**3. Molecular medicinal strategies and parasitic diseases**

Parasitic infections (protozoan and helminthic infections) affect more than a quarter world population and cause chronic illness primarily in developing countries of world. These diseases affect the quality of life and treatment costs possess economic burden on families leading to viscous circle of poverty.

Molecular medicine is a broad field that includes insight into the molecular aspect of diseases. Recombinant DNA and cloning technologies are the conventional tools for studying the disease associated molecular profiles. Recent technical advances have paved the way for utilization of several molecular strategies for treating infectious diseases. Molecular medicine aims to understand the molecular basis of disease pathogenesis and allows the utilization of the information in designing specific diagnostic, therapeutic and prophylactic options. Mainly molecular medicine relies on two strategies—targeting genome and targeting signaling pathways, as targeted approach of disease management. Thus, it aims to improve the human health through the understanding of mechanism in human diseases.

### **3.1 Targeting genome**

Apart from conventional approach of gene therapy (replacement of defective gene by exogenous DNA and editing mutated gene), recent technical advances have opened the arena for other strategies of manipulating the gene expression. Gene editing methods have gained limelight that involves the intrinsic molecular repair processes within the cell. The process of break repair in the DNA involves the homology-directed repair (HDR) and/or non-homologous end joining (NHEJ). The key step in gene-editing tool involves the precise introduction of double strand breaks. This process involves the use of engineered meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the recent CRISPR/Cas system [2]. Further, short antisense oligonucleotides potentially serve as tools for abrogating the transcription of target gene. As compared to other genome editing methods CRISPR/Cas system using guide RNA has shown immense potential for future of molecular medicine.

#### *3.1.1 Engineered meganucleases*

Although there remains plethora of meganucleases to choose from, however, most commonly used meganucleases include the ZFNs that have DNA binding zinc finger protein domain and nuclease domain. Cys2-His2 zinc finger domain is amongst the most abundant types of DNA binding motifs in eukaryotes [3, 4]. The ZFNs work by binding to the DNA and cleaving it, which then undergoes repair by either homologous recombination or error-prone NHEJ [5]. Site-specific cleavage is induced by manipulating the ZFN complex to recognize two sequences that are

on either side of target site. Upon identification, cleavage of genome is induced by restriction enzyme (FokI), thus generating double stranded breaks in genome that can be used for editing the region.

TALEN has been introduced as an alternative to ZFNs. These are similar to ZFNs in using restriction enzyme that incorporates with the DNA binding domain, but are of different origin. Similar to ZFNs, these protein structures lead to the double stranded breaks in DNA for genome editing [6]. Unfortunately, there are no evidences for the use of the aforementioned meganucleases for the development of molecular medicine for parasitic diseases.

#### *3.1.2 RNA-guided engineered nucleases (CRISPR-Cas9 system)*

After the discovery of clustered regularly interspaced palindromic repeats (CRISPR) (in 1987) as a part of bacterial immune system against invading viruses. This strategy has potential application in editing human chromosome with great accuracy. It is RNA guided gene editing tool that uses Cas9 endonuclease for generating double-stranded breaks at loci of interest, which are then repaired via. HDR (using a template) or NHEJ or error-prone microhomology-mediated end joining (MMEJ) [7]. Thus, leading to mutation (insertion, deletion or substitution) with no or minimal damage to host genome. Since the sequencing of the parasite genome, CRISPR/Cas9 genome editing tool accelerated the molecular research in parasitology.

Genome editing in malarial parasite (*P. falciparum* and *P. yoelii*) has provided the ground for development of molecular medicine [8, 9]. CRISPR system of genome editing has played crucial role in understanding the drug resistance and pathogen survival thus, genome editing of the parasite genome holds promises to trigger host immune responses while preventing disease pathology. There are several studies for CRISPR/Cas9 analyses in *T. gondii* however, the most remarkable one include the genome-wide screening that identified the genes involved in infection process [10, 11]. This has greatly accelerated the research to understand the parasite metabolic needs for survival and virulence, conversely, shedding light on dealing with drug resistance mechanisms. Similarly, CRISPR/Cas9 system for *T. cruzi* and *T. brucei* has facilitated functional studies of drug targets and/ or vaccine candidates [12, 13]. Genetic manipulation has always remained arduous for *Leishmania* however, CRISPR/Cas9 system has proven efficacy for rapid genome editing and understanding gene functions with therapeutic implication [14, 15]. Similarly, genome editing for *T. vaginalis* has been recently introduced with potential *in vivo* toxicity issues which has been dealt by using nucleofusion based transfection [16, 17]. Large-scale functional genomic screening cannot rely on conventional CRISPR/Cas9 approach thus, CRISPRi and CRISPRa approaches have gained significant attention for generating knock-in/knock-down libraries.

Genome editing using CRISPR/Cas9 system in *Schistosoma mansoni* eggs reduced the infection induced granulomas. Similarly, CRISPR/Cas9 mediated deletion of granulin gene from liver fluke (*O. viverrini*) significantly improved the disease symptoms [18]. This is just the start of the use of CRISPR/Cas9 system in parasitic diseases, it has broader potential in designing molecular medicine for disease intervention.

#### *3.1.3 RNA interference (RNAi)*

RNA interference is the approach of inhibiting the gene expression or translation by insertion of double-stranded RNA into the cells and/or organism that mediates targeted degradation of its homologous RNA. It can be done *via*. Short interfering RNA (siRNAs), micro RNA (miRNA) and piwi-interacting (piRNA). It mediates

**35**

*Molecular Medicines for Parasitic Diseases DOI: http://dx.doi.org/10.5772/intechopen.91956*

controlling parasitic infections.

cally active compounds against *T. brucei* [27].

to its genome homeostatic potential.

molecular medicine.

**3.2 Targeting cells and signaling pathways**

gene silencing by forming RNA-induced silencing complex (RISC) that eventually degrades target mRNA. This unique ability of silencing target gene holds hopes for

Malarial parasites lack the conventional RNAi pathway however, antisense oligodeoxynucleotides treatment (against parasite topoisomerase) has shown to significantly reduce the parasite multiplication [28]. Further, topoisomerase targeting antisense nanoparticles and chitosan-based nanoformulation have also been used to inhibit *P. falciparum* growth [29, 30]. Recently, synthetic siRNA targeting the β-actinin and cysteine protease served as potential molecular target for *T. vaginalis* infection [31]. Although still scrappy, better understanding of RNAi pathway in protozoan parasites is likely to revolutionize the molecular medicine due

RNAi based silencing of key genes involved in regulating the parasite survival and development have been potential candidates for therapeutics. Several miRNA have been known to regulate the nematode development and survival in the host microenvironment due to their immunoregulatory potential. These have also been crucial players of host-parasite interaction and have been used as diagnostic marker of infection. *Nippostrongylus brasiliensis* (rat parasite) was the first nematode where RNAi was reported [32]. Much evidences for RNAi based knock-down studies have been seen in *Schistosoma* [33, 34], *Brugia* [35], *Trichinella spiralis* [36], *Ascaris suum* [37], *Angiostrongylus cantonensis* [38], *Taenia saginata* [39], *Echinococcus* [40], *H. contortus* [41] and *Onchocerca volvulus* [42]. Unfortunately, RNAi has not achieved the expected success in parasitic nematodes (schistosomes being an exception) [43]. This could be attributed to the lack of several key components of RNAi pathway [44]. Thus, targeted therapy with RNAi based approach (especially miRNA) is still in its infancy, in context of helminthic infections, that needs orchestrated support from the investors as well as the scientific community in order to stand alone as potential candidate for development of

This area of molecular medicine remains the hottest area of research in the field of parasitic diseases after the World Health Organization (WHO) warning about the risk of post-antibiotic era. The search for novel therapeutic strategies intends to enhance pathogen killing by targeting regulatory molecules/pathways. Better understanding of disease immunobiology and cellular signaling will provide momentum to the identification of the pathways of therapeutic importance. This area of research towards the development of molecular medicine involves the use of genetically engineered antibodies, recombinant proteins, small molecules to alter signaling pathways, targeting the immunometabolic pathways, inflammasomes, etc.

*T. brucei* was the first protozoan parasite where RNAi based targeting of β-tubulin gene, changed parasite morphology [19]. Conversely, *T. cruzi* [20]*, L. donovani* [21] and *L. major* [22] lack the essential protein (Ago-1) for suppressing the gene expression. RNAi mediated targeting of cathepsin B reduced the disease progression [23]. RNAi mediated targeting of topoisomerases and farnesyl pyrophosphate synthase have proven efficacy of RNAi based molecular medicine for disease intervention [24, 25]. Later, in 2011, RNAi target sequencing (RIT) identified several potential targets for genome-scale functional analyses which could potentially therapeutic targets [26]. RNA aptamers (synthetic RNA and DNA molecules) binds the target ligands (RNA/DNA) with high-specificity, and have been developed as pharmaceuti-

#### *Molecular Medicines for Parasitic Diseases DOI: http://dx.doi.org/10.5772/intechopen.91956*

*Methods in Molecular Medicine*

can be used for editing the region.

molecular medicine for parasitic diseases.

*3.1.2 RNA-guided engineered nucleases (CRISPR-Cas9 system)*

on either side of target site. Upon identification, cleavage of genome is induced by restriction enzyme (FokI), thus generating double stranded breaks in genome that

TALEN has been introduced as an alternative to ZFNs. These are similar to ZFNs in using restriction enzyme that incorporates with the DNA binding domain, but are of different origin. Similar to ZFNs, these protein structures lead to the double stranded breaks in DNA for genome editing [6]. Unfortunately, there are no evidences for the use of the aforementioned meganucleases for the development of

After the discovery of clustered regularly interspaced palindromic repeats (CRISPR) (in 1987) as a part of bacterial immune system against invading viruses. This strategy has potential application in editing human chromosome with great accuracy. It is RNA guided gene editing tool that uses Cas9 endonuclease for generating double-stranded breaks at loci of interest, which are then repaired via. HDR (using a template) or NHEJ or error-prone microhomology-mediated end joining (MMEJ) [7]. Thus, leading to mutation (insertion, deletion or substitution) with no or minimal damage to host genome. Since the sequencing of the parasite genome, CRISPR/Cas9 genome editing tool accelerated the molecular research in parasitology. Genome editing in malarial parasite (*P. falciparum* and *P. yoelii*) has provided the ground for development of molecular medicine [8, 9]. CRISPR system of genome editing has played crucial role in understanding the drug resistance and pathogen survival thus, genome editing of the parasite genome holds promises to trigger host immune responses while preventing disease pathology. There are several studies for CRISPR/Cas9 analyses in *T. gondii* however, the most remarkable one include the genome-wide screening that identified the genes involved in infection process [10, 11]. This has greatly accelerated the research to understand the parasite metabolic needs for survival and virulence, conversely, shedding light on dealing with drug resistance mechanisms. Similarly, CRISPR/Cas9 system for *T. cruzi* and *T. brucei* has facilitated functional studies of drug targets and/ or vaccine candidates [12, 13]. Genetic manipulation has always remained arduous for *Leishmania* however, CRISPR/Cas9 system has proven efficacy for rapid genome editing and understanding gene functions with therapeutic implication [14, 15]. Similarly, genome editing for *T. vaginalis* has been recently introduced with potential *in vivo* toxicity issues which has been dealt by using nucleofusion based transfection [16, 17]. Large-scale functional genomic screening cannot rely on conventional CRISPR/Cas9 approach thus, CRISPRi and CRISPRa approaches have gained significant attention for generating knock-in/knock-down libraries. Genome editing using CRISPR/Cas9 system in *Schistosoma mansoni* eggs reduced the infection induced granulomas. Similarly, CRISPR/Cas9 mediated deletion of granulin gene from liver fluke (*O. viverrini*) significantly improved the disease symptoms [18]. This is just the start of the use of CRISPR/Cas9 system in parasitic diseases, it has broader potential in designing molecular medicine for disease intervention.

RNA interference is the approach of inhibiting the gene expression or translation by insertion of double-stranded RNA into the cells and/or organism that mediates targeted degradation of its homologous RNA. It can be done *via*. Short interfering RNA (siRNAs), micro RNA (miRNA) and piwi-interacting (piRNA). It mediates

**34**

*3.1.3 RNA interference (RNAi)*

gene silencing by forming RNA-induced silencing complex (RISC) that eventually degrades target mRNA. This unique ability of silencing target gene holds hopes for controlling parasitic infections.

*T. brucei* was the first protozoan parasite where RNAi based targeting of β-tubulin gene, changed parasite morphology [19]. Conversely, *T. cruzi* [20]*, L. donovani* [21] and *L. major* [22] lack the essential protein (Ago-1) for suppressing the gene expression. RNAi mediated targeting of cathepsin B reduced the disease progression [23]. RNAi mediated targeting of topoisomerases and farnesyl pyrophosphate synthase have proven efficacy of RNAi based molecular medicine for disease intervention [24, 25]. Later, in 2011, RNAi target sequencing (RIT) identified several potential targets for genome-scale functional analyses which could potentially therapeutic targets [26]. RNA aptamers (synthetic RNA and DNA molecules) binds the target ligands (RNA/DNA) with high-specificity, and have been developed as pharmaceutically active compounds against *T. brucei* [27].

Malarial parasites lack the conventional RNAi pathway however, antisense oligodeoxynucleotides treatment (against parasite topoisomerase) has shown to significantly reduce the parasite multiplication [28]. Further, topoisomerase targeting antisense nanoparticles and chitosan-based nanoformulation have also been used to inhibit *P. falciparum* growth [29, 30]. Recently, synthetic siRNA targeting the β-actinin and cysteine protease served as potential molecular target for *T. vaginalis* infection [31]. Although still scrappy, better understanding of RNAi pathway in protozoan parasites is likely to revolutionize the molecular medicine due to its genome homeostatic potential.

RNAi based silencing of key genes involved in regulating the parasite survival and development have been potential candidates for therapeutics. Several miRNA have been known to regulate the nematode development and survival in the host microenvironment due to their immunoregulatory potential. These have also been crucial players of host-parasite interaction and have been used as diagnostic marker of infection. *Nippostrongylus brasiliensis* (rat parasite) was the first nematode where RNAi was reported [32]. Much evidences for RNAi based knock-down studies have been seen in *Schistosoma* [33, 34], *Brugia* [35], *Trichinella spiralis* [36], *Ascaris suum* [37], *Angiostrongylus cantonensis* [38], *Taenia saginata* [39], *Echinococcus* [40], *H. contortus* [41] and *Onchocerca volvulus* [42]. Unfortunately, RNAi has not achieved the expected success in parasitic nematodes (schistosomes being an exception) [43]. This could be attributed to the lack of several key components of RNAi pathway [44]. Thus, targeted therapy with RNAi based approach (especially miRNA) is still in its infancy, in context of helminthic infections, that needs orchestrated support from the investors as well as the scientific community in order to stand alone as potential candidate for development of molecular medicine.

### **3.2 Targeting cells and signaling pathways**

This area of molecular medicine remains the hottest area of research in the field of parasitic diseases after the World Health Organization (WHO) warning about the risk of post-antibiotic era. The search for novel therapeutic strategies intends to enhance pathogen killing by targeting regulatory molecules/pathways. Better understanding of disease immunobiology and cellular signaling will provide momentum to the identification of the pathways of therapeutic importance. This area of research towards the development of molecular medicine involves the use of genetically engineered antibodies, recombinant proteins, small molecules to alter signaling pathways, targeting the immunometabolic pathways, inflammasomes, etc.

#### *3.2.1 Immunotherapeutic approach*

Immunotherapy is use of biological substances (antigen/antibody, immunomodulators administration) to regulate host immune system in order to fulfill prophylactic and/or therapeutic purpose. Immunotherapy aims to trigger the immune power by directly (antigen based or active immunotherapy) or indirectly (antibody based or passive immunotherapy) [45, 46]. This section describes various immunotherapeutic strategies of molecular medicine that have been reported for the parasitic diseases.

#### *3.2.1.1 Recombinant proteins/cytokine therapy*

Cytokines are the small molecular weight, chemical messengers that regulates the immune responses in autocrine and paracrine manner. There is plethora of evidences for the involvement of cytokines in determining the pathophysiological consequences. Recombinant protein (cytokine) based therapy aims to trigger T-cell immune responses and induces parasite clearance.

In *T. cruzi* infection, TGF-β (transforming growth factor-β) has been implicated to yield pathological consequences however, treatment with TGF-β receptor kinase, SB-431542, has shown to restrict the entrance of parasite in the cardiomyocytes and disease associated cardiomyopathy [47, 48]. Additionally, cytokine combination therapy with recombinant IFN-γ and TNF-α has anti-parasitic potential [49]. In context of leishmaniasis, Murray et al. first proved the significance of targeting the cytokine, they showed that monoclonal antibody-based treatment targeting the IL-10 receptor (anti-IL-10 receptor) instigated parasite clearance by inducing NO (nitric oxide) production [50]. Likewise, combination of recombinant IFN-γ therapy with conventional chemotherapy yielded promising results in controlling the disease pathology [51]. Not much has been reported about the cytokine-based therapy in other protozoan diseases.

Conversely, in helminthic infection, MAb (monoclonal antibody) based blocking of IL-4 and IL-10 has shown disease improvement by reducing parasitic burden and inducing TH1 immune responses [52]. Likewise, IL-4 based MAb therapy, in schistosomiasis, has shown to inhibit granuloma formation and hepatic fibrosis [53]. Similar findings of marked reduction in granuloma and hepatic fibrosis were reported upon treatment with *Schistosoma* eggs along with recombinant IL-12 treatment [54]. Exogenous IL-25 based therapy has shown to potentially modulate intestinal functions by regulating IL-13 mediated STAT6 signaling in order to favor protective immune responses in intestinal nematode infection by *N. brasiliensis* [55]. Conversely, exogenous treatment with IL-13 and IL-25 triggered ILCs (innate lymphoid cells) responses and conferred protection against helminthic infections [56]. In schistosomiasis, IL-13 inhibitor, sIL-13Ralpha2-Fc has proved therapeutic benefit by preventing tissue fibrosis due to TH2 dominated inflammatory responses [49] .

The significance of exogenous cytokine therapy has also been underlined in trichiasis, where IL-33 is known to induce thymic stromal lymphopoietin that generates polarized TH2 responses to confer protection against intestinal nematodes [57]. While, IL-25 treatment instigated TH2 responses and restricted infection induced gastrointestinal inflammation [58], MAb based blockade of IL-10 ameliorated disease pathology. There are evidences for the IL-27 mediated suppression of T-cell proliferation thus IL-27 receptor (WSX-1) knock down improved the mucosal immunity [59]. The use of immune triggering cytokines (IFN-γ, IL-12, GM-CSF) and/or blocking immunoregulatory cytokines that possesses pathological consequences holds hopes for the development of molecular medicine. Thus, therapeutic potential of cytokine therapy can be exploited alone

**37**

ventricular dysfunctions [77].

*Molecular Medicines for Parasitic Diseases DOI: http://dx.doi.org/10.5772/intechopen.91956*

for improving treatment outcomes.

*3.2.1.2 Immune checkpoint therapy*

inhibitory molecules).

and/or in combination with conventional chemotherapy opening up the avenues

Immune checkpoint molecules are involved in regulating the T-cell activation and functions. The expression of these molecules is enhanced during chronic infections as a result of immune subversion, thus, therapeutically targeting these molecules has shown promising results in cancer and infectious diseases vaccines [60, 61]. Indeed, T-cell dysfunctionality or exhaustion is the key for impaired T-cell responses during chronic infections; exhaustion is marked by loss of IL-2 production, reduced cytotoxicity, impaired production of pro-inflammatory mediators and reduced proliferative ability. The expression of multiple immune checkpoint molecules (PD-1, CTLA-4, LAG-3, Tim-3, TIGIT) remains the hallmark feature of exhausted cells; elevated expressions of these molecules are accompanied with progressive loss of T-cell functionality [62]. Immune checkpoint inhibitors have been novel strategy of reinvigorating the immune cell functions by abrogating the signaling by the immune checkpoint (or co-

A number of immune checkpoint molecules have been reported in leishmaniasis including—LAG-3, Tim-3, CTLA-4, PD-1, etc. that negatively regulates T-cell functionality [63–65]. MAb based blockade of PD-1 and LAG-3 in malaria triggered pro-inflammatory cytokine responses and relieved T-cell inhibition [66]. Likewise, therapeutically targeting LAG-3 and PD-L1 restored CD4+ T-cells functions, restored follicular helper

Unfortunately, this strategy of immune checkpoint therapy has been in its nascent stage for parasitic infections, and has yet not been used for HAT, Chagas

Immune cell manipulation offers another fascinating approach of molecular medicine to fight with parasitic diseases, when other treatment options fail to provide protective immunity [68, 69]. Direct transfer of immune cells has been holding great promises for conferring protection against protozoan, bacterial and viral infections [70]. Adoptive T-cell transfer therapy using tumor-infiltrating lymphocytes is the best example to clinical success of cellular therapy [71].

DC (dendritic cell) based vaccination approach using parasite peptide (KMP-11) elicited TH1 responses, reduced parasite load and induced lymphocyte proliferation in leishmaniasis infection [72]. Similarly, vaccination with DC along with histone H1 elicited pro-inflammatory responses (IFN-γ and IL-12), reduced the IL-10 and IL-4 producing cells and induced polarized TH1 responses [73]. Atypical progenitor cells (IL-7R+ c-kit+ cells) from malaria infected mice are potent fighters against infection, while transplantation of these cells had similar effects in disease recovery [74]. After the success of direct administration of MSCs (mesenchymal stromal cells) and antigen specific T-cells stem cell therapy has recently budded in the field of infectious diseases. MSCs have been shown to be equally important in conferring resistance against *P. berghei* infection, by suppressing IL-10, reducing the regulatory T-cells population and inducing the production of IL-12 [75, 76]. Likewise, autologous transplantation of MSCs and myoblasts has shown to significantly reduce the

T-cells, plasma cells eventually cleared the blood stage of *Plasmodium* [67].

disease, gastrointestinal protozoans as well as helminthic infections.

*3.2.1.3 Immune cells and stem cell-based therapy*

and/or in combination with conventional chemotherapy opening up the avenues for improving treatment outcomes.

## *3.2.1.2 Immune checkpoint therapy*

*Methods in Molecular Medicine*

*3.2.1 Immunotherapeutic approach*

*3.2.1.1 Recombinant proteins/cytokine therapy*

immune responses and induces parasite clearance.

Immunotherapy is use of biological substances (antigen/antibody, immunomodulators administration) to regulate host immune system in order to fulfill prophylactic and/or therapeutic purpose. Immunotherapy aims to trigger the immune power by directly (antigen based or active immunotherapy) or indirectly (antibody based or passive immunotherapy) [45, 46]. This section describes various immunotherapeutic strategies of molecular medicine that have been reported for the parasitic diseases.

Cytokines are the small molecular weight, chemical messengers that regulates the immune responses in autocrine and paracrine manner. There is plethora of evidences for the involvement of cytokines in determining the pathophysiological consequences. Recombinant protein (cytokine) based therapy aims to trigger T-cell

In *T. cruzi* infection, TGF-β (transforming growth factor-β) has been implicated to yield pathological consequences however, treatment with TGF-β receptor kinase, SB-431542, has shown to restrict the entrance of parasite in the cardiomyocytes and disease associated cardiomyopathy [47, 48]. Additionally, cytokine combination therapy with recombinant IFN-γ and TNF-α has anti-parasitic potential [49]. In context of leishmaniasis, Murray et al. first proved the significance of targeting the cytokine, they showed that monoclonal antibody-based treatment targeting the IL-10 receptor (anti-IL-10 receptor) instigated parasite clearance by inducing NO (nitric oxide) production [50]. Likewise, combination of recombinant IFN-γ therapy with conventional chemotherapy yielded promising results in controlling the disease pathology [51]. Not much has been reported about the cytokine-based therapy in other protozoan diseases.

Conversely, in helminthic infection, MAb (monoclonal antibody) based blocking of IL-4 and IL-10 has shown disease improvement by reducing parasitic burden and inducing TH1 immune responses [52]. Likewise, IL-4 based MAb therapy, in schistosomiasis, has shown to inhibit granuloma formation and hepatic fibrosis [53]. Similar findings of marked reduction in granuloma and hepatic fibrosis were reported upon treatment with *Schistosoma* eggs along with recombinant IL-12 treatment [54]. Exogenous IL-25 based therapy has shown to potentially modulate intestinal functions by regulating IL-13 mediated STAT6 signaling in order to favor protective immune responses in intestinal nematode infection by *N. brasiliensis* [55]. Conversely, exogenous treatment with IL-13 and IL-25 triggered ILCs (innate lymphoid cells) responses and conferred protection against helminthic infections [56]. In schistosomiasis, IL-13 inhibitor, sIL-13Ralpha2-Fc has proved therapeutic benefit by preventing tissue fibrosis due to TH2 dominated inflammatory responses [49] . The significance of exogenous cytokine therapy has also been underlined in trichiasis, where IL-33 is known to induce thymic stromal lymphopoietin that generates polarized TH2 responses to confer protection against intestinal nematodes [57]. While, IL-25 treatment instigated TH2 responses and restricted infection induced gastrointestinal inflammation [58], MAb based blockade of IL-10 ameliorated disease pathology. There are evidences for the IL-27 mediated suppression of T-cell proliferation thus IL-27 receptor (WSX-1) knock down improved the mucosal immunity [59]. The use of immune triggering cytokines (IFN-γ, IL-12, GM-CSF) and/or blocking immunoregulatory cytokines that possesses pathological consequences holds hopes for the development of molecular medicine. Thus, therapeutic potential of cytokine therapy can be exploited alone

**36**

Immune checkpoint molecules are involved in regulating the T-cell activation and functions. The expression of these molecules is enhanced during chronic infections as a result of immune subversion, thus, therapeutically targeting these molecules has shown promising results in cancer and infectious diseases vaccines [60, 61]. Indeed, T-cell dysfunctionality or exhaustion is the key for impaired T-cell responses during chronic infections; exhaustion is marked by loss of IL-2 production, reduced cytotoxicity, impaired production of pro-inflammatory mediators and reduced proliferative ability. The expression of multiple immune checkpoint molecules (PD-1, CTLA-4, LAG-3, Tim-3, TIGIT) remains the hallmark feature of exhausted cells; elevated expressions of these molecules are accompanied with progressive loss of T-cell functionality [62]. Immune checkpoint inhibitors have been novel strategy of reinvigorating the immune cell functions by abrogating the signaling by the immune checkpoint (or coinhibitory molecules).

A number of immune checkpoint molecules have been reported in leishmaniasis including—LAG-3, Tim-3, CTLA-4, PD-1, etc. that negatively regulates T-cell functionality [63–65]. MAb based blockade of PD-1 and LAG-3 in malaria triggered pro-inflammatory cytokine responses and relieved T-cell inhibition [66]. Likewise, therapeutically targeting LAG-3 and PD-L1 restored CD4+ T-cells functions, restored follicular helper T-cells, plasma cells eventually cleared the blood stage of *Plasmodium* [67].

Unfortunately, this strategy of immune checkpoint therapy has been in its nascent stage for parasitic infections, and has yet not been used for HAT, Chagas disease, gastrointestinal protozoans as well as helminthic infections.

### *3.2.1.3 Immune cells and stem cell-based therapy*

Immune cell manipulation offers another fascinating approach of molecular medicine to fight with parasitic diseases, when other treatment options fail to provide protective immunity [68, 69]. Direct transfer of immune cells has been holding great promises for conferring protection against protozoan, bacterial and viral infections [70]. Adoptive T-cell transfer therapy using tumor-infiltrating lymphocytes is the best example to clinical success of cellular therapy [71].

DC (dendritic cell) based vaccination approach using parasite peptide (KMP-11) elicited TH1 responses, reduced parasite load and induced lymphocyte proliferation in leishmaniasis infection [72]. Similarly, vaccination with DC along with histone H1 elicited pro-inflammatory responses (IFN-γ and IL-12), reduced the IL-10 and IL-4 producing cells and induced polarized TH1 responses [73]. Atypical progenitor cells (IL-7R+ c-kit+ cells) from malaria infected mice are potent fighters against infection, while transplantation of these cells had similar effects in disease recovery [74].

After the success of direct administration of MSCs (mesenchymal stromal cells) and antigen specific T-cells stem cell therapy has recently budded in the field of infectious diseases. MSCs have been shown to be equally important in conferring resistance against *P. berghei* infection, by suppressing IL-10, reducing the regulatory T-cells population and inducing the production of IL-12 [75, 76]. Likewise, autologous transplantation of MSCs and myoblasts has shown to significantly reduce the ventricular dysfunctions [77].

Transplantation of bone marrow mononuclear cells has marked effect on improving the inflammation and fibrosis in Chagas disease [78, 79]. Also, bone marrow transplantation holds promises for improving the quality of life in congestive heart failure due to Chagas disease [80, 81]. Adoptive immunotherapy in toxoplasmosis, by transferring CD8+ T-cells restricted parasite de-encystation; however, it failed to revert the T-cells exhaustion attributing to the short-lives of exhausted cells [82]. Further, MSCs therapy in toxoplasmosis has not been successful however, when used in combination with the spiramycin, pyrimethamine and folinic acid provided therapeutic benefits. Similarly, for coccidiosis, using adoptive transfer strategy, intraepithelial lymphocytes (IELs) and CD4+ T-cells from interferon gamma knock out (*Cryptosporidium parvum*-infected) mice has shown to provide protection against infection in naïve mice [83]. Likewise, adoptive transfer of sporozoites pulsed-DCs upon co-culture with CD4+ and CD8+ T-cells reduced parasite burden [84].

In helminthic diseases, MSC based therapy have been proven to be efficacious for reducing *Schistosoma japonicum* induced liver injury by using MSCs culture supernatant which inhibited macrophage activation by egg antigen. Macrophages primed with *N. brasiliensis* have been shown to clear the parasitic burden by neutrophil mediated mechanism of macrophage polarization [85] in strongyloidiasis. Filarial infections are associated with increased expression of Foxp3 expressing regulatory T-cells that impairs the CD4+ T-cell immunity. Regulatory T-cells targeted intervention using antibodies against CD25, glucocorticoid-induced TNF receptor familyrelated gene (GITR), provided cure for filarial infection [86]. In schistosomiasis, basophil depletion strategy has been shown to successfully ameliorate disease pathology and granulomatous lesions [87]. Similarly, *in vivo* DCs depletion has been an efficacious strategy to boost antigen specific T-cells expansion [88]. Antigen pulsed immune cell therapeutics has been extended to *F. hepatica* infection. DCs pulsed with parasite induces TH1 responses and has been a viable vaccination option that protects against disease associated hepatic damage [89]. Similarly, transfer of *Hymenolepis diminuta* pulsed bone marrow derived DCs cells ameliorated colitis pathology by IL-4 signaling [90]. Therefore, cell based therapeutic strategy serves as potential molecular medicinal approach for parasitic infections.

#### *3.2.1.4 Immunomodulators*

Immunomodulators are small molecular inhibitors of signaling pathways that serve as molecular medicine for disease intervention. Imatinib, an Abl/Arg tyrosine kinase inhibitor, induces cytoskeleton remodeling to facilitate leishmanial parasite phagocytosis in the macrophages and reduces disease associated lesions [91]. Another signaling pathway inhibitor, AS-605240 (PI3K gamma inhibitor) has shown to be as efficacious as sodium stibogluconate (SSG) in the treating of *L. mexicana* infection [92]. Similarly, another PI3K inhibitor CAL-101 and IC87114 are known to effectively reduce parasitic burden by reducing the B-cells and regulatory T-cells populations [93, 94]. Another tyrosine kinase inhibitor, ibrutinib has been shown to treat leishmaniasis by triggering TH1-polarized IFN-γ production [95]. Tellurium based immunomodulator (AS101) has shown to effectively revert t-cell anergy and promote NO production while inhibiting IL-10 signaling in *L. donovani* infection. In Chagas disease, inhibitors of GPCRs provide protection against the disease by preventing the parasite entry and infection [96]. Parasite derived thromboxane A2 signaling induces apoptosis, vasoconstriction and disease associated cardiomyopathy thus use of SQ29548, thromboxane A2 receptor antagonist abrogates the *T. cruzi* infection [97]. Conversely, platelet activating factor and leukotriene B4 induces NO production and effectively controls the parasite [98, 99].

**39**

*Molecular Medicines for Parasitic Diseases DOI: http://dx.doi.org/10.5772/intechopen.91956*

T-cell proliferation and IFN-γ production [103].

kinase (MAPK) and NF-κB signaling [104].

research is underway to explore other molecules.

**3.3 Nano-medicinal approach**

therapeutic options [118].

Further, β-adrenergic receptor blockade along with carvedilol has been an effective

Rosiglitazone, peroxisome proliferator-activator receptor gamma (PPARγ) agonist, is known to enhance phagocytic clearance of parasitized erythrocytes and reduce parasitic burden in malaria by inhibiting the mitogen-activated protein

potentially improved innate cytokine responses (IL-33 and IL-25) eventually causing parasite expulsion [105]. Therefore, small molecular have shown potential therapeutic benefits in parasitic infection, here is just the tip of huge iceberg,

In helminthic infection (Strongyloidiasis), anakinra (IL-1β receptor antagonist)

The application of nanomaterials in the field of medicine for diagnosis and treatment received considerable attention in recent decades for parasitic diseases. The diagnostic potential of nanomaterials has been seen in malaria [106, 107], toxoplasmosis [108], cryptosporiodiosis [109], amebiasis [110] and leishmaniasis [111, 112]. Considering the nanoparticles as treatment option for parasitic diseases, these particles have proven efficacy in targeting the infected macrophages for treatment of VL [113]. Silver alone or in combination with chitosan nanoparticles exhibited anti-toxoplasma effects by exacerbating serum IFN-γ levels and lowering the parasitic burden [114]. Spiramycin loaded chitosan nanoparticles have shown to effectively treat toxoplasmosis [115]. In giardiasis, combination nanotherapy with silver, chitosan and curcumin nanoparticles have been shown to effectively clear the parasites from intestine and stool without any adverse effects [116]. Chitosan as nanosuspension has also shown lethal effects on *Cryptosporidium* oocysts [117]. Similarly, silver nanoparticles have also been shown to effectively reduced oocyst burden by triggering IFN-γ, without any adverse events as seen with standard

The biodegradability and non-immunogenic properties of nanoparticles have made them suitable as delivery agents for drugs and vaccines. Nanoformulation of recombinant *P. falciparum* protein (Pfs25H) served as transmission blocking vaccine for malaria, by abrogating the parasite infectivity to mosquitoes. Similarly, polymeric vaccine using polymer poly(lactide-co-glycoside) acid (PLGA) nanoparticles with malaria antigen, VMP001, and immunostimulatory monophosphoryl A (MPL-A) triggered antigen-specific immune responses against *P. vivax* [119]. Furthermore, iron oxide nanoparticle conjugated with recombinant merozoite surface protein 1 (rMSP1) were efficiently engulfed by macrophages and DCs, that eventually triggered the pro-inflammatory responses [120]. In VL, conjugation of quercetin with gold nanoparticle [121], doxorubicin along with chitosan [122], amphotericin B as chitosan nanocapsule [123] and mannose-chitosan based nanoformulation of rifampicin served as effective delivery system for VL management [124]. Chitosan/poly (vinyl alcohol) based microspheres has also shown to abrogate the *Cryptosporidium* sporozoites attachment to the enterocytes thus served

as potential oral chemotherapy for *Cryptosporidium* infection [125].

Tyrosine kinase inhibitors (lapatinib) have proven their efficacies in controlling Human African Trypanosomiasis (HAT) pathogenesis by blocking parasite endocytosis [101]. Furthermore, PI3Kγ/mTOR signaling inhibitors as NVP-BEZ235 restricts the *T. brucei* infection [102]. Lectin based therapy using parasite galactose-N-acetyld-galactosamine inhibitable lectin (Gallectin) instigates IL-12 production from DCs,

strategy to improve clinical symptom of Chagas cardiomyopathy [100].

#### *Molecular Medicines for Parasitic Diseases DOI: http://dx.doi.org/10.5772/intechopen.91956*

*Methods in Molecular Medicine*

Transplantation of bone marrow mononuclear cells has marked effect on improv-

ing the inflammation and fibrosis in Chagas disease [78, 79]. Also, bone marrow transplantation holds promises for improving the quality of life in congestive heart failure due to Chagas disease [80, 81]. Adoptive immunotherapy in toxoplasmosis, by transferring CD8+ T-cells restricted parasite de-encystation; however, it failed to revert the T-cells exhaustion attributing to the short-lives of exhausted cells [82]. Further, MSCs therapy in toxoplasmosis has not been successful however, when used in combination with the spiramycin, pyrimethamine and folinic acid provided therapeutic benefits. Similarly, for coccidiosis, using adoptive transfer strategy, intraepithelial lymphocytes (IELs) and CD4+ T-cells from interferon gamma knock out (*Cryptosporidium parvum*-infected) mice has shown to provide protection against infection in naïve mice [83]. Likewise, adoptive transfer of sporozoites pulsed-DCs

upon co-culture with CD4+ and CD8+ T-cells reduced parasite burden [84].

potential molecular medicinal approach for parasitic infections.

Immunomodulators are small molecular inhibitors of signaling pathways that serve as molecular medicine for disease intervention. Imatinib, an Abl/Arg tyrosine kinase inhibitor, induces cytoskeleton remodeling to facilitate leishmanial parasite phagocytosis in the macrophages and reduces disease associated lesions [91]. Another signaling pathway inhibitor, AS-605240 (PI3K gamma inhibitor) has shown to be as efficacious as sodium stibogluconate (SSG) in the treating of *L. mexicana* infection [92]. Similarly, another PI3K inhibitor CAL-101 and IC87114 are known to effectively reduce parasitic burden by reducing the B-cells and regulatory T-cells populations [93, 94]. Another tyrosine kinase inhibitor, ibrutinib has been shown to treat leishmaniasis by triggering TH1-polarized IFN-γ production [95]. Tellurium based immunomodulator (AS101) has shown to effectively revert t-cell anergy and promote NO production while inhibiting IL-10 signaling in *L. donovani* infection. In Chagas disease, inhibitors of GPCRs provide protection against the disease by preventing the parasite entry and infection [96]. Parasite derived thromboxane A2 signaling induces apoptosis, vasoconstriction and disease associated cardiomyopathy thus use of SQ29548, thromboxane A2 receptor antagonist abrogates the *T. cruzi* infection [97]. Conversely, platelet activating factor and leukotriene B4 induces NO production and effectively controls the parasite [98, 99].

*3.2.1.4 Immunomodulators*

In helminthic diseases, MSC based therapy have been proven to be efficacious for reducing *Schistosoma japonicum* induced liver injury by using MSCs culture supernatant which inhibited macrophage activation by egg antigen. Macrophages primed with *N. brasiliensis* have been shown to clear the parasitic burden by neutrophil mediated mechanism of macrophage polarization [85] in strongyloidiasis. Filarial infections are associated with increased expression of Foxp3 expressing regulatory T-cells that impairs the CD4+ T-cell immunity. Regulatory T-cells targeted intervention using antibodies against CD25, glucocorticoid-induced TNF receptor familyrelated gene (GITR), provided cure for filarial infection [86]. In schistosomiasis, basophil depletion strategy has been shown to successfully ameliorate disease pathology and granulomatous lesions [87]. Similarly, *in vivo* DCs depletion has been an efficacious strategy to boost antigen specific T-cells expansion [88]. Antigen pulsed immune cell therapeutics has been extended to *F. hepatica* infection. DCs pulsed with parasite induces TH1 responses and has been a viable vaccination option that protects against disease associated hepatic damage [89]. Similarly, transfer of *Hymenolepis diminuta* pulsed bone marrow derived DCs cells ameliorated colitis pathology by IL-4 signaling [90]. Therefore, cell based therapeutic strategy serves as

**38**

Further, β-adrenergic receptor blockade along with carvedilol has been an effective strategy to improve clinical symptom of Chagas cardiomyopathy [100].

Tyrosine kinase inhibitors (lapatinib) have proven their efficacies in controlling Human African Trypanosomiasis (HAT) pathogenesis by blocking parasite endocytosis [101]. Furthermore, PI3Kγ/mTOR signaling inhibitors as NVP-BEZ235 restricts the *T. brucei* infection [102]. Lectin based therapy using parasite galactose-N-acetyld-galactosamine inhibitable lectin (Gallectin) instigates IL-12 production from DCs, T-cell proliferation and IFN-γ production [103].

Rosiglitazone, peroxisome proliferator-activator receptor gamma (PPARγ) agonist, is known to enhance phagocytic clearance of parasitized erythrocytes and reduce parasitic burden in malaria by inhibiting the mitogen-activated protein kinase (MAPK) and NF-κB signaling [104].

In helminthic infection (Strongyloidiasis), anakinra (IL-1β receptor antagonist) potentially improved innate cytokine responses (IL-33 and IL-25) eventually causing parasite expulsion [105]. Therefore, small molecular have shown potential therapeutic benefits in parasitic infection, here is just the tip of huge iceberg, research is underway to explore other molecules.

#### **3.3 Nano-medicinal approach**

The application of nanomaterials in the field of medicine for diagnosis and treatment received considerable attention in recent decades for parasitic diseases. The diagnostic potential of nanomaterials has been seen in malaria [106, 107], toxoplasmosis [108], cryptosporiodiosis [109], amebiasis [110] and leishmaniasis [111, 112].

Considering the nanoparticles as treatment option for parasitic diseases, these particles have proven efficacy in targeting the infected macrophages for treatment of VL [113]. Silver alone or in combination with chitosan nanoparticles exhibited anti-toxoplasma effects by exacerbating serum IFN-γ levels and lowering the parasitic burden [114]. Spiramycin loaded chitosan nanoparticles have shown to effectively treat toxoplasmosis [115]. In giardiasis, combination nanotherapy with silver, chitosan and curcumin nanoparticles have been shown to effectively clear the parasites from intestine and stool without any adverse effects [116]. Chitosan as nanosuspension has also shown lethal effects on *Cryptosporidium* oocysts [117]. Similarly, silver nanoparticles have also been shown to effectively reduced oocyst burden by triggering IFN-γ, without any adverse events as seen with standard therapeutic options [118].

The biodegradability and non-immunogenic properties of nanoparticles have made them suitable as delivery agents for drugs and vaccines. Nanoformulation of recombinant *P. falciparum* protein (Pfs25H) served as transmission blocking vaccine for malaria, by abrogating the parasite infectivity to mosquitoes. Similarly, polymeric vaccine using polymer poly(lactide-co-glycoside) acid (PLGA) nanoparticles with malaria antigen, VMP001, and immunostimulatory monophosphoryl A (MPL-A) triggered antigen-specific immune responses against *P. vivax* [119]. Furthermore, iron oxide nanoparticle conjugated with recombinant merozoite surface protein 1 (rMSP1) were efficiently engulfed by macrophages and DCs, that eventually triggered the pro-inflammatory responses [120]. In VL, conjugation of quercetin with gold nanoparticle [121], doxorubicin along with chitosan [122], amphotericin B as chitosan nanocapsule [123] and mannose-chitosan based nanoformulation of rifampicin served as effective delivery system for VL management [124]. Chitosan/poly (vinyl alcohol) based microspheres has also shown to abrogate the *Cryptosporidium* sporozoites attachment to the enterocytes thus served as potential oral chemotherapy for *Cryptosporidium* infection [125].

For helminthic infections, chitosan based albendazole formulation skewed the T-cell responses to TH1 type and reduced the parasitic burden, which led to parasite clearance in echinococcosis [126, 127] as well as in toxocariasis [128]. Similarly, silver assembled on fungal (*Trichoderma harzianum*) cell wall in the form of nanoformulation improved the anti-fascioliasis potential of triclabendazole [129]. Liposomal nanoformulations (nanoparticles and nanocapsules) have been widely used for enhancing the efficacies and bioavailability of oral drugs for disease intervention. These formulations have gained significance as nanocarriers in helminthic infection due to their ability to diffuse through the intestinal mucosal layers. In schistosomiasis, liposome encapsulated praziquantel has shown significant reductions in parasitic burdens and hepatic granulomas due to increased affinity for parasite phospholipids [130]. Liposome based nanocapsules of praziquantel (PZQ-LNCs) improved the drug efficacy and ameliorated disease pathology [131]. Further, liposomal nanocapsules of miltefosine exerted potential schistosomal effects by ameliorating hepatic histology (reducing the granuloma size, number and inflammation) in single dose [132].

Nanoformulation has also been used for vaccine development and as adjuvants, self-assembling protein nanoparticles (SAPN) have shown to trigger protective antibodies and long-lived memory responses to confer sterile protection against malarial parasites (*P. berghei* and *P. falciparum*) [133]. SAPNs have also been used for delivering the epitopes to induce CD4+ and CD8+ T-cell responses against *Toxoplasma gondii* [134]. Archaea based nanoformulations (archaeosomes) have been used as adjuvant as a part of prophylactic vaccine against *T. cruzi*, instigated humoral as well as cell-mediated immune responses (TH1 responses) leading to marked reduction in parasitic burdens [135]. Cationic solid lipid nanoparticles have been successfully used as adjuvant as part of prime-boost strategy to reduce the parasitic burdens during VL. The vaccination triggered IFN-γ production, NO production and high levels of immunoglobulins (IgG1 and IgG2a) [136]. Therefore, nanoparticles served as viable, safe and effective vaccine platform as well as development of molecular medicine for cost effective vaccine delivery.

### **4. Conclusion**

In this world where cost of developing medicine for parasitic infections remain the greatest challenge, drug developers are embracing molecular medicine approach that promises to deal with the parasitic infections and improves the chances of successful treatment. Molecular medicine has revolutionized the field of drug discovery/development however, there are significant hurdles in turning the promise into reality. Perhaps, gradually but it is shaping the future of medicine with the help of molecular platforms, better bioinformatics services and better pharmacogenomic analyses has greatly facilitated the scientific community and the stakeholders to come on common platform to fight against the parasitic diseases.

**41**

**Author details**

Bhawana Singh

Varanasi, India

Department of Medicine, Institute of Medical Sciences, Banaras Hindu University,

© 2020 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,

\*Address all correspondence to: bhavanasonali9@gmail.com

provided the original work is properly cited.

*Molecular Medicines for Parasitic Diseases DOI: http://dx.doi.org/10.5772/intechopen.91956* *Molecular Medicines for Parasitic Diseases DOI: http://dx.doi.org/10.5772/intechopen.91956*

*Methods in Molecular Medicine*

For helminthic infections, chitosan based albendazole formulation skewed the T-cell responses to TH1 type and reduced the parasitic burden, which led to parasite clearance in echinococcosis [126, 127] as well as in toxocariasis [128]. Similarly, silver assembled on fungal (*Trichoderma harzianum*) cell wall in the form of nanoformulation improved the anti-fascioliasis potential of triclabendazole [129]. Liposomal nanoformulations (nanoparticles and nanocapsules) have been widely used for enhancing the efficacies and bioavailability of oral drugs for disease intervention. These formulations have gained significance as nanocarriers in helminthic infection due to their ability to diffuse through the intestinal mucosal layers. In schistosomiasis, liposome encapsulated praziquantel has shown significant reductions in parasitic burdens and hepatic granulomas due to increased affinity for parasite phospholipids [130]. Liposome based nanocapsules of praziquantel (PZQ-LNCs) improved the drug efficacy and ameliorated disease pathology [131]. Further, liposomal nanocapsules of miltefosine exerted potential schistosomal effects by ameliorating hepatic histology (reducing the granuloma size, number and inflammation) in single dose [132].

Nanoformulation has also been used for vaccine development and as adjuvants, self-assembling protein nanoparticles (SAPN) have shown to trigger protective antibodies and long-lived memory responses to confer sterile protection against malarial parasites (*P. berghei* and *P. falciparum*) [133]. SAPNs have also been used for delivering the epitopes to induce CD4+ and CD8+ T-cell responses against *Toxoplasma gondii* [134]. Archaea based nanoformulations (archaeosomes) have been used as adjuvant as a part of prophylactic vaccine against *T. cruzi*, instigated humoral as well as cell-mediated immune responses (TH1 responses) leading to marked reduction in parasitic burdens [135]. Cationic solid lipid nanoparticles have been successfully used as adjuvant as part of prime-boost strategy to reduce the parasitic burdens during VL. The vaccination triggered IFN-γ production, NO production and high levels of immunoglobulins (IgG1 and IgG2a) [136]. Therefore, nanoparticles served as viable, safe and effective vaccine platform as well as devel-

In this world where cost of developing medicine for parasitic infections remain the greatest challenge, drug developers are embracing molecular medicine approach that promises to deal with the parasitic infections and improves the chances of successful treatment. Molecular medicine has revolutionized the field of drug discovery/development however, there are significant hurdles in turning the promise into reality. Perhaps, gradually but it is shaping the future of medicine with the help of molecular platforms, better bioinformatics services and better pharmacogenomic analyses has greatly facilitated the scientific community and the stakeholders to

opment of molecular medicine for cost effective vaccine delivery.

come on common platform to fight against the parasitic diseases.

**40**

**4. Conclusion**
