In vitro and in vivo protective efficacies of antibodies that neutralize the RNA N-glycosidase activity of Shiga toxin 2

<p>Abstract</p> <p>Backgound</p> <p>Shiga toxin 2 (Stx2), one of two Stx liberated by Stx-producing <it>Escherichia coli</it>, is composed of an A subunit monomer and a B subunit pentamer, and is directly linked with hemolytic uremic syndrome in children. The pentameric B subunit binds to its cell surface receptor Gb₃for toxin internalization, and the A subunit follows intracellular retrograde transport to the cytosol where its RNA <it>N</it>-glycosidase activity (RNA-NGA) shuts down the protein synthesis, and leads to cell death. The present study investigated the ability of 19 Stx2 A subunit-specific human monoclonal antibodies (HuMAbs) to neutralize the RNA-NGA, and the association this neutralizing activity with protection of HeLa cells and mice against Stx2-induced death.</p> <p>Results</p> <p>The HuMAbs that were stronger inhibitors of RNA-NGA were also better at neutralizing Stx2 mediated HeLa cell death, and those that were weaker inhibitors of RNA-NGA activity were also weaker in protecting HeLa cells. These results suggest that the ability of an A subunit-specific antibody to block the RNA-NGA of the toxin is directly related to its ability to neutralize Stx2-mediated HeLa cell death. However, with the exception of the best RNA-NGA blocking antibodies 5C12 and 2F10, the efficacies of antibody neutralization of RNA-NGA of Stx2 did not correlate with their <it>in vivo </it>protective efficacies. The HuMAb 6C3, which neutralized RNA N-glycosidase activity of Stx2 less effectively than the HuMAbs 6D8 and 6B7, protected 100% of the mice against Stx2 challenge at 50 μg/mouse dose. In contrast, the HuMAbs 6D8 and 6B7, which neutralized RNA N-glycosidase activity of Stx2 more effectively than 6C3, protected 20% and 0% mice at that dose, respectively.</p> <p>Conclusions</p> <p>The neutralization efficiency of the RNA-NGA of Stx2 by A subunit-specific antibodies correlate strongly with their abilities to protect HeLa cells against Stx2-mediated toxicity but only the strongest RNA-NGA-neutralizing antibodies correlate very well with both protecting HeLa cells and mice against Stx2 challenge.</p

Adenovirus vector expressing Stx1/Stx2-neutralizing agent protects piglets infected with Escherichia coli O157: H7 against fatal systemic intoxication

Hemolytic-uremic syndrome (HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), remains untreatable. Production of human monoclonal antibodies against Stx, which are highly effective in preventing Stx sequelae in animal models, is languishing due to cost and logistics. We reported previously that the production and evaluation of a camelid heavy-chain-only V(H) domain (VHH)-based neutralizing agent (VNA) targeting Stx1 and Stx2 (VNA-Stx) protected mice from Stx1 and Stx2 intoxication. Here we report that a single intramuscular (i.m.) injection of a nonreplicating adenovirus (Ad) vector carrying a secretory transgene of VNA-Stx (Ad/VNA-Stx) protected mice challenged with Stx2 and protected gnotobiotic piglets infected with STEC from fatal systemic intoxication. One i.m. dose of Ad/VNA-Stx prevented fatal central nervous system (CNS) symptoms in 9 of 10 animals when it was given to piglets 24 h after bacterial challenge and in 5 of 9 animals when it was given 48 h after bacterial challenge, just prior to the onset of CNS symptoms. All 6 placebo animals died or were euthanized with severe CNS symptoms. Ad/VNA-Stx treatment had no impact on diarrhea. In conclusion, Ad/VNA-Stx treatment is effective in protecting piglets from fatal Stx2-mediated CNS complications following STEC challenge. With a low production cost and further development, this could presumably be an effective treatment for patients with HUS and/or individuals at high risk of developing HUS due to exposure to STEC

Sensitivity and Specificity of a Monoclonal Antibody-Based Fluorescence Assay for Detecting Enterocytozoon bieneusi Spores in Feces of Simian Immunodeficiency Virus-Infected Macaques

Enterocytozoon bieneusi is clinically the most significant among the microsporidia causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. Microscopy with either calcofluor or modified trichrome stains is the standard diagnostic test for microsporidiosis and does not allow species identification. Detection of E. bieneusi infection based on PCR is limited to a few reference laboratories, and thus it is not the standard diagnostic assay. We have recently reported the development and characterization of a panel of monoclonal antibodies against E. bieneusi, and in this publication we evaluated the specificity and sensitivity of an immunofluorescence assay (IFA), compared with PCR, in simian immunodeficiency virus-infected macaques. The IFA, which correlated with the primary PCR method, with a detection limit of 1.5 ×10(5) spores per gram of feces, will simplify considerably the detection of E. bieneusi spores in clinical and environmental specimens and in laboratory and epidemiological investigations

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

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

<i>Cryptosporidium</i>-specific IgG in gnotobiotic post-immune sera and <i>C</i>. <i>hominis</i> infection in piglet gut.

3–5-day old gnotobiotic piglets received a primary infection with C. p. anthroponosum TU114 (n = 4) or C. hominis TU502 (n = 8) or remained uninfected (unprimed) and were challenged (secondary infection) with C. p. anthroponosum TU114 (n = 3) or C. hominis TU502 (n = 4) after recovery (~33–45 days after birth). (A) Cryptosporidium-specific IgG was determined from sera collected 10 days post-challenge. Some sera from primed and challenged piglets had elevated anti- C. p. parvum or C. hominis in contrast to undetectable anti-gp900 D3 response in all sera regardless of infections received (unprimed/primed). Each data point is the mean OD 450nm of duplicates at 1:1600 serum dilution obtained when tested against Cryptosporidium antigens. (B) Sections of terminal ilea of gnotobiotic piglets. The left image shows a normal villi coated with normal enterocytes. The right image shows the heavily infected enterocytes with C. hominis forms (arrows) embedded in the microvillus border. The tip of the infected section is covered by irregular and damaged layer of enterocytes (H & E; M– 400x). Scale indicates 20μm.</p

<i>Cryptosporidium</i>-specific IgG in post immune rat sera and the effect of these sera in <i>C</i>. <i>parvum</i> infection in vitro.

Rats were immunized 3 times on day 0, 35 and 56 with sporozoite lysates of C. hominis TU502 (n = 2), zoonotic C. parvum (n = 2) or C. p. anthroponosum TU114 (n = 1), with each immunization containing lysate of 106 excysted oocysts. Additional rats (n = 2) were immunized with or C. p. parvum derived gp900 D3 or PBS as control (n = 1). (A) shows the high level of Cryptosporidium-specific IgG (anti-C. hominis, anti-C. p. parvum IOWA and anti-gp900 D3) in post-immune sera in comparison to serum from the control rat. Values indicate optical densities (OD) measured at 450-nm absorbance of the average of technical duplicates at 1:1600 serum dilution. Post immune C. hominis, C. p. anthroponosum TU114 and control had low level of gp900 domain 3 IgG. (B) The effect of post-immune sera to inhibit intracellular C. parvum infection in vitro (using MDBK cells) was determined by adding sera at 1:100, 1:200, 1:400 and 1:800 dilutions. Infection control was a C. p. parvum IOWA cell culture maintained in growth medium alone. Rate of infection (%) in the presence of sera were normalized using data from infection control. Comparison of infection rate between cultures with post immune and age-matched PBS control was analyzed using 2-way ANOVA with Dunnett’s multiple comparisons test. Data shown are mean ±SD from duplicate assays.</p

<i>Cryptosporidium</i>-specific IgG in serum of <i>C</i>. <i>hominis</i> TU502 immunized alpaca.

Each data point is the mean OD 450nm of duplicates of specified serum dilutions obtained when tested against Cryptosporidium antigens (C. hominis, C. p. parvum IOWA and gp900 D3). An alpaca was immunized with C. hominis TU502 oocyst lysates subcutaneously with CpG and Alhydrogel adjuvants, 7 times at 2–4 weeks intervals. Two weeks after the last immunization serum was collected and the anti-C. hominis TU502, C. p. parvum IOWA and gp900 D3 IgG was determined by ELISA. Statistical comparisons: anti-C. hominis TU502 vs. anti-gp900-d3, p = 0.0021; anti-zoonotic C. p. parvum IOWA vs. anti-gp900-d3, p = 0.0008, Kruskal-Wallis test with Dunn’s multiple comparison test.</p

Rat immunization regimen and their serum antibody response against <i>Cryptosporidium</i> antigens determined by ELISA^a.

Rat immunization regimen and their serum antibody response against Cryptosporidium antigens determined by ELISAa.</p