DOI: 10.3724/SP.J.1206.2008.00415

Progress in Biochemistry and Biophysics (生物化学与生物物理进展) 2009/36:6 PP.736-742

Monoclonal Antibody S2C4 Neutralizes The Toxicity of Shiga Toxin 2 and Its Variants*

Shiga toxin 2 (Stx2) toxoid produced by formaldehyde treatment of purified toxin was used to immunize BALB/c mice for monoclonal antibody (MAb) production. The neutralizing activities of positive clones against Stx2 were screened by in vitro cytotoxicity assay. The isotype and specificity of resultant clone was determined, and its efficacy to neutralize the activity of purified Stx2 was evaluated by in vitro and in vivo toxicity model. Lastly, its spectrum of activity against Stx2 variants was also accessed by mouse toxicity model. It was demonstrated that one of the 12 positive MAb clones against Stx2, designating S2C4 had neutralizing activity. S2C4 belongs to the immunoglobulin G1 subclass and has a κ light chain, and it reacts with the A subunit of Stx2 and does not bind to Stx2 B subunit or to Stx1. S2C4 could efficiently neutralize the cytotoxicity of Stx2 to Vero cells and mice. It also protected mice against lethal doses of Stx2 variants challenge including Stx2c and Stx2vha. S2C4 is a promising candidate molecule in preventing the progression of hemolytic-uremic syndrome (HUS) mediated mainly by Stx2 in Stx-producing Escherichia coli (STEC) infection.

Key words:monoclonal antibody (MAb) S2C4, Shiga toxin 2 (Stx2), neutralization, Stx-producing Escherichia coli (STEC)

ReleaseDate:2014-07-21 14:49:47

Funds:This work was supported by a grant from The Natural Science Foundation of Jiangsu Province (BK2006242).

1 Tzipori S, Sheoran A, Akiyoshi D, et al. Antibody therapy in the management of shiga toxin-induced hemolytic uremic syndrome. Clin Microbiol Rev, 2004, 17(4): 926~941

2 Zheng H, Jing H, Wang H, et al. stx2vha is the dominant genotype of Shiga toxin-producing Escherichia coli O157:H7 isolated from patients and domestic animals in three regions of China. Microbiol Immunol, 2005, 49(12): 1019~1026

3 Donohue-Rolfe A, Jacewicz M, Keusch G T. Isolation and characterization of functional shiga toxin subunits and renatured holotoxin. Mol Microbiol, 1989, 3(9): 1231~1236

4 Furutani M, Ito K, Oku Y, et al. Demonstration of RNA N-glycosidase activity of a verotoxin (VT2 variant) produced by Escherichia coli 091:H21 from patient with the hemolytic uremic syndrome. Microbiol Immunol, 1990, 34(4): 387~392

5 Kozlov Yu V, Kabishev A A, Lukyanov E V, et al. The primary structure of the operons coding for Shigella dysenteriae toxin and temperature phage H30 shiga-like toxin. Gene, 1988, 67(2): 213~221

6 Jackson M P, Newland J W, Holees R K, et al. Nucleotide sequence analysis of structural genes for Shiga-like toxinⅠencoded by bacteriophage 933J from Escherichia coli. Microb Pathog, 1987, 2(2): 147~153

7 Tesh V L, Burris J A, Owens J W, et al. Comparison of the relative toxicities of Shiga-like toxins typeⅠ and typeⅡ for mice. Infect Immun, 1993, 61(8): 3392~3402

8 Wong C S, Jelacic S, Habeeb R L, et al. The risk of the hemolytic- uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N Engl J Med, 2000, 342(26): 1930~1936

9 Bitzan M, Klemt M, Steffens R, et al. Differences in verotoxin neutralizing activity of therapeutic immunoglobulins and sera from healthy controls. Infection, 1993, 21(3): 140~145

10 Mukherjee J, Chios K, Fishwild D, et al. Human Stx2-Specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection. Infect Immun, 2002, 70(2): 612~619

11 Nakao H, Kiyokawa N, Fujimoto J, et al. Monoclonal antibody to shiga toxin 2 which blocks receptor binding and neutralizes cytotoxicity. Infect Immun, 1999, 67(11): 5717~5722

12 Jiao Y, Zeng X, Guo X, et al. Purification and functional characterization of enterohemorrhagic Escherichia coli O157:H7 Shiga toxinⅡ. Chin Infect Dis, 2008, 26(4): 217~220

13 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London), 1975, 256(5517): 495~497

14 Xiao G H, Jeffers M, Bellacosa A, et al. Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/ Akt and mitogen-activated protein kinase pathways. Proc Natl Acad Sci USA, 2001, 98(1): 247~252

15 Friedrich A W, Bielaszewska M, Zhang W L, et al. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J Infect Dis, 2002, 185(1): 74~84

16 Wadolkowski E A, Burris J A, O’Brien A D. Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7. Infect Immun, 1990, 58(8): 2438~2445

17 Gannon V P, Teerling C, Masri S A, et al. Molecular cloning and nucleotide sequence of another variant of the Escherichia coli Shiga-like toxinⅡ family. J Gen Microbiol, 1990, 136(6): 1125~1135

18 Ito H, Terai A, Kurazono H, et al. Nishibuchi M. Cloning and nucleotide sequencing of Vero toxin 2 variant genes from Escherichia coli O91:H21 isolated from a patient with the hemolytic uremic syndrome. Microb Pathog, 1990, 8(1): 47~60

19 Schmitt C K, McKee M L, O’Brien A D. Two copies of Shiga-like toxinⅡ-related genes common in enterohemorrhagic Escherichia coli strains are responsible for the antigenic heterogeneity of the O157:H- strain E32511. Infect Immun, 1991, 59(3): 1065~1073

20 Pierard D, Muyldermans G, Moriau L, et al. Identification of new verocytotxin type 2 variant B-subunit genes in human and animal Escherichia coli isolates. J Clin Microbiol, 1998, 36(11): 3317~3322

21 Sheoran A S, Chapman S, Singh P, et al. Stx2-specific human monoclonal antibodies protect mice against lethal infection with Escherichia coli expressing Stx2 variants. Infect Immun, 2003, 71(6): 3125~3130

22 Mizutani S, Nakazono N, Sugino Y. The so-called chromosomal verotoxin genes are actually carried by defective prophages. DNA Res, 1999, 6(2): 141~143

23 Koudelka A P, Hufnagel L A, Koudelka G B. Purification and characterization of the repressor of the shiga toxin-encoding bacteriophage 933W: DNA binding, gene regulation, and autocleavage. J Bacteriol, 2004, 186(22): 7659~7669

24 Neely M N, Friedman D I. Functional and genetic analysis of regulatory regions of coliphage H-19B: location of Shiga-like toxin and lysis genes suggest a role for phage functions in toxin release. Mol Microbiol, 1998, 28(6): 1255~1267

25 Waldor M K. Bacteriophage biology and bacterial virulence. Trends Microbiol, 1998, 6(8): 295~297

26 Nishikawa K, Matsuoka K, Kita E, et al. A therapeutic agent with oriented carbohydrates for treatment of infections by Shiga toxin-producing Escherichia coli O157:H7. Proc Natl Acad Sci USA, 2002, 99(11): 7669~7674

27 Paton A W, Morona R, Paton J C. A new biological agent for treatment of Shiga toxigenic Escherichia coli infections and dysentery in humans. Nat Med, 2000, 6(3): 265~270

28 Yamagami S, Motoki M, Kimura T, et al. Efficacy of postinfection treatment with anti-Shiga toxin (Stx) 2 humanized monoclonal antibody TMA-15 in mice lethally challenged with Stx-producing Escherichia coli. J Infect Dis, 2001, 184(6): 738~742