b Shiga toxin-producing Escherichia coli (STEC) is a major cause of severe food-borne disease worldwide, and two Shiga toxins, Stx1 and Stx2, are primarily responsible for the serious disease consequence, hemolytic-uremic syndrome (HUS). Here we report identification of a panel of heavy-chain-only antibody (Ab) V H (VHH) domains that neutralize Stx1 and/or Stx2 in cell-based assays. VHH heterodimer toxin-neutralizing agents containing two linked Stx1-neutralizing VHHs or two Stx2-neutralizing VHHs were generally much more potent at Stx neutralization than a pool of the two-component monomers tested in cell-based assays and in vivo mouse models. We recently reported that clearance of toxins can be promoted by coadministering a VHHbased toxin-neutralizing agent with an antitag monoclonal antibody (MAb), called the "effector Ab," that indirectly decorates each toxin molecule with four Ab molecules. Decoration occurs because the Ab binds to a common epitopic tag present at two sites on each of the two VHH heterodimer molecules that bind to each toxin molecule. Here we show that coadministration of effector Ab substantially improved the efficacy of Stx toxin-neutralizing agents to prevent death or kidney damage in mice following challenge with Stx1 or Stx2. A single toxin-neutralizing agent consisting of a double-tagged VHH heterotrimer-one Stx1-specific VHH, one Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH-was effective in preventing all symptoms of intoxication from Stx1 and Stx2 when coadministered with effector Ab. Overall, the availability of simple, defined, recombinant proteins that provide cost-effective protection against HUS opens up new therapeutic approaches to managing disease.

The early immunoglobulin repertoire of neonatal foals comprised IgGa, IgG(T), and IgA; endogenous synthesis of IgGb could not be detected until 63 days after birth. The restricted repertoire of immunoglobulins in foals may influence humoral immune responses to vaccination.

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 g (؎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.

No abstract

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

No abstract

Summary Equine herpesvirus‐1 (EHV‐1) remains a frequent cause of upper respiratory tract infection and abortion in horses worldwide. However, little is known about the local antibody response elicited in the upper airways of horses following exposure to EHV‐1. This study analysed the mucosal humoral immune response of weanling foals following experimental infection with virulent EHV‐1, or vaccination with either of 2 commercial vaccines. Twenty weanlings were assigned to 5 groups and were inoculated with, or vaccinated against, EHV‐1 following different regimens. Finally, all weanlings were simultaneously challenged intranasally with virulent EHV‐1 Army 183 (A183). Nasal wash and serum samples were collected at regular intervals until 13 weeks after final challenge. Nasal washes were assayed for EHV‐1‐specific equine IgGa, IgGb, IgG(T), IgA, IgM and total virus‐specific antibody using an indirect, quantitative ELISA. Total serum antibody responses were also monitored, and clinical signs of EHV‐disease were recorded for each individual. Virus‐specific IgA dominated the mucosal antibody response elicited in weanlings inoculated with A183, being detectable at up to 3.1 μg/mg total IgA 13 weeks after challenge. Neither inactivated EHV‐1 administered i.m., nor attenuated EHV‐1 administered intranasally induced detectable mucosal antibodies. EHV‐1‐specific mucosal antibodies impeded EHV‐1 plaque formation in vitro. Such virus‐neutralising antibody probably contributes to a reduction of shedding of EHV‐1 from the respiratory tract of virus‐infected horses.

Infection of children with Shiga toxin (Stx)-producing Escherichia coli (STEC) is the leading cause of hemolyticuremic syndrome (HUS). Stx2, one of two toxins liberated by the bacteria, is directly linked with HUS. We have previously shown that Stx2-specific human monoclonal antibodies (HuMAbs) protect mice and piglets from fatal systemic complications of Stx2. The present study investigates the mechanisms by which our most efficacious A-and B-subunit-specific HuMAbs neutralize the cytotoxic effects of Stx2 in vitro. Whereas the B-subunit-specific HuMAb 5H8 blocked binding of Stx2 to its receptor on the cell surface, the A-subunit-specific HuMAb 5C12 did not interfere with the toxin-receptor binding. Further investigations revealed that 5C12 did not block endocytosis of Stx2 by HeLa cells as both Stx2 and 5C12 colocalized with early endosomes. However, 5C12 blocked the retrograde transport of the toxin into the Golgi and the endoplasmic reticulum, preventing the toxin from entering the cytosol where the toxin exerts its cytotoxic effect. The endocytosed 5C12/Stx2 complexes appear to be rapidly transported to the plasma membrane and/or to the slow recycling perinuclear compartments, followed by their slow recycling to the plasma membrane, and release into the extracellular environment.Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) can become life threatening if it induces systemic complications, mainly hemolytic-uremic syndrome (HUS), the leading cause of acute renal failure in children (2,11,21,25). Of Stx1 and Stx2, the two immunologically distinct Stxs produced by STEC, strains producing only Stx2 are more frequently associated with HUS (10, 27). Stx1 and Stx2 are similar in basic structure, binding specificity, and mode of action (9). The Stx molecule consists of an A-subunit monomer and a B-subunit pentamer. The pentameric B subunit binds to its cell surface receptor CD77, also called globotriaosylceramide (Gb 3 ). This triggers endocytosis of the holotoxin, mainly through clathrin-coated pits (16). Internalized Stx is then delivered to the trans-Golgi network, where it is carried by retrograde transport to the endoplasmic reticulum (ER), and then to the cytosol (28). During this process, the A subunit is nicked by the membrane bound protease furin, generating a catalytically active N-terminal A1 fragment, while a C-terminal A2 fragment remains linked by a disulfide bond (28). This disulfide bond is subsequently reduced to release the active A1 component. The released A1 fragment has RNA N-glycosidase activity that removes a single adenine from the 28S rRNA, thereby inhibiting protein synthesis in the intoxicated cells (7).Several therapeutic approaches that attempted to neutralize Stx either in the gut or in the circulation include the use of synthetic Gb 3 analogues, genetically manipulated bacteria expressing Gb 3 , and Stx-specific neutralizing antibodies (32). The systemic administration of Stx-specific neutralizing antibodies, we believe, is currently the most promising approach for...