Ferdowsi University of Mashhad

Document Type : Research Articles

Authors

1 Education and Extension Organization (AREEO), Agricultural Research, Razi Vaccine and Serum Research Institute, Mashhad Branch, Mashhad, Iran

2 Department of Microbiology and Immunology, Faculty of Veterinary Medicine University of Tehran, Tehran, Iran

Abstract

     Clostridium perfringens and novyi species are two important toxin-producing pathogens which pose a risk to the livestock health. Epsilon and alpha toxins are major toxins of these two pathogens, respectively. Advances in current vaccine industrialization lead to the utilization of toxin epitopes instead of the whole pathogen/toxoids to produce novel vaccines. In the present study, bioinformatics approaches were applied to design a fused protein containing both toxin fragments of interest with the highest antigenicity score for B-cells. To do so different specialized algorithms including I-TASSER, IEDB, ElliPro, PyDock and CLC Main Workbench were applied. The chimeric protein was successfully cloned, expressed, and purified using an immobilized-metal affinity chromatography for His-tagged proteins. During in vivo experiments on rabbits, the levels of immunization provided by the recombinant protein or native alpha and epsilon toxins were compared based on serological studies. Results indicated that the designed protein was able to stimulate effective immune responses against both alpha and epsilon toxins. This can be used as a proper strategy to design novel peptide-based subunit vaccines. Clostridium perfringens and novyi species are two important toxin-producing pathogens which pose a risk to the livestock health. Epsilon and alpha toxins are major toxins of these two pathogens, respectively. Advances in current vaccine industrialization lead to the utilization of toxin epitopes instead of the whole pathogen/toxoids to produce novel vaccines. In the present study, bioinformatics approaches were applied to design a fused protein containing both toxin fragments of interest with the highest antigenicity score for B-cells. To do so different specialized algorithms including I-TASSER, IEDB, ElliPro, PyDock and CLC Main Workbench were applied. The chimeric protein was successfully cloned, expressed, and purified using an immobilized-metal affinity chromatography for His-tagged proteins. During in vivo experiments on rabbits, the levels of immunization provided by the recombinant protein or native alpha and epsilon toxins were compared based on serological studies. Results indicated that the designed protein was able to stimulate effective immune responses against both alpha and epsilon toxins. This can be used as a proper strategy to design novel peptide-based subunit vaccines.
 

Keywords

 Adhikari R. P., Karauzum H., Sarwar J., Abaandou L., Mahmoudieh M., Boroun A. R., et al. (2012) Novel structurally designed vaccine for S. aureus α-hemolysin: protection against bacteremia and pneumonia. PloS one, 7:6, p.e38567.
Alves G. G., Machado de Avila R. A., Chavez-Olortegui C. D. and Lobato F. C. (2014) Clostridium perfringens epsilon toxin: the third most potent bacterial toxin known. Anaerobe 30:102-107.
Attasi M. Z. (1984) Antigenic structures of proteins: their determination has revealed important aspects of immune recognition and generated strategies for synthetic mimicking of protein binding sites. European journal of biochemistry, 145:1, 1-20.
Busch C., Schomig K., Hofmann F. and Aktories K. (2000) Characterization of the catalytic domain of Clostridium novyi alpha-toxin. Infection and immunity, 68:11, 6378-6383.
Chandran D., Naidu S. S., Sugumar P., Rani G. S., Vijayan S. P., Mathur D., et al.  (2010) Development of a recombinant epsilon toxoid vaccine against enterotoxemia and its use as a combination vaccine with live attenuated sheep pox virus against enterotoxemia and sheep pox. Clinical and Vaccine Immunology, 17:6, 1013-1016.
Chen J., Rood J. I. and McClane B. A. (2011) Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system. MBio 2:6. e00275-11
Karczewski J., Zorman J., Wang S., Miezeiewski M., Xie J., Soring K., et al. (2014) Development of a recombinant toxin fragment vaccine for Clostridium difficile infection. Vaccine, 32:24, 2812-2818.
Majidi B., Fathi Najafi M. and Saeedyan S. (2020) Expression and purification of Brucella spp lumazine synthase decameric carrier in fusion to extracellular domain of influenza M2E protein. Iranian Journal of Chemistry and Chemical Engineering (IJCCE).
Mehrvarz M., Najafi M. F., Salehi T. Z. and Majidi B. (2020) In silico analysis and selection of Clostridium perfringens type D epsilon toxin multiepitope fragment. International Journal of Scientific Research and Engineering Development 2:6, 434-440.
Noshari N. G., Najafi M. F., Kakhki A. m., Majidi B. and Mehrvarz M. (2016) Identification and cloning of highly epitopic regions of Clostridium novyi alpha toxin. Turkish Journal of Biology, 40:6, 1219-1226.
Oscherwitz J. (2016) The promise and challenge of epitope-focused vaccines. Human vaccines & immunotherapeutics, 12:8, 2113-2116.
Palatnik-de-Sousa C. B., Soares I. d. S. and Rosa D. S. (2018) Epitope discovery and Synthetic Vaccine design. Frontiers in immunology 9:826.
Pilechian L. R., AGHAEI P. K., Shamsara M., Jabbari A., Habibi G., Goudarzi H. et al. (2011) Fusion of Clostridium perfringens type D and B epsilon and beta toxin genes and it’s cloning in E. coli,  Archives of razavi institute, 66:1, 1-10.
Sambrook J., Green M., Sambrook J. and Sambrook J. (2012) Molecular Cloning: A Laboratory Manual, fourth ed. Cold Spring Harbor Laboratory Press, NewYork.
Schmidt S. R. (2009) Fusion-proteins as biopharmaceuticals–applications and challenges. Curr Opin Drug Discov Devel 12:2, 284-295.
Souza A. M., Reis J. K., Assis R. A., Horta C. C., Siqueira F. F., Facchin S., et al. (2010) Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization. Genetics and Molecular Research 9:1, 266-276.
Uppalapati S., Kingston J., Murali H. and Batra H. (2012) Generation and characterization of an inter‐generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications. Journal of applied microbiology, 113:2, 448-458.
Watts C. (2004) The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules. Nature immunology, 5:7, 685-692.
Weimer E. T., Lu H., Kock N. D., Wozniak D. J. and Mizel S. B. (2009) A fusion protein vaccine containing OprF epitope 8, OprI, and type A and B flagellins promotes enhanced clearance of nonmucoid Pseudomonas aeruginosa. Infection and immunity 77:6, 2356-2366.
CAPTCHA Image