis induces higher immunity* [24]. Probiotics such as *Lactobacillus* keep the environment of the intestine healthy and prevent enteric infections. Probiotic *Lactobacillus casei* is most commonly used for protection against *Trichinellosis*. *L. casei* is involved in the production of IL-4, IgA, and IgG (anti-*T. spiralis* antibodies) and has a preventive role against high infection of *T. spiralis*. Some strains of *L. casei* include *L. casei* ATCC 469, *L. casei* ATCC 7469, and *L. casei* Shirota have proven efficacy against *T. spiralis* infection. For the control of *Trichinellosis*, Probiotics and plants-based veterinary vaccines are a new approach and can be used as treatment and edible vaccines for various parasitic diseases in animals. Due to the low cost of plants production, sterile delivery, and transportation at a suitable temperature, plants are considered as a suitable vehicle for veterinary vaccines [1].

Antigens in the vaccines administered orally are subject to proteolysis by the proteolytic enzymes present in the digestive tract of the organism. It will decrease the bioavailability of the vaccines and will induce a low immune response [1]. On the other hand, in plant-based vaccines antigens are protected from proteolytic enzymes by the cell wall of the plant cells and enable antigens to reach their desired destination (gut-associated lymphoid tissue). Various plants and vegetable species such as potato, tomato, tobacco, alfalfa, rice, spinach, beans, maize, strawberries, and carrots can be used in the biotechnology of plants for the expression and production of recombinant proteins.

For the prevention and control of diseases in animals and their transmission from animals to humans, plant-based vaccines seem to be an excellent tool. More research is required to thoroughly understand the applications of medical plant extracts, probiotics, and other biological agents [24].

### **9. Conclusion**

At least twelve species and genotypes of *Trichinella* genus can cause veterinary or medical health hazards in a wide geographical range throughout the world. The main etiological agent of *Trichinellosis* in humans is only *T. spiralis* parasite and can result in mild to severe clinical signs and symptoms. Numerous antigens are used as *Perspective Chapter: Advances in the Development of Anti-*Trichinella spiralis *Vaccine… DOI: http://dx.doi.org/10.5772/intechopen.103027*

candidate vaccines from different stages of *T. spiralis and* can be used as DNA vaccines or recombinant protein vaccines. The role of progesterone and mifepristone against *T. spiralis* is also very helpful as they penetrate the vaccine into the target of *T. spiralis.* Altogether, we can get different strains for specific vaccines with molecular physiognomies of different Trichinella species.
