Can Cathepsin L Epitope Be an Effective Vaccine Candidate Against Fasciola gigantica via Bioinformatics?

Fasciolosis is an important helminthic disease caused by liver flukes, such as Fasciola gigantica, affecting a wide variety of domestic and wild animals, including cattle, sheep, goats, buffalo, deer, rabbit, and humans all around the world, especially in temperate and tropical climates. This host diversity contributes notably to the global spread of the infection (1). Fasciola gigantica is one of the most important parasitic helminths in Asia and Africa, affecting individual and small farming communities. It causes severe economic losses by decreasing animal production and livestock mortality. It is estimated that fasciolosis inflicts over US$3 billion in losses on the global animal industry (2). Fasciolosis has also been reported in humans in numerous countries. Iran is one of the countries where fasciolosis is a serious issue (3). Juvenile stages of Fasciola migrate through the circulatory system to the liver and mature into adults in the biliary ducts. The major cause of pathogenesis and clinical features of the infection is related to the migration of the parasite to the liver and the presence of adults in the biliary ducts (4).

Today, anti-parasitic drugs against fasciolosis are less effective due to the emergence of drug resistance. It has become a drastic problem in veterinary and medical sciences (5); thus, the attempts focus on designing a novel vaccine against the infection. Some candidates, including protease enzymes, have been proposed as the vaccine target (6). Proteases are secreted by different forms of parasites in order to facilitate parasitism and help the pathogens survive in their host for a long time (7). Cathepsin L (Cat L) enzyme, found in all stages of the liver flukes, plays a significant role in the biological functions of the parasites, such as migration, nutrition, and immune evasion (2). Vaccination with Cat L resulted in different protection rates against fasciolosis (8).

Bioinformatics has become a significant part of biology. Bioinformatics tools are irrefutable in comparing, analyzing, and interpreting genetic and genomic data and understanding evolutionary aspects of molecular biology and protein structure prediction. Increasing the knowledge about various aspects of the epitopes will result in the development and construction of novel epitope-based vaccines against pathogens.

As far as we know, there is no published report regarding bioinformatics analysis of the Cat L sequences of FgNEJ. Therefore, the current study used bioinformatics methods to predict and identify B and T-cell epitopes and general features of Cat L of FgNEJ.

Parasitic infections are a major threat to humans and animals, leading to serious diseases (22, 23). The control strategy for Fasciola infection is currently based on using antihelmintic drugs. However, due to the long-term use of drugs, drug resistance has been reported in many countries, including Australia, Ireland, and Argentina (24). Vaccination seems to be the best way to control fasciolosis infection (25) Vaccines are more sustainable, cost-effective, and environmentally friendly tools that significantly boost immunity against pathogens. They mostly consist of killed or attenuated pathogens, which may sometimes be dangerous for the host due to problems in its preparation process. Thus, it is safer to apply an epitope-based vaccine, inducing and stimulating an immune response against the specific pathogen (26). Different antigens have been described as vaccine targets against fasciolosis. It has been reported that cysteine proteinases are promising vaccine antigens as they play a vital role in host-parasite interaction. Cathepsins are from the family of cysteine proteases expressed in Fasciola species. Several isoforms of Cat L and B are expressed in different stages of Fasciola parasites (27).

One of the most valuable ways to design and deliver an epitope-based vaccine is by analyzing the peptide signal and antigen properties using bioinformatics tools (20, 28). Introductory bioinformatics analysis of potential vaccine candidates causes a significant reduction in the number of used animals in vaccine trials and provides important and essential data about the protein of interest in an easy and simple way (29). In order to construct an ideal vaccine with perfect protection against pathogens, it is crucial to understand the entire structure of their antigens (30). Therefore, computational analysis can be pivotal in the vaccine design process. The present study employed various bioinformatics tools to design the vaccine to learn more about the Cat L of FgNEJ. The signal peptide is the most common characteristic researchers analyze when their target is to find secreted or membrane-bound proteins in the amino acid sequence (31). In the present study, the Cat L analysis revealed an N-terminal signal peptide using the bioinformatics software.

The prediction of protein structures improves our knowledge regarding the function and biological aspects of the protein. The secondary structure plays a significant role in epitope function (32). In the current study, the maximum parts of the secondary structure belonged to the random coil, which is introduced on the protein surface and may be a potential epitope (33). Additionally, alpha-helix and beta-turn are commonly found in the internal part of the protein, and they retain proteins by high chemical-bond energy. It does not seem to work as an epitope.

The epitope is part of the antigen, detected by B-cell and T-cell molecules of the mammalian immune system. Only a few amino acids of a protein (instead of the total protein) can provide enough protective response. Therefore, predicting or identifying this part of the amino acid can be vital to understanding the mechanisms of immunity and pathogenesis of pathogens (32). Most importantly, these epitopes can be used to design epitope-based vaccines (34). Prediction of B-cell and T-cell epitopes is a key step for vaccine design, development of diagnostic reagents, and understanding the antigen-antibody interactions on a molecular level (20). Predicting linear B-cell epitopes in Cat L of FgNEJ was performed using reliable bioinformatics software to find potential targets for vaccines against Fasciola infection.

The current study applied bioinformatics tools such as BCPREDS, ABCpred, Bcepred, and IEDB to predict B-cell epitopes on Cat L of FgNEJ. The results of BCPREDS identified 5 potential antigenic epitopes on the sequence. Also, based on the data from ABCpred software, of 32 epitopes predicted on Cat L, 10 high-score epitopes can be used as targets in vaccine research. Additionally, we applied Bcepred to predict linear B-cell epitopes. The software used physical and chemical properties such as hydrophilicity, accessibility, flexibility/mobility, exposed surface, and turns in order to predict B-cell epitopes. Each of these properties predicted several B-cell epitopes on Cat L. Some protein parameters, such as flexibility, hydrophilicity, turns, accessibility, polarity, exposed surface, and antigenic propensity, have been associated with the localization of continuous epitopes. In the present study, the data obtained from IEDB disclosed some potential epitopes on Cat L of FgNEJ. The results from these tools indicated potential epitopes on this protein that can be strong candidates for designing an effective vaccine against fasciolosis (after testing in animal models). In a study performed on antibody recognition of cathepsin L, the characteristics of known linear B-cell epitopes on F. hepatica Cat L protein using the sera of F. hepatica infected or vaccinated cattle in two independent experiments were identified, and these results were confirmed by other studies (35-37). The study also demonstrated that vaccinated animals with Cat L showed fluke burden reduction with eliciting antibodies (8). Another study on the Plasmodium vivax AMA-1 protein found similar results using the same bioinformatics tools, reported several promising epitopes on AMA-1, and suggested that these bioinformatics tools were valid to predict and identify epitopes on proteins (20).

The MHC molecules represent T-cell epitopes to T-cells. Peptide binding to MHC is a major factor in the selection of potential epitopes and is an essential step in presenting the antigen to T-cells. Prediction of peptide-MHC binding epitopes could be vital for constructing a reliable epitope-based vaccine against infections (38). The significant information to understand the process of infection pathogenesis was provided by the prediction and characterization of both CD8 and CD4 T-cell epitopes on a protein (39). Another major step in vaccine development is the identification of CTL-stimulating peptides. In this study, 10 epitopes were predicted using CTLpred software. Responses derived from cytotoxic T-cells are induced during a pathway composed of intracellular antigens refining upon even epitopes as common targets.