INFECTION AND IMMUNITY, Feb. 2002, p. 612–619 0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.2.612–619.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 2
Human Stx2-Specific Monoclonal Antibodies Prevent Systemic Complications of Escherichia coli O157:H7 Infection Jean Mukherjee,1 Kerry Chios,1 Dianne Fishwild,2 Deborah Hudson,2 Susan O’Donnell,2 Stephen M. Rich,1 Arthur Donohue-Rolfe,1 and Saul Tzipori1* Tufts University School of Veterinary Medicine, North Grafton, Massachusetts,1 and Medarex, San Jose, California2 Received 2 May 2001/Returned for modification 7 August 2001/Accepted 12 November 2001
Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC), such as E. coli O157:H7. EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits. In piglets and mice, Stx can induce fatal neurological symptoms. Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing E. coli O157:H7 strain 86-24. Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS. Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1 A-subunit-specific and 3 IgG1 and 1 IgG3 B-subunit-specific antibodies. Six IgG1 A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1 B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of >95% activity of 1 ng of Stx2 in the presence of 0.04 g of HuMAb in vitro and significant prolongation of survival of mice given 50 g of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously. When administered i.p. to gnotobiotic piglets 6 or 12 h after infection with E. coli O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions. The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including E. coli O157:H7. of B subunits to globotriaosylceramide (39) followed by A-subunit inactivation of the 60S ribosomal subunit, resulting in inhibition of protein synthesis (10, 33, 34). Although there is no animal model which mimics HUS in humans, the gnotobiotic piglet model of E. coli O157:H7 infection and murine models of Stx toxicosis have proved useful for studying the in vivo effects of Stx (4, 7, 38). Pigs are the only species other than humans naturally susceptible to the systemic effects of Stx produced by E. coli proliferating in the gastrointestinal tract. A variant of Stx2, designated Stx2e, is responsible for edema disease in swine (22, 24). In piglets and humans, EHEC strains, including those which produce Stx, cause attaching and effacing lesions within the gastrointestinal tract (13, 37). Tzipori et al. have postulated that the injured mucosa may facilitate systemic Stx absorption (37). Although HUS does not occur in pigs, the clinical signs and lesions observed in pigs given Stx2e intravenously (i.v.) (14, 22) and in those infected with Stx2 or Stx2e-producing E. coli are similar and include ataxia, convulsions, paddling of limbs, tremors, and coma along with cerebral hemorrhage and edema (4, 23). Currently there is no effective treatment or prophylaxis for HUS. As in many toxin-mediated diseases, such as tetanus and botulism, little endogenous serum antibody against Stx is induced following EHEC infection (2, 21, 32). Nonetheless, passively administered toxin-specific antibodies have been shown to be highly effective at preventing toxin-mediated diseases (32). The gnotobiotic piglet model of E. coli O157:H7 infection with the Stx2-producing strain 86-24 has been used to demonstrate that administration of polyclonal porcine Stx2 antiserum can prevent development of the neurological signs and lesions
Hemolytic-uremic syndrome (HUS) occurs in 5 to 10% of reported cases of Escherichia coli O157:H7 infection and is the leading cause of renal failure in children (16). Development of HUS is epidemiologically associated with infection by enterohemorrhagic E. coli (EHEC) (19; M. A. Karmali, M. Petric, C. Lim, P. C. Fleming, and B. T. Steele, Letter, Lancet 2:1299– 1300, 1983). There are many serotypes of EHEC; however, O157:H7 is the serotype most frequently associated with HUS in children and the elderly in the United States (16). Typically, within 24 h following food-borne or waterborne infection with EHEC, hemorrhagic colitis, characterized by abdominal pain and watery and then bloody diarrhea, occurs (16). HUS, characterized by nonimmune microangiopathic hemolytic anemia, thrombocytopenia, and acute renal dysfunction, can develop several days following the onset of diarrhea (15). The risk of an individual child developing HUS following a bout of sporadic Shiga toxin-producing E. coli gastroenteritis has been estimated to be 3 to 26% (26, 29, 31; W. R. Grandsen, M. A. Damm, J. D. Anderson, J. E. Carter, and H. Lior, Letter, Lancet 2:150, 1985). EHEC strains produce one or two toxins, designated Shiga toxin 1 (Stx1) and Stx2, of which Stx2 is most frequently associated with HUS (3, 21, 27). Both Stx1 and Stx2 are cytotoxins comprised of one enzymatically active (A) subunit and five binding (B) subunits. Stx mediates HUS via endothelial cell injury, predominantly within the kidney, via successive binding * Corresponding author. Mailing address: Tufts University School of Veterinary Medicine, Division of Infectious Diseases, 200 Westboro Rd., Building 20, North Grafton, MA 01536. Phone: (508) 839–7955. Fax: (508) 839–7977. E-mail:
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associated with Stx2 activity (7). Similarly, passively administered Stx2e antiserum has been shown to prevent edema disease in swine (18). Several murine Stx2-specific monoclonal antibodies (MAbs) have been developed, and many have been shown to neutralize the activity of Stx2 in vitro and/or in vivo in mice (5, 9, 25, 30, 35). Here we describe development of a panel of Stx2 A- and B-subunit-specific human MAbs (HuMAbs), several of which similarly neutralize Stx2-mediated activity in vitro and in vivo. The availability of Stx2-specific HuMAbs provides an opportunity to administer a safe, immunotherapeutic reagent soon after presentation with bloody diarrhea in an effort to prevent development of HUS in susceptible individuals.
Western blot analysis. The subunit specificity of each Stx2 HuMAb was determined by Western blotting. Stx2 was cross-linked by using the homobifunctional cross-linking agent dimethylpimelimidate (Pierce Chemical Company, Rockford, Ill.) to create a mixture containing the A subunit bound to one to five B subunits and B subunit alone. The cross-linked Stx2 was electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 15% acrylamide slab gel and then electrophoretically transferred to a nylon membrane (Immobilon-P; Millipore, Bedford, Mass.) (6). Membranes were washed five times with PBS plus 0.05% Tween 20 between the following steps. Following electrophoretic transfer, membranes were soaked 1 h in PBS plus 0.3% Tween 20 and then 2 h in PBS plus 0.05% Tween 20 and 1% bovine serum albumin. A Surf Blot (models 10 and 10.5; Idea Scientific Company, Minneapolis, Minn.) apparatus was used to divide each membrane into discrete lanes. Stx2-specific HuMAbs were incubated at 10 g/ml in PBS in individual lanes in the presence of membrane-bound Stx2 for 2 h at room temperature, as were the previously undescribed Stx2-specific mouse IgG1 MAb 3D1 and polyclonal rabbit Stx2 antiserum (positive controls). A 1:1,000 dilution of alkaline phosphatase-labeled goat antimouse IgG1 (Southern Biotechnology Associates) or goat anti-human IgG (Sigma-Aldrich Co.) or IgM (Southern Biotechnology Associates) was added to each lane. Blots were developed with NBT-BCIP (nitroblue tetrazolium–5-bromo-4chloro-3-indolylphosphate) substrate (Promega, Inc., Madison, Wis.). Identification of specific A-B-subunit complexes and B-subunit multimers was performed as described previously (6). In vitro HeLa cell cytotoxicity assays. Two variations of an in vitro cytotoxicity assay were used to evaluate the ability of each HuMAb to neutralize the toxic effects of Stx2 exerted against HeLa cells. For each assay, HeLa cells were plated at 2 ⫻ 105/ml on 96-well plates in McCoy’s 5A medium (Mediatech, Inc., Herndon, Va.) plus 10% fetal calf serum (Harlan Bioproducts for Science, Inc., Madison, Wis.) and incubated overnight at 37°C in 5% CO2. Medium was removed prior to addition of Stx2–HuMAb mixtures. The proportion of surviving cells following exposure to Stx2 with or without HuMAb was determined spectrophotometrically following crystal violet staining (20). Each assay was performed independently a minimum of three times; the results at a selected data point were averaged. Assay I involved examining the effects of limiting HuMAb in the presence of Stx2. Each HuMAb was serially diluted 1:2 from 12.5 to 0.0061 g/ml; each dilution was incubated for 30 min at room temperature with 10 ng of Stx2 per ml. Stx2–HuMAb mixtures were transferred to HeLa cell monolayers and incubated overnight at 37°C. The relative percent neutralization of 1 ng of Stx2 in the presence of 39.1 ng of HuMAb was determined by using a standard curve based on the effects of Stx2 alone (Table 1). Assay II involved examining the effects of HuMAb in the presence of limiting Stx2. Stx2 was serially diluted 1:2 from 100 to 0.049 ng/ml; each dilution was incubated for 30 min at room temperature with 1 g of HuMAb per ml. Stx2–HuMAb mixtures were transferred to HeLa cell monolayers and incubated overnight at 37°C. The relative percent neutralization of 0.3125 ng of Stx2 in the presence of 100 ng of HuMAb was determined by using a standard curve based on the effects of Stx2 alone (data not shown). Murine toxin neutralization assay. A murine Stx2 neutralization assay was used to examine the ability of each HuMAb to neutralize the effects of Stx2 in vivo. Dose-response curves were conducted in groups of five 3- to 4-week-old female Swiss Webster mice (Taconic) to determine the amount of Stx2 required to induce 100% mortality in untreated animals (data not shown). HuMAb efficacy was evaluated by administering either 50 g of Stx2-specific HuMAb in 1 ml of PBS or 1 ml of PBS alone (control) i.p. to each of 8 to 10 3- to 4-week-old Swiss Webster mice followed by i.v. administration of 25 ng of Stx2, ⬃18 h later. Mice were observed twice daily for survival. Gnotobiotic piglet model of E. coli O157:H7 infection. A gnotobiotic piglet model of E. coli O157:H7 infection was used to examine the efficacy of selected passively administered HuMAbs in preventing the clinical signs and lesions associated with Stx2 during infection. Colostrum-deprived gnotobiotic piglets were derived by cesarean section and maintained in sterile microisolators (Class Biologically Clean, Ltd., Madison, Wis.). Piglets were divided into groups with approximately equal average weights. Within 24 h following derivation, piglets were orally infected with ⬃1010 cells of the Stx2-producing E. coli O157:H7 strain 86-24. This high inoculum usually induces neurological signs and lesions associated with Stx2 activity in ⬎85% of untreated piglets within 48 to 96 h postinfection (8). For each experiment, a single colony grown on Luria-Bertani (LB) agar from a frozen glycerol stock was used to inoculate 50 ml of LB broth. Cultures were incubated 9 h at 37°C with shaking at 200 rpm. Log-phase organisms were obtained by subculturing a 1:100 dilution in LB broth at 37°C with shaking at 200 rpm for an additional 3 to 4 h until an optical density at 600 nm of ⬃0.8 was reached. Six or twelve hours following infection, piglets were treated with 3 mg of HuMAb or an equal volume of PBS (control) administered i.p. Piglets were
MATERIALS AND METHODS Stx2 and Stx2 toxoid. Stx2 was isolated, purified, and quantitated as described previously (5). Stx2 toxoid was prepared by formalin treatment of Stx2. Briefly, 100 g of Stx2 was incubated overnight in 2% formalin and then dialyzed extensively against phosphate-buffered saline (PBS). Inactivation was confirmed by comparing cytotoxicity of the toxoid and that of active Stx2 by using HeLa cells (5). Hybridomas and HuMAbs. Murine hybridomas producing HuMAbs were generated with the HuMAb___Mouse (GenPharm, a subsidiary of Medarex; San Jose, Calif.), which contains transgenes bearing human immunoglobulin loci. Three distinct sets of HuMAb___Mouse mice containing human heavy chain transgenes designated HC2 (12), HCo7, and HCo12 (Longren et al., unpublished data) and the human light chain transgene, KCo5 (12), were used. Depending on the transgene sequences present, these mice are capable of expressing fully human immunoglobulin M kappa chain (IgM) and IgG1 ⫾ IgG3 antibodies. Stx2-specific HuMAbs were generated by immunizing HC2, HCo7, or HCo12 mice with 10 to 50 g of Stx2 toxoid emulsified in Freund’s complete (initial immunization only) or incomplete (all subsequent immunizations) adjuvant intraperitoneally (i.p.) at biweekly intervals a minimum of three times. Serum anti-Stx2 titers were determined by enzyme-linked immunosorbent assay (ELISA) on microtiter plates (Falcon 353912; Becton-Dickinson, Bedford, Mass.) coated with 1.5 g of Stx2 per ml and developed with horseradish peroxidase (HRP)-labeled goat anti-human IgG. The spleens of mice with titers of ⱖ1:800 were fused to the nonproductive murine myeloma P3X63-Ag8.653 by standard methods (11). Supernatants from hypoxanthine-aminopterin-thymidine-selected hybridomas were successively screened by ELISA on microtiter plates coated with 1.5 g of Stx2 per ml and developed with HRP-labeled goat anti-human IgG (Jackson Laboratory, Bar Harbor, Maine) or HRP-labeled goat anti-human kappa (Bethyl Laboratories, Inc., Montgomery, Tex., or SigmaAldrich Co., St. Louis, Mo.). Stable, positive clones were selected by subcloning twice by limiting dilution and finally by soft-agar cloning (11). HuMAb-containing ascitic fluid was prepared by injecting hybridoma cells into the peritoneal cavities of pristane (Sigma-Aldrich Co.)-primed ICR-SCID mice (Taconic, Germantown, N.Y.). HuMAb concentrations in ascitic fluid were determined relative to those of isotype-matched concentration standards (Sigma, St. Louis, Mo.) by ELISA (36). Characterization of HuMAbs by ELISA. The isotype of each HuMAb was determined by ELISA. Briefly, microtiter plates (9018; Costar, Corning, N.Y.) were coated with a 1:1,000 dilution of goat anti-human kappa (Southern Biotechnology Associates, Birmingham, Ala.) and blocked with 1% bovine serum albumin (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.) in PBS. Hybridoma culture supernatants or ascitic fluid containing individual HuMAbs was plated in each of eight wells. The wells were developed with alkaline phosphatase-labeled anti-human IgM, IgG1, IgG2, IgG3, IgG4, IgA, kappa, or lambda. (Southern Biotechnology Associates, Inc.) followed by addition of 1 mg of p-nitrophenyl phosphate (Sigma-Aldrich Co.) per ml. Absorbance at 405 nm was determined. The Stx binding specificity of each HuMAb was determined with a sandwich ELISA in which microtiter plates coated with 5 g of the murine Stx1- and Stx2-specific MAb 4D3 or 3D1 per ml were used to capture 1-g/ml solutions of Stx1 or Stx2, respectively. HuMAbs were plated in duplicate on pairs of plates containing Stx1 or Stx2. Assays were developed with alkaline phosphatase-labeled anti-human kappa (Southern Biotechnology Associates, Inc.) followed by the addition of 1 mg of p-nitrophenyl phosphate (Sigma-Aldrich Co.) per ml. Absorbance at 405 nm was determined.
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TABLE 1. Stx2 HuMAb isotypes, epitope specificities, and in vitro and in vivo Stx2 neutralization Group and MAba
Isotype
Stx2 subunit specificity
% Neutralization of Stx2 (avg ⫾ SD)b
No. of days (avg ⫾ SD)
P value
3.35 ⫾ 0.412 10.5 ⫾ 3.16 12.0 ⫾ 0 12.0 ⫾ 0 6.1 ⫾ 4.095 10.25 ⫾ 3.691 8.7 ⫾ 4.27 12.0 ⫾ 0 11.2 ⫾ 2.53 12.0 ⫾ 0 3.1 ⫾ 0.81 2.7 ⫾ 0.35 7.65 ⫾ 4.59 5.375 ⫾ 4.095 4.35 ⫾ 2.87 4.3 ⫾ 2.72
0.0101e ⱕ0.001d ⱕ0.001d ⱕ0.001d 0.0035e 0.0018f 0.0011d ⱕ0.001c ⱕ0.001d ⱕ0.001c 0.4680d 0.0071d 0.0156d 0.2190c 0.3737d 0.1579d
Marine survivalc
High 1G1 1G3 2F10 3E9 4G7 4H9 5A4 5C12 5H8 6G3 6H5 6H7 7C4 9F9 9H9 14C12
IgG1 IgG1 IgG1 IgG1 IgG3 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1
A A A A B A A A B B A A A A A A
92.0 ⫾ 7.2 98.6 ⫾ 1.2 99.6 ⫾ 0.3 99.2 ⫾ 0.7 99.3 ⫾ 0.2 94.5 ⫾ 3.1 97.7 ⫾ 2.1 99.7 ⫾ 0.3 96.0 ⫾ 2.2 98.4 ⫾ 2.1 94.7 ⫾ 2.2 90.4 ⫾ 3.8 99.5 ⫾ 0.2 95.7 ⫾ 1.7 94.3 ⫾ 1.1 97.9 ⫾ 2.4
Medium 1C3 2G5 4H10 5A8 5B11 5E12 6C3 6D8 6E6 7G2
IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1
A A A A A A A A A A
86.2 ⫾ 4.5 87.2 ⫾ 11.7 73.7 ⫾ 12.4 82.2 ⫾ 6.2 81.3 ⫾ 1.5 84.2 ⫾ 2.4 81.3 ⫾ 14.2 78.7 ⫾ 12.1 86.7 ⫾ 4.1 75.0 ⫾ 11.5
2.9 ⫾ 0.61 0.2380d 5.1 ⫾ 3.78 0.4047d 8.5 ⫾ 4.52 0.0012c 3.7 ⫾ 2.96 0.3495d 3.3 ⫾ 0.89 0.9004d 4.0 ⫾ 2.88 0.7591d 12.0 ⫾ 0 ⱕ0.001c 3.22 ⫾ 0.363 0.0254e 2.8 ⫾ 0.63 0.1989d 3.2 ⫾ 0.350 1.000f
Low 1G12 3A2 3F6 5F2 6B7 6C12 7B3 7F2 10E9 11B12
IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1
A A B A A A A A A A
46.4 ⫾ 22.0 47.3 ⫾ 41.5 22.0 ⫾ 38.1 62.7 ⫾ 0.6 60.5 ⫾ 52.6 51.4 ⫾ 40.6 69.2 ⫾ 11.1 64.3 ⫾ 28.1 62.0 ⫾ 15.1 52.0 ⫾ 45.0
3.1 ⫾ 0.211 3.85 ⫾ 2.868 2.78 ⫾ 0.258 4.15 ⫾ 2.78 3.45 ⫾ 0.956 3.7 ⫾ 0.63 3.11 ⫾ 0.70 5.95 ⫾ 4.186 2.75 ⫾ 0.354 4.15 ⫾ 2.769
0.0292c 0.5983f 0.1888c 0.0129e 0.0227e 0.0012d 0.4664d 0.0047e 0.0230c 0.0041c
a HuMAbs were sorted into three groups based on average percent neutralization of Stx2 in vitro: high,ⱖ90%; medium, 70 to 89%; and low, ⬍70%. HuMAbs which prolonged survival an average of ⬎ 10 days are in bold. b Average percent neutralization of 1 ng of Stx2 in presence of 39.1 ng of HuMAb in Hela cell cytotoxicity assays. Each value is the average of three values obtained from independent experiments. c Experiments were terminated on day 12; n ⫽ 8 to 10 mice per group. d Survival of PBS control was 3.3 ⫾ 0.35 days. e Survival of PBS control was 2.85 ⫾ 0.24 days. f Survival of PBS control was 3.9 ⫾ 2.85 days.
monitored several times each day for development of severe diarrhea or central nervous system (CNS) signs (paddling, head-pressing, fore- and hind-limb paresis, seizures, opisthotonos, and/or ventrally fixed eye deviation) associated with Stx2 activity. Piglets which developed such signs or which were alive at the termination of the experiment (6 to 10 days postinfection) were euthanized, and brain tissue (cerebral cortex and cerebellum) was obtained and formalin-fixed for histopathological examination for the presence of lesions (hemorrhage and edema) associated with Stx2 activity; blood was obtained for determination of serum HuMAb concentrations. Serum HuMAb concentrations were determined relative to those of isotype-matched concentration standards (Sigma) by ELISA (36). Statistical analyses. Survival data for mice and piglets were analyzed by both parametric (log rank test) and nonparametric (Wilcoxon test) methods with the
software program Statistica (v. 4.1; Statsoft, Inc., Tulsa, Okla.). The methods yielded comparable P values. Comparisons with a P value of ⬍0.05 were considered significant.
RESULTS Isotype and specificity of HuMAbs. Thirty-seven stable murine hybridomas secreting Stx2-specific HuMAbs were isolated from transgenic mice (HuMAb___Mouse; Medarex) bearing human immunoglobulin heavy and light chain transgenes (12; Longren et al., unpublished) and immunized with Stx2 toxoid. Thirty-six of the 37 hybridomas isolated secrete IgG1 HuMAbs; one, designated 4G7, secretes an IgG3 HuMAb (Table 1). As determined by ELISA, each hybridoma secretes HuMAb specific for Stx2; no cross-reactivity with Stx1 was observed with ELISA against Stx1 antigens (data not shown). The Stx2 subunit specificity of each HuMAb was determined by Western blot analysis (Fig. 1) (6). Stx2 is comprised of one A subunit of ⬃32 kDa and five B subunits of ⬃7.8 kDa each (17). The A-versus-B subunit specificity of the anti-Stx2 HuMAbs was determined based on binding to covalently crosslinked Stx2 comprised of the A subunit bound to zero to five B subunits and B-subunit monomers and multimers. MAbs with specificity for the B subunit bind the B-subunit monomers and multimers and the A-B complexes; MAbs with specificity for the A subunit bind the A-B complexes but do not bind Bsubunit monomers or multimers (6). The relative intensity of binding is determined not only by whether the particular Stx2 entity is recognized by a MAb but also by the percentage of each complex present within the preparation of cross-linked Stx2. Consistent with either A- or B-subunit specificity, all 37 HuMAbs clearly bound two A-B complexes which based on approximate molecular weights correspond to complexes of the A subunit and one or two B subunits. Four HuMAbs (3F6, 4G7, 5H8, and 6G3) bound entities corresponding to the Bsubunit monomer (1B), dimer (2B), and/or trimer (3B), indicating specificity for the B subunit. Although faint, the pattern of 3F6 and 4G7 binding to the 2B and 3B complexes is similar to that of the B-subunit-specific murine MAb 3D1. 5H8 and 6G3, however, exhibit different patterns of binding to the B subunit entities: 5H8 binds only the 2B complex, while 6G3 binds the 1B, 2B, and 3B complexes. The lack of binding to B-subunit monomers and multimers by the other 33 HuMAbs is indicative of specificity for the A subunit. Consistent with this specificity, these 33 HuMAbs do indeed bind an entity with a molecular weight corresponding to the A-subunit monomer. The binding patterns of the A-subunit-specific HuMAbs are similar; thus, differences in epitope specificity cannot be delineated. Consistent with B-subunit specificity, HuMAbs 4G7 and 3F6 do not bind the A-subunit monomer. However, unexpectedly, the B-subunit-specific HuMAbs 5H8 and 6G3 and the murine MAb 3D1 do bind the A-subunit monomer. Because we have seen similar reactions to the A subunit with antibody not specific for the toxin, this is likely due to a nonspecific interaction between the antibody and the A subunit rather than specific binding. In vitro and in vivo neutralization of Stx2. The ability of each HuMAb to neutralize the activity of purified Stx2 was studied with both in vitro HeLa cell cytotoxicity assays and an in vivo murine model of Stx2 neutralization (Table 1). Two
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FIG. 1. Western blot analysis of HuMAb binding to Stx2. Covalently cross-linked Stx2 was electrophoresed, transferred to a nylon membrane, and then divided into individual lanes. As indicated, each HuMAb was incubated in a separate lane. Polyclonal rabbit anti-Stx2 serum and the Stx2 B-subunit-specific murine MAb 3D1 were included as positive controls. The identities of significant major bands were determined from published molecular weights of the A and B subunits of Stx2 (17) and are indicated to the left of each panel. Anti-Stx2 contains antibodies which bind both the A and B subunits of Stx2; MAb 3D1 binds the B subunit and thus binds A-B complexes in addition to B-subunit monomers and multimers. The relative intensity of binding is determined not only by whether the particular Stx2 entity is recognized by a MAb but also by the percentage of each complex present within the preparation of cross-linked Stx2. With the exception of HuMAbs 3F6, 4G7, 5H8, and 6G3, all are specific for the A subunit based on the ability to bind A-B complexes but not B-subunit monomers or multimers. HuMAbs 3F6, 4G7, 5H8, and 6G3 are specific for the B subunit of Stx2 based on the ability to bind B-subunit monomers and multimers in addition to A-B-subunit complexes. Similar to the murine MAb 3D1, HuMAbs 3F6 and 4G7 bind the 2B and 3B multimers, whereas 5H8 binds the 2B multimer only and 6G3 binds the 1B monomer and 2B and 3B multimers, suggesting differential epitope specificity.
variations of an in vitro cytotoxicity assay were used to determine the amount of Stx2 neutralized by a given amount of each HuMAb. In one assay, HuMAb concentration was varied in the presence of a constant amount of Stx2 and the percentage of Stx2 neutralized was determined at a single concentration for each HuMAb (Table 1). In the second assay, Stx2 concentration was varied in the presence of a constant amount of HuMAb and the percentage of Stx2 neutralized by each HuMAb was determined at a single Stx2 concentration (data not shown). Similar results were obtained with both assays. Based on the data obtained with the former assay, each
HuMAb was assigned to one of three categories (high, medium, or low) based on relative percent neutralization at single HuMAb and Stx2 quantities (39.1 and 1 ng, respectively). Sixteen HuMAbs neutralized ⱖ90% of the Stx2 present (high), 11 neutralized 70 to 89% of the Stx2 present (medium), and 10 neutralized ⬍70% of the Stx2 present (low) (Table 1). A murine neutralization assay was used to assess the ability of each HuMAb to neutralize Stx2 in vivo. Approximately 18 h following i.p. administration of 50 g of HuMAb, mice were challenged i.v. with 25 ng of Stx2. Eight HuMAbs (1G3, 2F10, 3E9, 4H9, 5C12, 5H8, 6C3, and 6G3) (Table 1) significantly
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TABLE 2. Effect of Stx2 HuMAb administration on gnotobiotic piglets infected with E. coli O157:H7 strain 86-24 No. of piglets with result/no. tested
HuMAb Expt
a
Treatment
Survivald
Dose (mg)
Time (h post-infection)
CNS signsb
CNS lesionsc
No. of days (avg ⫾ SD)
P value
n
Hu-IgG level in serum (avg ⫾ SD, g/ml)
0.044 0.083
5 5 2
NDg 4.91 ⫾ 2.8 3.84 ⫾ 0
104
PBS 3E9 5H8
0 3 3
6 6 6
3/4 0/5 0/2
3/4 0/5 0/2
3.8 ⫾ 2.08f 6.7 ⫾ 0.67f 7.0 ⫾ 0f
117
PBS 3E9 5H8
0 3 3
12 12 12
2/2 0/4 0/3
2/3 0/4 0/3
6.67 ⫾ 1.53 10.0 ⫾ 0f 10.0 ⫾ 0f
0.0157 0.0287
3 4 3
ND 5.16 ⫾ 2.8 2.82 ⫾ 0.4
115
PBS 2F10
0 3
12 12
3/3 0/3
3/3 1/4e
3.83 ⫾ 1.04 6.0 ⫾ 1.35f
0.0683
3 4
ND 8.85 ⫾ 2.1
128
PBS 5C12
0 3
12 12
5/5 0/4
5/5 0/4
3.2 ⫾ 0.98 8.6 ⫾ 3.13
0.005
5 5
ND 4.81 ⫾ 6.97
a
Experiments 104, 115, 117, and 128 were terminated on days 7, 8, 10, and 10, respectively. CNS signs included paddling, head-pressing, seizures, opisthotonos, and/or ventrally fixed eye deviation. Only piglets observed immediately prior to death or euthanasia were included in the observations. c CNS lesions included hemorrhage and edema in histopathological sections of the cerebrum and/or cerebellum. d Average survival of gnotobiotic piglets following administration of 3 mg of HuMAb 6 or 12 h after oral infection with E. coli O157:H7 strain 86-24. All piglets which died or were euthanized due to experimental manipulations were included in the survival data. P values were calculated for the comparison of average survival of PBS control groups and HuMAb-treated groups by parametric (log rank) and nonparametric (Wilcoxon) analyses. Comparable P values were obtained with both analyses. The P values shown were obtained by log rank analysis. e The lesions in the CNS tissue of one piglet in this group were very mild and thus not conclusive. Nonetheless, the piglet was included with those that had definite lesions. f Average includes censored data points, i.e., animals alive at the termination of the experiment. Analysis accounted for censored observations (animals alive at termination of experiment), and thus, the estimate of the mean in these groups is biased against prolongation of survival. g ND, not detectable. b
prolonged average survival to ⬎10 days (experiments were terminated at day 12), relative to the PBS control groups, which had average survival values of 2.85 to 3.9 days. Of the 16 HuMAbs with high (⬎90%) in vitro neutralization values, 7 prolonged survival to ⬎10 days, 4 significantly prolonged survival for ⬍10 days, and 5 did not significantly prolong survival. Further, one of the eight HuMAbs which prolonged murine survival to ⬎10 days had an average in vitro neutralization of 81%. Thus, the in vivo murine Stx2 neutralization assay provided, with the exception of HuMAb 6C3 (Table 1), a more stringent assessment of the Stx2-neutralizing ability of the HuMAbs. Protection of gnotobiotic piglets infected with E. coli O157: H7. Four (2F10, 3E9, 5C12, and 5H8) of the eight Stx2-specific HuMAbs most effective at prolonging murine survival were studied in a gnotobiotic piglet model of E. coli O157:H7 infection (Table 2). In this model, 80% of untreated piglets develop neurological signs (8). Six to twelve hours following oral infection with the Stx2-producing E. coli O157:H7 strain 86-24, piglets were treated with 3 mg of HuMAb administered i.p. Three parameters were used to examine the effect of HuMAb administration relative to untreated PBS control piglets: (i) prevention of neurological signs (paddling, head-pressing, ataxia, and convulsions); (ii) ability to prolong survival; and (iii) prevention of neurological lesions (hemorrhage and edema) within the cerebral cortex and/or cerebellum. Constant monitoring was not possible, and as a result piglets occasionally died without the opportunity to observe them preceding death. For these piglets it is not known whether CNS signs developed, and furthermore, examination of brain tissue was not possible. Thus, only piglets observed up until the time of death or
euthanasia were included in determinations of presence or absence of CNS signs and lesions; however, all piglets which died or were euthanized due to experimental manipulations were included in the survival data. Piglets which died or were euthanized due to unrelated causes (esophageal puncture, extreme weakness, insufficient nourishment, and/or severe dehydration due to diarrhea) were excluded from the experimental data altogether. A total of nine experiments were performed to evaluate the efficacy of administering HuMAb 2F10, 3E9, 5C12, or 5H8 6 or 12 h following infection (results of four representative experiments are shown in Table 2). Nineteen of 21 (90%) control piglets observed immediately prior to death or euthanasia developed neurological signs, and 22 of 23 (96%) available for histologic examination had evidence of neurological lesions. In contrast, administration of HuMAb 2F10, 3E9, 5C12, or 5H8 6 or 12 h postinfection prevented development of neurological signs and lesions in 38 of 42 (90%) treated piglets in these experiments. Two treated piglets which did exhibit convincing neurological signs and lesions had serum HuMAb levels of ⬍0.01 g/ml, in contrast to the levels of 0.488 to 15.2 g/ml in piglets which did not develop neurological signs and/or lesions (data not shown). Interestingly, two additional piglets, one with both neurological signs and lesions and one with only mild neurological lesions, had serum HuMAb levels of 2.0 and 8.9 g/ml, respectively (data not shown). In addition to preventing development of fatal CNS signs and lesions, HuMAb administration also resulted in a trend toward prolongation of survival (Table 2). Due to the small sample size in each experimental group, prolongation of survival of HuMAb-treated groups versus PBS control groups was
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not always statistically significant. Nonetheless, the average survival of HuMAb-treated groups was greater than that in control groups in each experiment with the exception of one 3E9 group containing a single piglet which developed fatal neurological signs (data not shown). Significant prolongation of survival was observed at least twice following administration of HuMAb 3E9, 5H8, or 5C12 6 or 12 h postinfection. Comparison of all 44 HuMAb-treated pigs versus all 31 PBS control pigs indicates that HuMAb administration does indeed result in prolongation of survival (P ⱕ 0.0001).
not yet been undertaken, one can infer that fine epitope specificity rather than isotype or subunit specificity likely determines relative protective efficacy. Delineating the relative contribution of epitope specificity and isotype to protective efficacy is the focus of further studies. Four of the eight HuMAbs most effective at prolonging murine survival were further studied in a gnotobiotic piglet model of E. coli O157:H7 infection. This model differs significantly from the murine Stx2 neutralization assay: (i) instead of receiving purified Stx2, piglets are infected with an Stx2-producing strain of E. coli O157:H7, and thus, similar to humans, they develop diarrhea and can become dehydrated and/or unable to absorb nutrients, and (ii) HuMAbs are given 6 or 12 h following infection rather than prior to challenge, thereby simulating the situation likely to occur in humans, in which HuMAbs would be administered after exposure, which is normally after the onset of diarrhea, in an effort to prevent HUS. Three parameters were used to assess the effect of HuMAb administration: (i) prevention of neurological signs (paddling, head-pressing, ataxia, and convulsions), (ii) ability to prolong survival, and (iii) prevention of neurological lesions (hemorrhage and edema) within the cerebral cortex and/or cerebellum. Although 2 of 42 piglets had serum HuMAb levels of ⬍0.01 g/ml and 2 had serum HuMAbs levels of 2 and 9 g/ml, administration of HuMAbs 2F10, 3E9, 5C12, or 5H8 prevented development of neurological signs and histopathological lesions in the remaining 38 piglets (90%). Although HuMAb dose studies have not yet been carried out, based on the finding that piglets with HuMAb levels of 3.4 to 11.71 g/ml were protected against development of neurological signs and/or lesions, whereas those with levels of ⬍0.01 g/ml were not, one can infer that a dose of 3 mg per piglet was reasonable and levels of ⬃3 g/ml in serum are needed for protection. Both A-subunit-specific (2F10, 3E9, and 5C12) and B-subunit-specific (5H8) HuMAbs were effective at preventing development of neurological signs and lesions associated with Stx2 activity. The efficacy of these HuMAbs is likely due to binding of systemically absorbed Stx2. Binding of B-subunitspecific HuMAbs likely prevents access of Stx2 to target endothelial cells, whereas binding of A-subunit-specific HuMAbs likely either prevents Stx2 ribosomal inactivation or sterically hinders binding of the toxin to its target. Thus, administration of a cocktail comprised of protective Stx2 HuMAbs with specificity for the A and B subunits may provide better protection than administration of either HuMAb alone. Although Stx2 does not induce HUS in piglets, prevention of Stx2-mediated neurological signs and lesions indicates that Stx2-specific HuMAbs will likely be effective at preventing HUS in humans. Previously only polyclonal porcine antiserum and murine MAbs against Stx2 were available. Although polyclonal porcine Stx2 antiserum has been shown to prevent the fatal neurological signs associated with Stx2 activity in piglets, in contrast to the HuMAbs, its efficacy decreases when administered ⬎6 h postinfection (7). Unlike the HuMAbs, which are comprised of a homogeneous population of protective antibodies with a single affinity and epitope specificity, the polyclonal antiserum contains a broad spectrum of Stx2 antibodies with a variety of affinities and epitope specificities, each differing in its protective efficacy. Thus, within a given amount of
DISCUSSION Here we describe the development and functional characterization of 37 stable murine hybridomas which secrete Stx2specific HuMAbs. The predominance of A-subunit-specific HuMAbs is consistent with previous findings in which the majority of murine Stx2-specific MAbs were also specific for the A subunit (6, 25, 28, 29). Isolation of Stx2-specific human IgG1 and IgG3 HuMAbs with differential epitope specificity confirms previous findings that HuMAb___Mouse contains a sufficiently diverse repertoire of human heavy chain immunoglobulin loci capable of responding to a variety of antigens and undergoing isotype switching and affinity maturation (12). The availability of this large panel of HuMAbs not only facilitated identification of HuMAbs with potential therapeutic utility but also provided insight into the antibody characteristics which determine protective efficacy. Initially each HuMAb was screened in vitro by using a cytotoxicity assay to determine the relative ability to neutralize Stx2. Based on relative percent neutralization at a single HuMAb and Stx2 concentration, each HuMAb was assigned to one of three categories: high (ⱖ90% neutralization), medium (70 to 89% neutralization), or low (⬍70% neutralization). By using an in vivo murine assay, each HuMAb was further examined for the ability to neutralize Stx2. Interestingly, the ability to neutralize Stx2 in vivo did not necessarily correlate with the ability to neutralize Stx2 in vitro. Only 7 of the 16 HuMAbs with high in vitro neutralization also prolonged murine survival to ⬎10 days. Furthermore, one HuMAb (6C3) was found to have medium in vitro neutralizing activity but also prolonged murine survival to ⬎10 days. Thus, the murine neutralization assay served as a secondary filter for determining potential efficacy. Of the eight HuMAbs which prolonged murine survival to ⬎10 days, two were specific for the B subunit of Stx2 and six were specific for the A subunit. Of the 29 HuMAbs which failed to prolong survival ⬎10 days, 28 are of the IgG1 isotype and 1 is of the IgG3 isotype; 2 are specific for the B subunit of Stx2, and 27 are specific for the A subunit. Given that HuMAbs of both subunit specificities can be either highly or poorly protective, subunit specificity alone does not determine neutralizing activity. Although our panel of HuMAbs does not represent the entire repertoire of human antibody isotypes, given that the HuMAbs are predominantly of the IgG1 isotype and a broad spectrum of protective efficacy is observed, one can infer that association with the IgG1 isotype alone also does not determine protective efficacy. Detailed analysis of the contribution of isotype would require examining the functional activity of the Fab portion of a protective HuMAb in the context of various Fc regions. Although epitope mapping other than determining subunit specificity has
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total antibody, the effective dose present within the polyclonal preparation is less than that in a comparable HuMAb preparation. Although some of the murine MAbs have been shown to prevent the cytotoxic effects of Stx2 in vitro, of those which have been studied in vivo, none have been shown to prevent the systemic effects of Stx2-mediated activity (6, 9, 25, 28, 30). Thus, the advent of species-compatible, protective Stx2 HuMAbs provides the opportunity for immunotherapy of individuals infected with Stx2-producing bacteria and prevention of subsequent HUS development. HUS typically develops several days after the onset of bloody diarrhea, thereby providing an opportunity to administer such HuMAbs in an effort to prevent HUS development. The half-life of human IgG1 antibodies is 23 days (1). Thus, a single dose of a protective Stx2 HuMAb administered soon after the onset of bloody diarrhea would be sufficient to prevent development of HUS. The fact that these are fully human MAbs of the IgG1 isotype obviates further molecular manipulation for removing or replacing human-incompatible portions of the immunoglobulin molecule or isotype switching, further enhancing the potential of their development for clinical use. ACKNOWLEDGMENTS This work was supported by Public Health Service grants RO1AI41326 and P30-DK-34928 (from the Center for Gastroenterology Research on Absorptive and Secretory Processes) from the National Institutes of Health. We thank Jessica Brisbane and Tammy Richards for their expert technical assistance. REFERENCES 1. Benjamini, E., G. Sunshine, and S. Leskowitz. 1996. Immunology: a short course, 3rd ed., p. 77–92. John Wiley & Sons, Inc., New York, N.Y. 2. Bitzan, M., M. Klemt, R. Steffens, and D. E. Muller-Wiefel. 1993. Differences in verotoxin neutralizing activity of therapeutic immunoglobulins and sera from healthy controls. Infection 21:140–145. 3. Boerlin, P., S. A. McEwen, F. Boerlin-Petzold, J. B. Wilson, R. P. Johnson, and C. L. Gyles. 1999. Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J. Clin. Microbiol. 37:497–503. 4. Dean-Nystrom, E., J. F. L. Pohlenz, H. W. Moon, and A. D. O’Brien. 2000. Escherichia coli O157:H7 causes more severe systemic disease in suckling piglets than in colostrum-deprived neonatal piglets. Infect. Immun. 68:2356– 2358. 5. Donohue-Rolfe, A., D. W. Acheson, A. V. Kane, and G. T. Keusch. 1989. Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies. Infect. Immun. 57:3888–3893. 6. Donohue-Rolfe, A., G. T. Keusch, C. Edson, D. Thorley-Lawson, and M. Jacewicz. 1984. Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies. J. Exp. Med. 160:1767–1781. 7. Donohue-Rolfe, A., I. Kondova, J. Mukherjee, K. Chios, D. Hutto, and S. Tzipori. 1999. Antibody-based protection of gnotobiotic piglets infected with Escherichia coli O157:H7 against systemic complications associated with Shiga toxin 2. Infect. Immun. 67:3645–3648. 8. Donohue-Rolfe, A., I. Kondova, S. Oswald, D. Hutto, and S. Tzipori. 2000. Escherichia coli 0157:H7 strains that express Shiga toxin (Stx) 2 alone are more neurotropic for gnotobiotic piglets than are isotypes producing only Stx1 or both Stx1 and Stx2. J. Infect. Dis. 181:1825–1829. 9. Downes, F. P., T. J. Barrett, J. H. Green, C. H. Aloisio, J. S. Spika, N. A. Strockbine, and I. K. Wachsmuth. 1988. Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies. Infect. Immun. 56:1926–1933. 10. Endo, Y., K. Tsurugi, T. Yutsudo, Y. Takeda, T. Ogasawara, and K. Igarashi. 1988. Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins. Eur. J. Biochem. 171:45–50.
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