**4. Transcriptomic approaches to vaccine development for helminth diseases**

Transcriptomic analysis with a view toward vaccine design circumvents some of the challenges posed by relying on genomic analysis alone. Traditionally, to annotate a transcriptome, the transcriptome of interest is run using a pairwise homologybased analysis with other known curated and annotated genome sequence data sets from other organisms. Initially, the transcripts and genes of parasitic helminths were not able to be annotated in this manner as they did not correlate with data that were publicly available [16]. Analysis of transcriptomic data for various parasites identified several categories of genes that encode proteins without similarity to other organisms. It is likely that these genes are exclusive to the parasite they are found in and likely play a role in parasite survival and adaptation. The uniqueness of these genes

#### *Perspective Chapter: Multi-Omic Approaches to Vaccine Development against Helminth Diseases DOI: http://dx.doi.org/10.5772/intechopen.102621*

found in the transcriptome at various life stages may also provide targets for vaccine development [41]. Mangiola *et. al* sought to centralize these unique genes in annotated parasite transcriptomes through the creation of HelmDB [16]. This database was initially created by annotating the transcriptomes of 11 parasitic helminths with socioeconomic importance. Though HelmDB is no longer functional, transcriptomes for numerous species can be freely accessed via WormBase [35].

The creation of annotated transcriptome databases and the relative availability of transcriptome sequencing has created an opportunity for researchers to explore the difference in gene expression across the life cycle of various helminths. Vaccine development targeting multiple life stages of many parasitic helminths can be pursued by understanding the changes in gene expression throughout the life cycle [41–45]. These analyses have been carried out with different species of parasitic helminths and have been able to identify differentially expressed genes throughout the life cycle related to parasite infection, survival, and immune evasion. Genes that are differentially expressed in transcriptome analysis between life-cycle stages in relation to their role in the host infection process may be relevant to the survival of the parasite and can serve as targets for vaccine development that will prevent against infectious stages, or therapeutics that will protect against pathologic life stages [42]. The importance of this is apparent with the success of Barbervax®. The complex life cycle of *H. contortus* lasts 3 weeks. The first larval stage (L1) develops within an egg and hatches to molt to the second larval stage (L2) followed by a third larval stage (L3). It is the L3 stage that is ingested by the host and develops into the fourth larval stage (L4) to become adults [44]. Barbervax® consists of two adult-stage proteins present in the worm gut and is effective because worms ingest antibodies with each blood meal. The antibodies bind the proteins and disrupt gut function, leading to starvation and detachment (**Figure 1**) [46, 47]. While effective at reducing worm burden,

#### **Figure 1.**

*Mechanistic view of worm burden reduction in BarberVax®-immunized hosts. Vaccinated individuals raise IgG antibodies against the* H. contortus *intestinal proteins H11 and H-gal-GP. Upon infection and taking a blood meal, antibodies in the blood of vaccinated hosts disrupt the intestinal surface of the worm (lower inset) and interfere with normal nutrient uptake (upper inset). Adult worms in vaccinated animals produce fewer ova, eventually, succumb to starvation, and detach.*

vaccine-derived immunity does not protect immunized animals from infection with L3 parasites. The worms must mature through the L4 stage and into adulthood for protection to manifest. Schwartz *et al.* found that once ingested, the transition from L3 to L4 and adult is accompanied by a massive alteration of differentially transcribed genes [44]. These changes in gene expression notably did not inform the design of Barbervax®. It is plausible that subunit vaccines targeting L3 stage antigens could prevent the establishment of infection. A polyvalent vaccine consisting of L3 antigens, L4 antigens, and adult phase gut proteins would be maximally effective at both preventing infection and reducing worm burden should it occur (**Figure 2A**).

Transcriptomic analysis can also be used to examine the host–parasite interactions. On the helminth side, transcriptomic analysis can identify specific gene expression patterns in locations of interest in the parasite body. For example, Foth et al described transcripts found in the anterior region of *Trichuris muris*, which likely facilitate host–parasite interactions, nutrient uptake, and digestion [48]. An understanding of the relationship between the host and the parasite can identify vaccine candidates that

#### **Figure 2.**

*Potential omics-guided vaccine design. The current vaccination strategy against* H. contortus *(A) can be expanded to include antigens expressed in the L3 and/or L4 life stages, potentially preventing infection in addition to reducing worm burden should it occur. A proteomics-guided approach to vaccination against a hypothetical cestode (B) could target antigens present in the infectious stage (the cysticercus) and the adult worms, immunizing hosts against the infection or persistence of parasitic helminths. This approach could also include immunomodulatory effector proteins as antigens, maximizing the potential of a robust response to vaccination.*

#### *Perspective Chapter: Multi-Omic Approaches to Vaccine Development against Helminth Diseases DOI: http://dx.doi.org/10.5772/intechopen.102621*

target transcription products that play a role in immunomodulation or metabolism [49]. Transcriptomic analysis can also illuminate host responses by examining gene expression changes in host tissue during infection. Most notably, these analyses can aid in the understanding of the immunomodulation that allows for chronic parasite infections to occur. Parasitic effects on the host immune system have made vaccine development difficult, and it is, therefore, critical to understand mechanisms of immunomodulation exhibited by each parasitic helminth. For example, *Fasicola hepatica* infection was shown to inhibit natural killer cells and IgE production at the transcriptomic level, likely aiding *Fasciola hepatica* in evading cytotoxicity [50]. Vaccines targeting *F. hepatica* must, therefore, be designed and suitably adjuvanted in anticipation of the parasite's ability to strongly downregulate these protective activities post-challenge. Taken together, an ideal vaccine formulation would include not only protective *F. hepatica* antigens but antigens from the immunosuppressive effector proteins as well so that they are neutralized immediately upon infection of a vaccinated host.

A newer area of interest in vaccine development for parasitic helminths is the analysis of excretory/secretory products. These are various molecules released at the host–parasite interface and likely play a role in the manipulation of the host response. These products can be proteins, lipids, nucleic acids, metabolites, and extracellular vessels [51]. The microRNA (miRNA) present in extracellular vessels appears to play a role in the regulation of gene expression and immunomodulation of the host response. Understanding this miRNA will aid in identifying the ways that helminth infections are able to induce differing expressions within the host [52]. The ability of concentrated, purified versions of this miRNA may be able to be used to augment responses to subunit antigen vaccines.

Transcriptomic analysis from parasite life cycles and infected hosts is a useful tool in the development of anti-helminth vaccines. These analyses can contribute to all aspects of vaccine design, from identification of antigens to identifying (and thus circumventing) mechanisms with which parasitic helminths are able to evade adaptive immunity.

## **5. Proteomic approaches to vaccine development for helminth diseases**

Proteomic analysis is among the most powerful tools for the identification of potential protective antigens against helminth diseases. The advent of proteomic technologies provided the opportunity not only to identify potential antigens but to detect any post-translational modifications as well. In addition, proteomic analyses identify all potential antigens, not simply those targeted by patient immune responses during infection. To ensure long-term survival, helminths tend to modulate and subdue immune responses, and the ability of these organisms to undergo host immune evasion poses a challenge for vaccine development [53]. Evaluating the adaptive immune responses of infected patients to identify potential antigens may be misleading, because these responses may be directed at non-neutralizing or variable antigens. Proteomic analyses can identify secreted proteins (*i.e.*, the secretome) expressed by helminths that modulate host immune responses and promote parasite survival [18, 54]. Anti-helminthic vaccine design guided by proteomics holds the promise to target both protective helminth body antigens and to neutralize immune evasion proteins generated by the parasite (**Figure 2B**).

A small number of vaccines designed following proteomics, immunomics, and reverse vaccinology analyses have been described; however, few have moved into

animal trials to evaluate their efficacy. Potential antigens have been identified for *Schistosoma* spp. [19, 55], *Ascaris lumbricoides* [56, 57], *Trichuris trichiura*, *Necator americanus*, *Ancylostoma duodenale* [57], *Strongyloides stercoralis* [58], *Taenia solium* [59], *Toxocara canis* [60], *Onchocerca volvulus*, *Brugia malayi* [61], and *Echinococcus granulosus* [62, 63]. The number of experimental vaccines developed and tested for both immunogenicity and protection against challenge following *in vivo* proteomic analyses is vanishingly small. A recombinant protein vaccine targeting two surface glycoproteins of adult *Fasciola hepatica* lead to robust production of IgG antibodies, but failure to protect vaccinated cattle against infectious challenge [64]. A similarly designed recombinant protein vaccine targeting *T. canis* also resulted in seroconversion of immunized mice, and in this instance, worm burdens were significantly reduced compared to sham-vaccinated controls [60]. An experimental vaccine targeting secreted effector molecules of *Cooperia oncophora* initially seemed to provide some protection to vaccinated cattle, though subsequent studies found protection to be minimal [65]. However, another vaccine targeting secreted proteins of *Ostertagia ostertagi* resulted in a significant reduction of egg shedding by experimentally infected cattle [66]. These variable approaches across vaccine design strategies indicate that ideal formulations may require combining approaches and/or tailoring strategies as well as antigens to each parasitic helminth species. Consistent with this notion is the experimental vaccine against *Teladorsagia circumcincta*, which is a cocktail of larval stage antigens identified by reverse vaccinology, secreted immunomodulatory effector proteins, and adult-stage antigen identified by multi-omic analysis [65]. Vaccinated sheep showed significant reductions in both egg shedding and worm burden [67]. Though proteomic-guided development of immunizations against helminth diseases is a field in its infancy, it holds outstanding promise to craft vaccines that feature precise alignment with parasite life stages and the potential to raise immune responses that can neutralize immunomodulatory effector molecules.
