IN-SILICO MULTI-EPITOPE VACCINE CANDIDATE DESIGN AGAINST CHICKEN COCCIDIOSIS

Author:
Osuji Charles and Abubakar Salisu

Doi: 10.26480/mahj.01.2025.40.45

This is an open access article distributed under the Creative Commons Attribution License CC BY 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

The economic burdens of coccidiosis pose a very significant challenge to commercial poultry farmers. Not only does it threaten human nutritional and pecuniary endeavors, but efforts to control the infection through hygienic measures and biosecurity are insufficient to contain it. Drug treatments using anti-coccidiosis agents are somewhat ineffective owing to frequent resistance concerns which prompted exploration into chemoprophylaxis as a more viable solution. However, challenges of the low immune response, side effects, and cost, are undermining factors in using the existing vaccines. By adopting an immunoinformatics-aided procedure, this study designed a potential broad-spectrum vaccine for chicken coccidiosis, mining from the various chicken Eimeria apical membrane antigens (AMAs) and other key sporozoite surface antigens (SSAs). Standard structural Bioinformatics tools were utilized to identify antigenic epitopes from the important proteins involved in the chicken Eimeria pathogenesis and conjugate them into a multi-epitope subunit vaccine. A potential broad-spectrum sub-unit vaccine construct consisting of 26 selected epitopes was designed through stringent analyses of immunological, physicochemical, structural, and molecular validations. While appropriate linkers were used for the conjugation, Beta defensin-3 adjuvant, and Padre Sequences were included to enhance the immune response. The resulting construct is a stable and promising vaccine candidate for further analysis and wet laboratory validation.

1. INTRODUCTION

Chickens are undeniably one of the primary sources of animal protein for human consumption and are known for their high-quality protein products (Mesa-Pineda et al., 2021; Britez et al., 2023). Coccidiosis in chickens is an enteric pathogenic disease by Eimeria species that results in substantial global economic losses (Blake et al., 2020). As part of the Apicomplexan family, these parasites have a unique mechanism of invading host cells through membrane proteins aided by specialized organelles like micronemes which are crucial to their successful hosts’ cell invasion (Suarez et al., 2017; Burrell et al., 2020). The Eimeria pathogens that cause chicken coccidiosis thrive in the intestinal epithelial cells after being transmitted through oocysts, leading to varying degrees of morbidity and mortality in poultry (Zaheer et al., 2022). These pathogenic species of Eimeria which include tenella, acervulina, praecox, among others, are known to cause chicken coccidiosis (Clark et al., 2017; Abbas et al., 2019). Coccidiosis pathophysiology begins primarily with the pathogen’s invasion, which involves interactions between sporozoites and host cells, then migration to enterocytes, and subsequent proliferation. This leads to disruption of the normal mucosal cellular function resulting in increased permeability of the intestinal wall and poor nutrient absorption (Madlala et al., 2021). Consequential symptoms like fluid loss, diarrhea, weight loss, and intestinal hemorrhaging follow suit and most times culminate in death (Vrba et al., 2010). The severity and clinical outcomes depend on various factors like host-pathogen interactions, the species involved, the dose of infection, and the environment (Britez et al., 2023). Therefore, pathogenicity in chicken Eimeria infections varies from mild to severe (Tewari et al., 2011). Developing broad-spectrum vaccines that target various species of Eimeria pathogens in chickens is essential due to the negative impacts of coccidiosis on global economic and nutritional well-being. For many years, anti-coccidial drugs have been the primary method of control, but frequent cases of drug resistance have negatively affected its effectiveness (Zhang et al., 2012). Vaccines are considered the most effective method due to their efficacy and low likelihood of resistance development. However, the drawbacks of current vaccines include their limited broad-spectrum effectiveness, side effects, and costs, as most are from live and attenuated organism sources. The low efficiency associated with these vaccine preparations is primarily due to the antigenic variation among Eimeria species, driven by retro-transposons, resulting in limited cross-protection (Hinsu et al., 2018). Currently, there is rarely any chicken coccidiosis vaccine that provides protection against a wide range of Eimeria species. Immunoinformatics-designed multi-epitope vaccines are gaining recognition for their cost-effectiveness and flexibility in combating various pathogens. Subunit vaccines, unlike conventional ones, are safer because they do not contain infectious particles, reducing the risk of reinfection. Computationally optimized subunit vaccines are being recommended for disease prevention due to their overwhelming advantages over conventional vaccines (Sharma et al., 2021). Given the characteristic features of surface antigens as immune targets and the role of sporozoites in coccidiosis, surface proteins from sporozoite antigens were selected for this study. These proteins were chosen for their strong host-parasite interaction, ability to facilitate attachment and invasion, immune protection, and low variability (Vo et al., 2021). They include; the AMA-1 and other surface antigens like the Microneme and Immune-mapped proteins (MIC and IMP) (Liu et al., 2019; Wang et al., 2023). This in-silico study, therefore aimed at mining multiple viable epitopes of immunological interest from the various known chicken coccidiosis pathogens and designing a potential broad-spectrum vaccine construct via a stringent evaluation methodology.

2.METHODOLOGY

2.1 Target sequence Retrieval, Epitopes Prediction and Selection

Surface protein sequences from the known pathogenic chicken Emeiria, species were selected. This was followed by retrieval of sequences of immunological importance from UniprotKb repository in FASTA format. The retrieved sequences were subjected to standard immunoinformatics web tool of NetMHCI v 4.0, NetMHCII v2.3 and Immune Epitope Database (IEDB) for the various epitopes mining processes. At default thresholds, strong antigenic epitopes with relatively lower threshold were preferably selected on the basis of their major histocompatibility complex classes I and II binding abilities for the T-lymphocytes. Epitopes’ percentile ranks and locations were considered for the Linear B-Lymphocyte mining process (Tarrahimofrad, et al., 2021; Khan et al., 2023).

2.2 Epitopes Screening

The selected predicted epitopes were subjected to screening pipelines to eliminate the poor immunologic and unsafe sequences by running them through antigenicity, allergenicity, immunogenicity and toxicity analyses. Antigenic potentials of the epitopes were analyzed using VaxiJen 2.0 web server whereas allergenicity and toxicity were evaluated with AllerTop 2.0 and Toxinpred web tools. The predicted HTL epitopes were further subjected to interleukin and cytokine inducing capacity evaluations with IFN-γ, IL-4 and IL-10 tools (Kumar et al 2021) for final selection. The selected CTLs, HTLs and LBC epitopes were shown in Tables 1, 2, and 3 respectively.

2.3 Vaccine Construct Design

The selected vaccine-fit epitopes of the linear B-lymphocytes and T-lymphocytes from the mined pool in conjunction with adjuvants and linkers were subjected to optimization process to design the potential vaccine construct shown in Figure 1 and 2 as primary and secondary structures respectively. Beta- defensing-3 and PADRE sequences were retrieved and used as adjuvants while EAAK, AAY, GPGPG and KK sequences were the linkers used. Linkers AAY and GPGPG were used separately for HTLs in different constructs while LBL epitopes were flanked by KK sequences. The connecting bond used for adjuvant-epitope linkage was the EAAK sequence (Madanagopal, 2023).

2.4 Vaccine Properties Prediction

The resulting construct was assessed for immunological, physicochemical and structural potentials (Bin-sayed et al., 2020). Double evaluations of the immunological parameters were performed. Antigenicity and allergenicity analyses were carried out with VaxiJen v2.0 / AntigenPro and AllerTop v2.0 / AllergenFP servers respectively. Toxicity evaluations were performed with ToxinPred2 and ToxDL web tools. The physicochemistry of the construct was analyzed with ExPASy-ProtParam web tool for quality and stability determination (Mahmud et al., 2021; Bashir et al., 2023). Key physiochemical factors like instability index, amino acid composition, aliphatic index, molecular formula, molecular mass, isoelectric potential (pI), extinction coefficient and the Grand Average of Hydropathicity (GRAVY), values were all analyzed (Pandey et al., 2021).

2.5 Vaccine Construct’s Structural analysis

The secondary structure of the vaccine construct was predicted using the PSIPRED 4.0 server through the Psi-BLAST algorithm (McGuffin et al., 2000). For 3-D structure determination and modeling, the PHYRE 2.0 tool was employed (Roy et al., 2010). Refinement of the 3-D structures was conducted using GalaxyRefine web server, which uses CASP10 refining method, to assess structural stability (Kumar et al., 2023). The refined structures were then downloaded and the selected model was chosen based on overall quality assessments (Yang et al., 2022). Structural authentication of the vaccine constructs was done with Procheck and ProSA tools, which provided the Z-score and Ramachandran values, respectively (Wiederstein and Sippl, 2007). The construct’s structural validation and molecular docking were also carried out. Chicken Toll-like receptor-15 (7-YLG) was used for docking the vaccine construct to assess their binding interactions. ClusPro v2.0 webserver which operates on PIPER algorithm was used for docking and data generation (Kozakov et al., 2017).

3.RESULTS

3.1 Epitopes Screening and Conjugation

A total of 26 epitopes consisting of 6 Linear B-lymphocytes, 7 Cytotoxic T-lymphocytes, and 13 Helper T-lymphocytes were conjugated to form the final construct which was consequently evaluated for antigenicity, allergenicity, and toxicity using two different web tools for each analysis, as detailed in

4.DISCUSSION

The development of prophylactic agents that can address the antigenic diversity challenge in chicken coccidiosis pathogenesis and overcome the low immunogenicity of current vaccines is necessary for the effective prevention of the disease. Advancements in structural and functional vaccinomics, which integrates ‘Omics’ technologies with reverse vaccinology, have significantly enhanced vaccine-likeness screening and development, especially against a variety of pathogens (Soltan et al., 2021). In-silico epitope mining and evaluation involve the use of computational tools to identify peptide sequences within antigens that can trigger immune responses.

In this study, mining and conjugation of potentially effective multi-epitope were carried out using several computational algorithms after which the final vaccine candidate was subjected to stringent immunological, structural, and physichochemical evaluations. The strong antigenicity, non-allergenicity, and non-toxicity of the construct were affirmed by the immunological analysis results. The physicochemical characterizations also revealed the excellent vaccine-likeness of the constructs as shown in Table 4. The molecular weights and instability indices of 51.743 kDa, and 38.07 which are less than 110 kDa and 40.0 respectively are in agreement with Kumar et al., (2023). The values of 56.92 and -0.570 recorded for GRAVY and aliphatic index respectively indicate good thermo-stability and hydrophilicity, preferable for good water molecule interaction in conformity with Atapour et al., (2020) and Kumar et al., (2023). The construct exhibited good solubility, isoelectric points, and extinction coefficients values of 0.9784, 5.74, and 36330 M-1 cm-1 which also conform with Habib et al., (2023) and sarvmeili et al., (2024). The predicted half-life for the construct is 1 hour in-vitro, in mammalian reticulocytes, 30 minutes, and over 10 hours in-vivo in yeast and E coli respectively. This is good for further pre-clinical investigative analysis. The secondary structure determination and examination using PSIPRED identified regions consisting of helices, β-strands, and random coils.

From the five refined 3-D models generated by GalaxyRefine, model-3 was selected based on its Clash scores, GDT-HA, RMSD, Poor rotamer, and Ramachandran favored scores as shown in Table 5. The chosen refined model of interest was downloaded and validated using Prosa and Procheck web tools to produce its Z-score and Ramachandran plots as shown in Figure 5.0. Docking results from the ClusPro server demonstrated the binding stability and structural compactness of different conformations as a measure of the cluster sizes and binding energy values as shown in Table 6. The selection of Complex-‘ 0’ as the best vaccine-receptor complex was primarily based on cluster size, as ClusPro ranks structures according to cluster population rather than solely energy values, in line with the approach of Desta et al. (2020).

5.CONCLUSION

Bioinformatics skills application in the exploration of immune components of living organisms has led to faster, cheaper, specific, and effective vaccine development process. Using computational optimization as a precursor to the wet lab validation process is an approach that is revolutionizing ‘Vaccinology’ in modern vaccine development. The results of in-silico guided mining and designing of potential broad-spectrum chicken coccidiosis vaccine construct from this current study produced a promising vaccine candidate based on vaccinomics evaluations. This research therefore provides a solid foundation for further molecular immunoinformatics analyses and wet lab validation using animal models for the development of an effective broad-spectrum chicken coccidiosis vaccine.

AUTHORS’ CONTRIBUTION

O.C designed and carried out the in-silico experimentation while A.S performed the data analysis and proofreading.

CONFLICTS OF INTEREST

“We hereby make a declaration no conflicts of interest regarding this work”.

REFERENCES

Abbas.,A., Abbas, R. Z., Khan, M. K., Raza, M. A., Mahmood, M. S., Saleemi, M. K., Hussain, T., Khan, J. A., and Sindhu, Z. U. ,2019. Anticoccidial effects of Trachyspermum ammi (Ajwain) in broiler chickens. Pak Vet Journal 39,301–304. DOI: https://doi.org/10.29261/pakvetj/2019.056

Atapour.,A., Negahdaripour, M., Ghasemi, Y., Razmjuee, D., Savardashtaki, A., Mousavi, S. M., et al.,2020. In silico designing a candidate vaccine against breast cancer. Int. J. Pept. Res. Ther. 26, 369–380. DOI: https://doi.10.1007/s10989-01909843-1

Bashir., Z., Ahmad, S. U., Kiani, B. H., Jan, Z., Khan, N., and Khan, U.,2021. Immunoinformatics Approaches to Explore B and T Cell Epitope-based Vaccine Designing for SARS-CoV-2 Virus. Pak J Pharm Sci. 34, 28-49.

Bin-Sayed., S., Nain, Z., Khan, M. S. A., Abdulla, F., Tasmin, R., and Adhikari, U. K.,2020. Exploring Lassa Virus Proteome to Design a Multi-epitope Vaccine through Immunoinformatics and Immune Simulation Analyses. Int. J Pept Res Ther. 26, 2089–2107. DOI: https://doi.org/10.1007/S10989-019-10003-8

Blake., D. P., Vrba, V., Xia, D., Jatau, I. D., Spiro, S., Nolan, M. J., and Tomley, F. M.,2020. Genetic and biological characterization of three cryptic Eimeria operationaltaxonomic units that infect chickens (Gallus gallus domesticus). Int J Parasitol. 51, 621–34. DOI: https://doi.10.1016/j.ijpara.2020.12.004

Britez., J. D., Rodriguez, A. E., Di Ciaccio, L., Marugán-Hernandez, V., and Tomazic, M.L.,2023. What Do We Know about Surface Proteins of Chicken Parasites Eimeria? Life, 13,1295. DOI: https://doi.org/10.3390/life13061295

Burrell., A., Tomley, F. M., Vaughan, S., and Marugan-Hernandez, V..,2020. Life cycle stages, specific organelles and invasion mechanisms of Eimeria species. Parasitology, 147(3)Pp., 263-278. DOI: https://doi.org/10.1017/S0031182019001562

Clark., E. L., Tomley, F. M., Blake, D. P.,2017. Are Eimeria genetically diverse, and does it matter? Trends Parasitol 33(3)Pp.,231–241. DOI: https://doi.org/10.1016/j.pt.2016.08.007
Desta.,I. T., Porter, K. A., Xia, B., Kozakov, D., Vajda, S.,2020. Performance and Its Limits in Rigid Body Protein-Protein Docking. Structure. 28(9)Pp.,1071-1081. DOI: https://doi.org/10.1016/istr.2020.06.006

Habib.,A., Liang, Y., Xu, X., Zhu, N., and Xie, J.,2023. Immunoinformatic Identification of Multiple Epitopes of gp120 Protein of HIV-1 to Enhance the Immune Response against HIV-1 Infection. International Journal of Molecular Sciences, 25(4)Pp., 24-32. https://doi.org/10.3390/ijms25042432

Hinsu., A. T., Thakkar, J. R., Koringa, P. G., Vrba, V., Jakhesara, S. J., and Psifidi, A.,2018. Next generation sequencing for the analysis of Eimeria populations in commercial broilers and indigenous chickens. Front Vet Sci. 5, 176. DOI: http://doi.10.3389/fvets.2018.00176

Khan.,M. S., Khan, I. M., Ahmad, S. U., Rahman, I., Khan, M. Z., Khan, M. S., Abbas, Z., Noreen, S., and Liu, Y.,2023. Immunoinformatics Design of B and T-cell Epitope-based SARS-CoV-2 Peptide Vaccination. Frontiers in Immunology,13,1-17 1001430. DOI: https://doi.org/10.3389/fimmu.2022.1001430

Kozakov.,D, Hall DR, Xia B, Porter KA, Padhorny D, Yueh C, Beglov D, Vajda S. The ClusPro web server for protein-protein docking. Nature Protocols. 2017 Feb;12(2)Pp.,:255-278

Kumar., A., Rathi, E., and Kini, S. G. (2021). Computational Design of a Broad-spectrum Multi-epitope Vaccine Candidate against Seven Strains of Human Coronaviruses. 3 Biotech (Springer), 12 (240), 1-17.DOI: https://doi.org/10.1007/s13205-022-03286-0

Kumar KM.,Karthik Y, Ramakrishna D, Balaji S, Skariyachan S, Murthy TPK, Sakthivel KM, Alotaibi BS, Shukry M, Sayed SM & Mushtaq M .,2023)Immunoinformatic exploration of a multi epitopebased peptide vaccine candidate targeting emerging variants of SARSCoV . Front. Microbiol. 14(1251716), 1-19. DOI:https://doi.org/1.3389/fmicb.2023.1251716

Liu.,G., Zhu, S., Zhao, Q., Dong, H., Huang, B., Zhao, H., Li, Z., Wang, L., and Han, H. ,2019. Molecular Characterization of Surface Antigen 10 of Eimeria tenella. Parasitol. Res. 118, 2989–2999

Madanagopal.,P., Muthusamy, S., Pradhan, S.N., and Prince, P. R.,2023. Construction and Validation of a Multi-epitope In-silico Vaccine Model for Lymphatic Filariasis by Targeting Brugia malayi: A Reverse Vaccinology Approach. Bull Natl Res Cent. 47(1),Pp., 47. DOI: https://doi.org/10.1186/s42269-023-01013-0.

Madlala.,T., Okpeku, M., and Adeleke, M. A.,2021. Understanding the interactions between Eimeria infection and gut microbiota, towards the control of chicken coccidiosis, A review; Parasite. 28, 1–10. DOI: https://doi.10.1051/parasite/2021047

Mahmud., S., Rafi, M. O., Paul, G. K., Promi, M. M., Shimu, M. S., Biswas, S., Emran, T. B., Dhama, K., Alyami, S. A., Moni, M. A., and Saleh, M. A.,2021. Designing a Multi-epitope Vaccine Candidate to Combat MERS-CoV by Employing an Immunoinformatics Approach. Scientific Reports, 11(1),Pp., 1-20. DOI: https://doi.org/10.1038/s41598-021-92176-1

McGuffin.,L. J., Bryson, K., and Jones, D. T. (2000). The PSIPRED Protein Structure Prediction Server. Bioinformatics, 16, 404–405. DOI: https:// doi. org/ 10. 1093/ BIOIN FORMA TICS/ 16.4. 404

Mesa-Pineda., C., Navarro-Ruíz, J. L., López-Osorio, S., Chaparro-Gutiérrez, J. J., & Gómez-Osorio, L. M.,2021. Chicken Coccidiosis: From the Parasite Lifecycle to Control of the Disease. Front. Vet. Sci. 8, 787653. DOI: https://doi.10.3389/fvets.2021.7876531

Pandey., R.K., Ojha, R., Dipti, K., Kumar, R., and Prajapati, V. K. ,2020. Immunoselective algorithm to devise multi-epitope subunit vaccine fighting against human cytomegalovirus infection. Infect. Genet. Evol., 82, 104282 DOI: https://doi.10.1016/j.meegid.2020.104282 .

Roy., A., Kucukural, A, Zhang, Y.,2010. I-TASSER: A Unified Platform for Automated Protein Structure and Function Prediction. Nat Protoc. 5,725–738. DOI: https://doi.org/10. 1038/ nprot.2010.5

Sarvmeili.,J., Baghban Kohnehrouz, B., Gholizadeh, A., Shanehbandi, D., and Ofoghi, H.,2024. Immunoinformatics design of a structural proteins driven multi-epitope candidate vaccine against different SARS-CoV-2 variants based on fynomer. Scientific Reports, 14(1)Pp.,1-26. DOI: https://doi.org/10.1038/s41598-024-61025-2

Sharma., S., Kumari, V., Kumbhar, B. V., Mukherjee, A., Pandey, R., and Kondabagil, K.,2021. Immunoinformatics approach for a novel multi-epitope subunit vaccine design against various subtypes of Influenza A virus. Immunobiology, 226(2)Pp., 152053. 1-10. DOI:https://doi.org/10.1016/j.imbio.2021.152053

Soltan.,M. A., Abdulsahib, W. K., Amer, M., Refaat, A. M., Bagalagel, A. A., Diri, R. M.,, Albogami, S., Fayad, E., Eid, R. A., Sharaf, S. M., Elhady, S. S., Darwish, K. M., and Eldeen M. A. ,2022. Mining of Marburg Virus Proteome for Designing an Epitope-BasedVaccine. Front. Immunol. 13, (907481) 1-19. DOI: http://doi.10.3389/fimmu.2022.907481

Suarez.,C. E., Bishop, R. P., Alzan, H. F., Poole, W. A. and Cooke B. M.,2017. Advances in the application of genetic manipulation methods to apicomplexan parasites. Int J Parasitol. 47, 701–10. DOI: https://doi.10.1016/j.ijpara.2017.08.002

Tarrahimofrad.,H., Rahimnahal, S., Zamani, J., Jahangirian, E., and Aminzadeh, S.,2021. Designing a multi-epitope vaccine to provoke the robust immune response against influenza A H7N9. Scientific Reports, 11(1)Pp.,1-22. https://doi.org/10.1038/s41598-021-03932-2

Tewari.,A. K, and Maharana, B. R. (2011). Control of poultry coccidiosis: changing trends. J Parasit Dis. 3,:10–17. DOI: https://doi.10.1007/s12639-011-0034-7

Võ, T.C., Naw, H., Flores, R.A., Lê, H.G., Kang, J.M., Yoo, W.G., Kim, W.H., Min, W., & Na, B. K.,2021. Genetic Diversity of Microneme Protein 2 and Surface Antigen 1 of Eimeria tenella. Genes, 12(9), Pp.,1418 1-12. DOI: https://doi.org/10.3390/genes12091418

Vrba.,V., Blake, D. P., and Poplstein, M.,2010. Quantitative real-time PCR assays for detection and quantification of all seven Eimeria species that infect the chicken. Vet Parasitol. 174, 183–90. DOI: https://doi.10.1016/j.vetpar.2010.09.006

Wang.,F., Zhang, A., Fan, X., Feng, Q., Zhang, Z., Liu, D., Su, S., Hou, Z., Xu, J., Kang, X., Pan, Z., Hu, H., and Tao, J.,2023. Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection. Vet Parasitol., 324, 110060. DOI: https://doi.10.1016/j.vetpar..110060 .

Wiederstein.,M., and Sippl, M. J.,2007. ProSA-web: Interactive Web Service for the Recognition of Errors in Three-dimensional Structures of Proteins. Nucleic Acids Res., 35 (W407), 1-4. DOI: https://doi.org/10.1093/NAR/GKM290

Yang.,C., Chen, E. A., and Zhang, Y.,2022. Protein–Ligand Docking in the Machine-Learning Era. Molecules, 27(14)Pp.,1-24. DOI: https://doi.org/10.3390/molecules27144568

Zhang.,J., Wang, L., Ruan, W., and An, J.,2012. Investigation into the prevalence of coccidiosis and maduramycin drug resistance in chickens in China. Veterinary parasitology. 191, 29-34. DOI: https://.doi.10.1016/j.vetpar.2012.07.027

Pages 40-45
Year 2025
Issue 1
Volume 5