Perspective Chapter: Multi-Omic Approaches to Vaccine Development against Helminth Diseases

Abstract

Though the past three decades have led to a renaissance in vaccine design, the development of vaccines that protect against helminth diseases remains elusive. The need for protective vaccines for humans and livestock remains urgent because of the side-effect profiles of anti-helminthic drugs and the growing incidence of antimicrobial resistance and declining efficacy. The “-omics” era has led to renewed interest in vaccine development against helminth diseases, as candidate vaccines can now be designed, evaluated, and refined in a fraction of the time previously required. In this chapter, we describe and review genomic, transcriptomic, and proteomic approaches to the design of vaccines against helminth diseases.

1. Introduction

Parasitic helminths that cause human and veterinary diseases can be found in two phyla: Nematoda and Platyhelminthes. Helminth diseases carry a significant global burden and collectively infect over 1 billion people [1], and cause a disproportionate number of neglected tropical diseases (NTDs). They are a significant cause of morbidity, and often result in permanent disabilities, impaired responses to other infections leading to worse outcomes, and significant social and economical burden upon patients [2, 3, 4, 5]. Helminth diseases of livestock further threaten human health and economic development by adversely affecting food security. Antimicrobial treatments are available for helminth infections; however, they come with significant challenges. The number of available drug classes is small, and the ones that are available cause significant side effects and do not protect against reinfection [6]. As drug resistance has been observed in animal nematode models, the catastrophic potential for treatment-refractory infections exists [7, 8]. These challenges indicate that the ideal strategy for helminth disease control is prevention rather than treatment. Despite this, there are no known effective vaccines to protect humans against diseases, such as filariasis which carry a high morbidity rate [9, 10]. While practical measures, such as skin coverings (i.e., shoes, waders) and vector control, aid in prevention; these strategies would optimally be coupled with vaccination to ultimately meet the goals of reducing or eliminating disease burden. Continuity of care, treatment side effects, and the potential for drug resistance underscore the urgent need for anti-helminth vaccine development.

Vaccine development against helminth diseases has historically been challenging for a variety of reasons. Helminths are diploid organisms with multiple life stages that are notoriously immunomodulatory. They are able to migrate to multiple tissues and possess numerous immune evasion strategies. The combination of the transient antigen profiles and complex Type 2 immune responses have rendered efforts to immunize patients with killed organisms, attenuated organisms, or single immunogens unsuccessful [11, 12, 13]. Many experimental vaccines for ruminant helminth diseases, such as echinococcosis and fascioliasis, have been described, and a vaccine that protects sheep and goats from Barber’s pole worm (Barbervax®, developed by the Moredun Foundation) has been licensed in the United Kingdom, Australia, and South Africa. The development of Barbervax® was a lengthy process because of the technology available at the time. Additionally, Barbervax® and other experimental vaccines suffer from modest efficacy and at times complicated dosing regimens. Vaccines for human helminth diseases have yet to be licensed due to failures of traditional vaccine design approaches.

The advent of the “-omics” era has led to renewed enthusiasm for vaccine development against helminth diseases and other NTDs. Vaccines similar to Barbervax® can now be designed and modified in a fraction of the time required. Research efforts utilizing genomics, transcriptomics, and proteomics have been undertaken to identify potential antigens and evaluate their expression kinetics during infection and chronic disease as well as their potential to evolve in response to vaccinated populations. Ultimately, multi-omics approaches to vaccine design for helminth infections have the potential to address a multitude of complex factors that are involved in the host–parasite interaction, the intricacies of vaccine design, and the evolutionary implications that follow the introduction of any and all selective pressures. In this chapter, we explore genomic, transcriptomic, and proteomic approaches to the design of vaccines against helminth diseases.

2. “Omic” technologies and reverse vaccinology

Vaccine design was historically approached by manipulating whole infectious agents or their toxins, either by inactivating them or attenuating them. Next-generation vaccines (i.e., those deriving from molecular and synthetic biology) are rooted in reverse vaccinology, wherein design begins by examining the complete genomes, transcriptomes, or proteomes of pathogens. Advances made toward anti-helminth vaccines will undoubtedly rely on reverse vaccinology via multi-omic analysis.

The field of genetic and genomic studies has significantly progressed in the last few decades. Scientists have progressed from analyzing single genes and their functions to studying the entire genetic complements—genomes—of organisms. The field of pathogen genomics has facilitated the development of numerous precise diagnostics and vaccines. These vaccines almost exclusively target viral or bacterial pathogens, however [14]. While it is possible to identify potential antigens based on gene sequences, actual transcribed and translated epitopes may look vastly different, and may not elicit the expected immune response. As such, genomics alone may not be the most reliable informant of a potential vaccine target, due to variations in transcription and protein processing that take place. Section 3 of this chapter aims to review genomic approaches to vaccine development against helminth diseases and elucidate critical concepts and issues related to this approach.

As opposed to a genome, a transcriptome is a collection of all non-ribosomal RNA within a cell type, tissue, or organism under a specific set of circumstances or at a specific stage of the life cycle. The study of transcriptomics allows for the focus to be placed on gene expression throughout various steps of the life cycle and under different conditions [15]. Recently, the availability of sequencing technologies has made both genomics and transcriptomics relatively low-cost analyses that can be routinely performed in many laboratories. Transcriptomic analysis of helminths suffered from a bottleneck due to a lack of publicly available genomic databases for parasitic helminths until recently. Some of these challenges still persist, however, because helminths contain many unique sequences that have not previously been annotated with correlation to an associated protein in other organisms [16]. Additionally, transcriptomics can be used to provide insight into immunomodulation and thus vaccine interference mechanisms by being used as profiling tools to screen infected hosts. While transcriptomic analysis provides greater sensitivity in predicting potential antigens that will be expressed during infection, it cannot account for post-transcriptional regulation of protein expression or any non-canonical post-translational modifications. Section 4 of this chapter aims to review transcriptomic approaches to vaccine development against helminth diseases and elucidate critical concepts and issues related to this approach.

Thematically similar to a transcriptome, a proteome is the full complement of mature, modified proteins present under specific conditions within specific cells or tissues [17]. The proteomic analysis allows target-based approaches to parasite interventions, including the development of anti-helminth vaccines. Previously, transcriptomes of pathogens have been used to identify vaccine targets; however, proteomics allows for a greater likelihood of true representation of potential antigens present during infection. This is especially important for helminths and other parasites because protein expression varies greatly based on the life-cycle stage [18, 19]. By describing a parasitic helminth’s proteome, we can gain a better insight into antigenic targets that are present at each life stage of the parasite. Similar to transcriptomic analysis, proteomic studies of infected hosts can also aid in understanding and circumventing helminth immunomodulatory mechanisms that could adversely affect vaccine efficacy. These studies can be critical in aiding complex vaccine designs such that poor or adverse responses can be avoided. Section 5 of this chapter aims to review proteomic approaches to vaccine development against helminth diseases and elucidate critical concepts and issues related to this approach.

3. Genomic approaches to vaccine development for helminth diseases

The advent of high-throughput genome sequencing has fundamentally changed the approach to vaccine design, enabling the evaluation and fine-scale targeting of potential vaccine antigens throughout the parasite life cycle. Structural genomic, functional genomic, and epigenomic approaches allow for the identification of an estimated 10- to 100-fold more new antigens for vaccine design and drug target candidates as compared to conventional methods in the same time frame [20]. Furthermore, the completion of the Human Genome Project allows for the evaluation of potential antigens for molecular mimicry by parasites that could cause pathological responses to vaccines, and for a thorough understanding of host-pathogen interactions during active infection that could impact vaccine-derived protection [21].

The use of genome-wide applications for human vaccine development has already been observed for bacterial and viral pathogens. The complete genome sequence of Neisseria meningitidis Group B, the agent of meningococcal meningitis, was used to identify several candidate vaccine antigens [22]. Potential antigens were later successfully narrowed down using reverse vaccinology approaches [23]. More recently, the development of both mRNA and Adenovirus-vectored vaccines for the viral pathogen SARS-CoV-2 relied exclusively on viral genomics [24].

The first parasitic nematode, whose genome was sequenced, was Brugia malayi [25], and technological advances have allowed for the sequencing of several more human and animal parasites over the past two decades [26, 27, 28, 29, 30, 31]. The continually expanding amount of genomic data is available in numerous public databases, including generalized repositories, such as GenBank and EBI, and specialized resources, such as WormBase and HelmDB [16, 32, 33, 34]. Additionally, veterinary parasites, such as Haemonchus contortus, serve as a model for genomically-based vaccine development due to its status as the only helminth with a commercially available vaccine and its phylogenetic position that makes it an excellent candidate to be compared to Caenorhabditis elegans, a model organism closely related to numerous human parasites [35, 36].

Despite their numerous advantages, genomic analyses have several drawbacks. Genomic analyses allow for the identification of numerous potential vaccine antigens; however, antigen target selection for vaccine development can be clouded by the immense number of options, many of which may be nonfunctional or promote regulatory responses in helminths and should be eliminated from vaccine formulations [37]. This was previously observed in the development of candidate vaccines against Schistosoma mansoni, where genomic analyses and reverse vaccinology yielded multiple antigen sites and peptides for vaccine development, none of which were protective [38, 39]. Genome sequences also include noncoding intron sections that have to be eliminated during the development process leading to more time-consuming than necessary. Similarly, genomic technology may recommend the creation of monovalent vaccines for helminths that may prove ineffective, as the vaccines may only confer partial immunity [12], or may prove ineffective in human candidates [40].

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 homology-based 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 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, 42, 43, 44, 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, 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).

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.

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.

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 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.

6. Conclusions

Vaccines that protect against helminth diseases remain largely elusive in human and veterinary medicine. The successful licensure and deployment of the subunit vaccine Barbervax® provide evidence that the development of next-generation vaccines against parasitic helminths is an attainable goal. Multi-omic approaches allow for the design and evaluation of rationally designed subunit vaccines. The development of successful candidate vaccines has enormous potential to provide protection for the billions of people impacted by helminth diseases.