Production of a Human Recombinant Polyclonal Fab Antivenom against Iranian Viper Echis carinatus

Document Type: Original Articles

Authors

1 Department of Immunization and Plasma Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

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

3 Department of Medical Biotechnology, School of Medicine, Tarbiat Modares University, Tehran, Iran

4 Department of Venomous Animals, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

5 Department of Foot and Mouth Disease, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

6 Department of Virology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

7 Department of Biotechnology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

Abstract

Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (P<0.05). The neutralization potency of the product against the venom of Echis carinatus (E. carinatus) was about 7 LD50/ml (54.6 µg/ml) when tested on mice. Based on the results, the Fab fragment antivenom had the ability to neutralize the in vivo biological activity of the venom of Iranian E. carinatus. However, further studies are recommended to reach a suitable concentration of antivenom fragment.

Keywords

Main Subjects


Article Title [French]

Production d'un de Sérum Recombinant Humain de Fab Polyclonal contre Echis carinatus Iranien

Abstract [French]

La morsure de serpent venimeux est une blessure mortelle dans de nombreuses régions tropicales et subtropicales, y compris Iran. L'etraitement de référence en cas d'envenimation humaine est l'utilisation d'anti-venirs. Malgré l'efficacité uniques des sérums antivenimeux dérivés du cheval sur le traitement des morsures de serpents, ils présent néanmoins des limitations et doivent être améliorés. Dans cette étude, un fragment de sérum recombinant humain de Fab Polyclonal a été produit dans la bacterie Rosetta-g en utilisant une bibliothèque de gènes construite dans l’étude précédente. Le Fab préparé a été purifié en plusieurs étapes, dessalé et appauvri en lipopolysaccharide en utilisant une solution de sulfate d'ammonium et une dialyse contre un tampon phosphate et une solution de Triton X-114, respectivement. Par la suite, le produit a été analysé par électrophorèse sur gel de polyacrylamide de dodécyl sulfate de sodium (SDS-PAGE) et par dosage immuno-enzymatique (ELISA), respectivement. Enfin, à capacité de neutralisation du produit a été étudiée chez des souris syriennes de laboratoire. Les résultats obtenus ont montré des bandes réduites correspondantes au fragment Fab avec un poids moléculaire d'environ 28 kDa à une concentration de 3,1 mg / ml. Il y avait une différence significative entre les groupes en termes de test ELISA (p <0,05). La capacité de neutralisation de ce produit contre le venin Echis carinatus était d'environ 7 LD50 / ml (54,6 µg / ml) lorsqu'il a été testé sur des souris. Sur la base des résultats, l'antivenin du fragment Fab avait la capacité de neutraliser l'activité biologique in vivo du venin E. carinatus d'Iran. Cependant, des études complémentaires sont recommandées pour atteindre une concentration appropriée de fragment antivenin.

Keywords [French]

  • Echis carinatus
  • Fragment Fab
  • Bibliothèque de Gènes
  • Antivenin
  • Polyclonal
Al-Maliki, S., Al-Fartosi, K., R Ali, B., 2015. Effect of crude venom of Echis carinatus sochureki snake on hematological parameters of male and female rats. World J Pharm Sci 3, 1687-1692

Ariaratnam, C.A., Meyer, W.P., Perera, G., Eddleston, M., Kuleratne, S.A., Attapattu, W., et al., 1999. A new monospecific ovine Fab fragment antivenom for treatment of envenoming by the Sri Lankan Russell's viper (Daboia Russelii Russelii): a preliminary dose-finding and pharmacokinetic study. Am J Trop Med Hyg 61, 259-265.

Aubrey, N., Muzard, J., Christophe Peter, J., Rochat, H., Goyffon, M., Devaux, C., et al., 2004. Engineering of a recombinant Fab from a neutralizing IgG directed against scorpion neurotoxin AahI, and functional evaluation versus other antibody fragments. Toxicon 43, 233-241.

Bugli, F., Graffeo, R., Paroni Sterbini, F., Torelli, R., Masucci, L., Sali, M., et al., 2008. Monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice. Toxicon 51, 547-554.

Carmen, S., Jermutus, L., 2002. Concepts in antibody phage display. Brief Funct Genomic Proteomic 1, 189-203.

Dantas-Barbosa, C., de Macedo Brigido, M., Maranhao, A.Q., 2012. Antibody phage display libraries: contributions to oncology. Int J Mol Sci 13, 5420-5440.

de Haard, H.J., van Neer, N., Reurs, A., Hufton, S.E., Roovers, R.C., Henderikx, P., et al., 1999. A Large Non-immunized Human Fab Fragment Phage Library That Permits Rapid Isolation and Kinetic Analysis of High Affinity Antibodies. J Biol Chem 274, 18218-18230.

Dehghani, R., Fathi, B., Shahi, M.P., Jazayeri, M., 2014. Ten years of snakebites in Iran. Toxicon 90, 291-298.

Fu, Y.F., Feng, M., Ohnishi, K., Kimura, T., Itoh, J., Cheng, X.J., et al., 2011. Generation of a neutralizing human monoclonal antibody Fab fragment to surface antigen 1 of Toxoplasma gondii tachyzoites. Infect Immun 79, 512-517.

Griffiths, A.D., Williams, S.C., Hartley, O., Tomlinson, I.M., Waterhouse, P., Crosby, W.L., et al., 1994. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J 13, 3245-3260.

Hammers, C.M., Stanley, J.R., 2014. Antibody Phage Display: Technique and Applications. J Investig Dermatol 134, 1-5.

Humphreys, D.P., Carrington, B., Bowering, L.C., Ganesh, R., Sehdev, M., Smith, B.J., et al., 2002. A plasmid system for optimization of Fab′ production in Escherichia coli: importance of balance of heavy chain and light chain synthesis. Protein Expr Purif 26, 309-320.

Itoh, K., Inoue, K., Hirooka, K., Maruyama, K., Ohkawa, M., Matsui, K., et al., 2001. Phage display cloning and characterization of monoclonal antibody genes and recombinant Fab fragment against the CD98 oncoprotein. Jpn J Cancer Res 92, 1313-1321.

Kamyab, M., Kim, E., Hoseiny, S.M., Seyedian, R., 2017. Enzymatic Analysis of Iranian Echis carinatus Venom Using Zymography. Iran J Pharm Res 16, 1155.

Koh, D.C., Armugam, A., Jeyaseelan, K., 2006. Snake venom components and their applications in biomedicine. Cell Mol Life Sci 63, 3030-3041.

Kwong, K.Y., Rader, C., 2009. E. coli expression and purification of Fab antibody fragments. Curr Protoc Protein Sci 55, 6.10. 11-16.10. 14.

Malakar, P., Venkatesh, K., 2012. Effect of substrate and IPTG concentrations on the burden to growth of Escherichia coli on glycerol due to the expression of Lac proteins. Appl Microbiol Biotechnol 93, 2543-2549.

Morais, V., Massaldi, H., 2009. Snake antivenoms: adverse reactions and production technology. Journal of Venomous Animals and Toxins including Trop Dis 15, 2-18.

Motedayen, M.H., 2015. Production of recombinant Fab fragment of polyclonal antibody against venom of poisonous snakes, using a phage display library technique and evaluation of its antivenom activity in Syrian laboratory mouse. University of Tehran, Faculty of Veterinary Medicine, Tehran, Iran, p. 499.                     

Nalbantsoy, A., Karabay-Yavasoglu, N., Sayim, F., Deliloglu-Gurhan, I., Gocmen, B., Arikan, H., et al., 2012. Determination of in vivo toxicity and in vitro cytotoxicity of venom from the Cypriot blunt-nosed viper Macrovipera lebetina lebetina and antivenom production. Journal of Venomous Animals and Toxins including Trop Dis 18, 208-216.

Pansri, P., Jaruseranee, N., Rangnoi, K., Kristensen, P., Yamabhai, M., 2009. A compact phage display human scFv library for selection of antibodies to a wide variety of antigens. BMC Biotechnology 9, 6.

Peterson, N.C., 2005. Advances in Monoclonal Antibody Technology: Genetic Engineering of Mice, Cells, and Immunoglobulins. ILAR Journal 46, 314-319.

Rahbarizadeh, F., Rasaee, M.J., Frozandeh, M.M., Allameh, A.A., 2003. Production of a recombinant VHH antibody against MUC1 with phage display method and determination of its characteristics. Tarbiat Modarres University., Tehran, Iran.

Restaino, O.F., Bhaskar, U., Paul, P., Li, L., De Rosa, M., Dordick, J.S., et al., 2013. High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production. Appl Microbiol Biotechnol 97, 3893-3900.

Roncolato, E.C., Campos, L.B., Pessenda, G., e Silva, L.C., Furtado, G.P., Barbosa, J.E., 2015. Phage display as a novel promising antivenom therapy: a review. Toxicon 93, 79-84.

Sapsutthipas, S., Leong, P.K., Akesowan, S., Pratanaphon, R., Tan, N.H., Ratanabanangkoon, K., 2015. Effective equine immunization protocol for production of potent poly-specific antisera against Calloselasma rhodostoma, Cryptelytrops albolabris and Daboia siamensis. PLoS Negl Trop Dis 9, e0003609.

Schofield, D.J., Pope, A.R., Clementel, V., Buckell, J.,
Chapple, S., Clarke, K.F., et al., 2007. Application of phage display to high throughput antibody generation and characterization. Genome Biol 8, R254.

Shukra, A., Sridevi, N., Chandran, D., Maithal, K., 2014. Production of recombinant antibodies using bacteriophages. Akadémiai Kiadó, co-published with Springer Science+ Business Media BV, Formerly Kluwer Academic Publishers BV.

Theakston, R., Reid, H., 1983. Development of simple standard assay procedures for the characterization of snake venoms. Bull World Health Organ 61, 949.

Theakston, R.D.G., Warrell, D.A., Griffiths, E., 2003. Report of a WHO workshop on the standardization and control of antivenoms. Toxicon 41, 541-557.

Turunen, L., Takkinen, K., Soderlund, H., Pulli, T., 2009. Automated panning and screening procedure on microplates for antibody generation from phage display libraries. J Biomol Screen 14, 282-293.

Wu, B.-P., Xiao, B., Wan, T.-M., Zhang, Y.-L., Zhang, Z.-S., Zhou, D.-Y., et al., 2001. Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer. World J Gastroenterol 7, 811.

Yap, M.K.K., Tan, N.H., Sim, S.M., Fung, S.Y., Tan, C.H., 2015. The Effect of a Polyvalent Antivenom on the Serum Venom Antigen Levels of Naja sputatrix (Javan Spitting Cobra) Venom in Experimentally Envenomed Rabbits. Basic Clin Pharmacol Toxicol 117, 274-279.

Zhang, J., Zhu, C., Fan, D., 2013. Endotoxin Removal from Recombinant Human-like Collagen Preparations by Triton X-114 Two-phase Extraction. Biotechnology 12, 135.

Zhu, Z., Dimitrov, D.S., 2009. Construction of a large naive human phage-displayed Fab library through one-step cloning. Therapeutic Antibodies, Springer, pp. 129-142.