Arthur Donohue-Rolfe

Antibody Therapy in the Management of Shiga Toxin-Induced Hemolytic Uremic Syndrome

SUMMARY Hemolytic uremic syndrome (HUS) is a disease that can lead to acute renal failure and often to other serious sequelae, including death. The majority of cases are attributed to infections with Escherichia coli, serotype O157:H7 strains in particular, which cause bloody diarrhea and liberate one or two toxins known as Shiga toxins 1 and 2. These toxins are thought to directly be responsible for the manifestations of HUS. Currently, supportive nonspecific treatment is the only available option for the management of individuals presenting with HUS. The benefit of antimicrobial therapy remains uncertain because of several reports which claim that such intervention can in fact exacerbate the syndrome. There have been only a few specific therapies directed against neutralizing the activities of these toxins, but none so far has been shown to be effective. This article reviews the literature on the mechanism of action of these toxins and the clinical manifestations and current management and treatment of HUS. The major focus of the article, however, is the development and rationale for using neutralizing human antibodies to combat this toxin-induced disease. Several groups are currently pursuing this approach with either humanized, chimeric, or human antitoxin antibodies produced in transgenic mice. They are at different phases of development, ranging from preclinical evaluation to human clinical trials. The information available from preclinical studies indicates that neutralizing specific antibodies directed against the A subunit of the toxin can be highly protective. Such antibodies, even when administered well after exposure to bacterial infection and onset of diarrhea, can prevent the occurrence of systemic complications.

Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection.

ABSTRACT 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 κ light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1κ A-subunit-specific and 3 IgG1κ and 1 IgG3κ B-subunit-specific antibodies. Six IgG1κ A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1κ B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of >95% activity of 1 ng of Stx2 in the presence of 0.04 μg of HuMAb in vitro and significant prolongation of survival of mice given 50 μg 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.

Human Antibody against Shiga Toxin 2 Administered to Piglets after the Onset of Diarrhea Due to Escherichia coli O157:H7 Prevents Fatal Systemic Complications

ABSTRACT Infection of children with Shiga toxin (Stx)-producing Escherichia coli (STEC) can lead to hemolytic-uremic syndrome (HUS) in 5 to 10% of patients. Stx2, one of two toxins liberated by the bacterium, is directly linked with HUS. We have previously shown that Stx-specific human monoclonal antibodies protect STEC-infected animals from fatal systemic complications. The present study defines the protective antibody dose in relation to the time of treatment after the onset of diarrhea in infected gnotobiotic piglets. Using the mouse toxicity model, we selected 5C12, an antibody specific for the A subunit, as the most effective Stx2 antibody for further characterization in the piglet model in which piglets developed diarrhea 16 to 40 h after bacterial challenge, followed by fatal neurological symptoms at 48 to 96 h. Seven groups of piglets received doses of 5C12 ranging from 6.0 mg/kg to 0.05 mg/kg of body weight, administered parenterally 48 h after bacterial challenge. The minimum fully protective antibody dose was 0.4 mg/kg, and the corresponding serum antibody concentration in these piglets was 0.7 μg (±0.5)/ml, measured 7 to 14 days after administration. Of 40 infected animals which received Stx2 antibody treatment of ≥0.4 mg/kg, 34 (85%) survived, while only 1 (2.5%) of 39 placebo-treated animals survived. We conclude that the administration of the Stx2-specific antibody was protective against fatal systemic complications even when it was administered well after the onset of diarrhea. These findings suggest that children treated with this antibody, even after the onset of bloody diarrhea, may be equally protected against the risk of developing HUS.

Maturational regulation of globotriaosylceramide, the Shiga-like toxin 1 receptor, in cultured human gut epithelial cells.

Differentiated villus intestinal epithelial cells express globotriaosylceramide, the Shiga-like toxin 1 (SLT-1) receptor, and are sensitive to toxin-mediated cytotoxicity, whereas undifferentiated crypt cells neither express Gb3 nor respond to toxin. To investigate if SLT-1 receptors are maturationally regulated in human intestinal cells, we examined the effect of butyrate, a known transcriptional regulator of differentiation genes in many cell types, using cultured colonic cancer-derived epithelial cell lines. Exposure to butyrate increased villus cell marker enzymes such as alkaline phosphatase, sucrase, and lactase, expression of toxin receptors, and sensitivity to SLT-1 in villus-like CaCo-2A and HT-29 cells. These effects were reversibly inhibited by preincubation of CaCo-2A cells with actinomycin D or cycloheximide. Butyrate-treated CaCo-2A cells unable to bind fluoresceinated SLT-1 B subunit were undifferentiated as assessed by alkaline phosphatase activity. HT-29 cells induced to differentiate by another signal, glucose deprivation, upregulated receptor content and response to toxin. Crypt-like T-84 cells responded to butyrate with a modest increase in alkaline phosphatase and toxin binding, but no induction of sucrase or lactase, and no change in sensitivity to toxin. The results demonstrate that expression of SLT-1 toxin receptors and toxin sensitivity are coregulated with cellular differentiation in cultured intestinal cells.

A comparison of HEp‐2 cell invasion by enteropathogenic and enteroinvasive Escherichia coli

In order to further characterize cellular invasion by enteropathogenic Escherichia coli (EPEC), we compared invasion of HEp-2 cells by EPEC and enteroinvasive E. coli (EIEC). We used a gentamicin HEp-2 cell assay and measured bacterial recovery under conditions of varying incubation time and temperature, and in the presence or absence of inhibitors of cellular microfilaments and microtubules. We found that, unlike EIEC, EPEC did not rapidly multiply within HEp-2 cell but invaded well at 32 degrees C. While microfilament inhibitors reduced invasion by both EIEC and EPEC, microtubule inhibitors reduced invasion by EPEC only. These results suggest that EPEC and EIEC differ in their mechanisms of epithelial cell invasion.

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

Infection with Escherichia coli O157:H7 can lead to hemolytic uremic syndrome (HUS) in some children. Epidemiologic data suggest that Shiga toxin (Stx) 2-producing strains are more frequently associated with HUS than are Stx1-producing strains. Less clear is whether strains that express Stx2 alone are more frequently associated with HUS than strains that express Stx1 and Stx2. Isogenic mutants 933stx1- and 933stx2- were produced from strain 933 (Stx1 and Stx2 producer), and 86-24stx2- was produced from strain 86-24 (Stx2 producer). Neurologic lesions or symptoms developed in 18 (90%) of 20 gnotobiotic piglets orally infected with strain 86-24, in 15 (85%) of 18 infected with mutant 933stx1-, in 9 (31%) of 29 infected with strain 933, in 0 of 5 infected with mutant 86-24stx2-, and in 0 of 6 infected with mutant 933stx2-. It was concluded that strains expressing Stx2 alone are more neurotropic for piglets when fed orally than are those strains expressing Stx1 and 2, whereas Stx1-producing strains induce only diarrhea. It is also conceivable that strains that produce Stx2 may constitute a significant predictive risk factor for HUS in humans.

Production and Characterization of Protective Human Antibodies against Shiga Toxin 1

ABSTRACT Hemolytic-uremic syndrome (HUS) is a serious complication which is predominantly associated in children with infection by Shiga toxin-producing Escherichia coli (STEC). By using HuMAb-Mouse (Medarex) animals, human monoclonal antibodies (Hu-MAbs) were developed against Shiga toxin 1 (Stx1) for passive immunotherapy of HUS. Ten stable hybridomas comprised of fully human heavy- and light-chain immunoglobulin elements and secreting Stx1-specific Hu-MAbs (seven immunoglobulin M(κ) [IgM(κ)] elements [one specific for the A subunit and six specific for the B subunit] and three IgG1(κ) elements specific for subunit B) were isolated. Two IgM(κ) Hu-MAbs (2D9 and 15G9) and three IgG1(κ) Hu-MAbs (5A4, 10F4, and 15G2), all specific for subunit B, demonstrated marked neutralization of Stx1 in vitro and significant prolongation of survival in a murine model of Stx1 toxicosis.

Isolation and characterization of functional Shiga toxin subunits and renatured holotoxin

Shiga toxin is a potent protein toxin produced by Shigella dysenteriae type I strains. In this report we present a procedure for the separation of functionally intact toxin A and B chains and for their reconstitution to form biologically active molecules. In agreement with the findings of others, the isolated A chain was shown to be a potent in vitro inhibitor of eukaryotic protein synthesis. The isolated B chain bound to HeLa cells and competitively inhibited the binding and cyto‐toxic activity of holotoxin. These findings show that the functional role of the B chain is to recognize cell surface functional receptors. By labelling the B sub‐unit alone, prior to renaturatlon of holotoxin, the polypeptide chains were shown to associate non‐covalently with a stoichiometry of one A chain and five B chains.

Stx2-Specific Human Monoclonal Antibodies Protect Mice against Lethal Infection with Escherichia coli Expressing Stx2 Variants

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains are responsible for causing hemolytic-uremic syndrome (HUS), and systemic administration of Shiga toxin (Stx)-specific human monoclonal antibodies (HuMAbs) is considered a promising approach for prevention or treatment of the disease in children. The goal of the present study was to investigate the ability of Stx2-specific HuMAbs to protect against infections with STEC strains that produce Stx2 variants. Dose-response studies on five HuMAbs, using the mouse toxicity model, revealed that only the three directed against the A subunit were protective against Stx2 variants, and 5C12 was the most effective among the three tested. Two HuMAbs directed against the B subunit, while highly effective against Stx2, were ineffective against Stx2 variants. In a streptomycin-treated mouse model, parenteral administration of 5C12 significantly protected mice up to 48 h after oral bacterial challenge. We conclude that 5C12, reactive against the Stx2 A subunit, is an excellent candidate for immunotherapy against HUS and that antibodies directed against the A subunit of Stx2 have broad-spectrum activity that includes Stx2 variants, compared with those directed against the B subunit.

Increased adherence and actin pedestal formation by dam‐deficient enterohaemorrhagic Escherichia coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) are highly infectious pathogens capable of causing severe diarrhoeal illnesses. As a critical step during their colonization, EHEC adhere intimately to intestinal epithelial cells and generate F‐actin ‘pedestal’ structures that elevate them above surrounding cell surfaces. Intimate adhesion and pedestal formation result from delivery of the EHEC type III secretion system (TTSS) effector proteins Tir and EspFU into the host cell and expression of the bacterial outer membrane adhesin, intimin. To investigate a role for DNA methylation during the regulation of adhesion and pedestal formation in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC O157:H7 and demonstrate that this mutation results in increased interactions with cultured host cells. EHECΔdam exhibits dramatically elevated levels of adherence and pedestal formation when compared with wild‐type EHEC, and expresses significantly higher protein levels of intimin, Tir and EspFU. Analyses of GFP fusions, Northern blotting, reverse transcription polymerase chain reaction, and microarray experiments indicate that the abundance of Tir in the dam mutant is not due to increased transcription levels, raising the possibility that Dam methylation can indirectly control protein expression by a post‐transcriptional mechanism. In contrast to other dam‐deficient pathogens, EHECΔdam is capable of robust intestinal colonization of experimentally infected animals.