Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation.

BACKGROUND: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. OBJECTIVE: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. METHODS: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. RESULTS: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. CONCLUSION: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

Hyperimmune Bovine Colostral Anti-CS17 Antibodies Protect Against Enterotoxigenic Escherichia coli Diarrhea in a Randomized, Doubled-Blind, Placebo-Controlled Human Infection Model.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) commonly cause diarrhea in children living in developing countries and in travelers to those regions. ETEC are characterized by colonization factors (CFs) that mediate intestinal adherence. We assessed if bovine colostral IgG (bIgG) antibodies against a CF, CS17, or antibodies against CsbD, the minor tip subunit of CS17, would protect subjects against diarrhea following challenge with a CS17-expressing ETEC strain. METHODS: Adult subjects were randomized (1:1:1) to receive oral bIgG against CS17, CsbD, or placebo. Two days prior to challenge, subjects began dosing 3 times daily with the bIgG products (or placebo). On day 3, subjects ingested 5 × 109 cfu ETEC strain LSN03-016011/A in buffer. Subjects were assessed for diarrhea for 120 hours postchallenge. RESULTS: A total of 36 subjects began oral prophylaxis and 35 were challenged with ETEC. While 50.0% of the placebo recipients had watery diarrhea, none of the subjects receiving anti-CS17 had diarrhea (P = .01). In contrast, diarrhea rates between placebo and anti-CsbD recipients (41.7%) were comparable (P = 1.0). CONCLUSIONS: This is the first study to demonstrate anti-CS17 antibodies provide significant protection against ETEC expressing CS17. More research is needed to better understand why anti-CsbD was not comparably efficacious. Clinical Trials Registration. NCT00524004.

Leaf-Encapsulated Vaccines: Agroinfiltration and Transient Expression of the Antigen Staphylococcal Endotoxin B in Radish Leaves.

Transgene introgression is a major concern associated with transgenic plant-based vaccines. Agroinfiltration can be used to selectively transform nonreproductive organs and avoid introgression. Here, we introduce a new vaccine modality in which Staphylococcal enterotoxin B (SEB) genes are agroinfiltrated into radishes (Raphanw sativus L.), resulting in transient expression and accumulation of SEB in planta. This approach can simultaneously express multiple antigens in a single leaf. Furthermore, the potential of high-throughput vaccine production was demonstrated by simultaneously agroinfiltrating multiple radish leaves using a multichannel pipette. The expression of SEB was detectable in two leaf cell types (epidermal and guard cells) in agroinfiltrated leaves. ICR mice intranasally immunized with homogenized leaves agroinfiltrated with SEB elicited detectable antibody to SEB and displayed protection against SEB-induced interferon-gamma (IFN-γ) production. The concept of encapsulating antigens in leaves rather than purifying them for immunization may facilitate rapid vaccine production during an epidemic disease.

A preliminary study on topical ozonated oil in the therapeutic management of atopic dermatitis in murine.

OBJECTIVE: To explore whether ozonated oil recovery atopic dermatitis (AD) via immunoregulation. METHODS: Mice were repeatedly challenged with the triplex allergens of staphylococcal enterotoxin B, ovalbumin and calcipotriol ointment on the back to develop AD lesions, and were treated with ozonated oil. The lesional skins were scanned by reflectance confocal microscopy to measure the thickness of epidermis. The skin tissues were stained. Th1-type and Th2-type cytokines in serum and in tissues were detected by ELISA and real-time PCR, respectively. RESULTS: Ozonated oil significantly inhibited inflammation and healed the lesions in 7 d. Ozonated oil inhibited NGF expression as compared to the groups treated with vehicle or PBS (p < .01).The serum proteins and lesional transcripts of Th2 cytokines including IL-4 and IL-31 were lower in the ozonated oil treated group than the groups treated with vehicle or PBS (p < .05). The IL-10 level was increased with treatment of ozonated oil (p < .01). On the other hand, the expressions of Th1 cytokines including IL-2, TNF-α, and IFN-γ in the serum were not regulated by ozonated oil. CONCLUSIONS: Our results showed that ozonated oil could suppress inflammation in an AD murine via decreasing Th2-dominant cytokines response and increasing IL-10 expression. These suggest that ozonated oil may be a potential remedy for AD.

Entrapment of LTB protein in alginate nanoparticles protects against Enterotoxigenic Escherichia coli.

Vaccine delivery vehicles are just as important in vaccine efficiency. Through the progress in nanotechnology, various nanoparticles have been evaluated as carriers for these substances. Among them, alginate nanoparticles are a good choice because of their biodegradability, biocompatibility, ease of production, etc. In this study, feasibility of alginate nanoparticles (NPs) such as recombinant LTB from Enterotoxigenic Escherichia coli (ETEC) carrier was investigated. To do this, the eltb gene was cloned and expressed in E. coli BL21 (DE3) host cells, and a Ni-NTA column purified the protein. NPs were achieved through ion gelation method in the presence of LTB protein and CaCl2 as the cross-Linker and NPs were characterized physicochemically. Balb/C mice groups were immunized with LTB-entrapped NPs or LTB with adjuvant and immunogenicity was assessed by evaluating IgG titer. Finally, the neutralization of antibodies was evaluated by GM1 binding and loop assays. LTB protein was expressed and efficiently entrapped into the alginate NPs. The size of NPs was less than 50 nm, and entrapment efficiency was 80%. Western blotting showed maintenance of the molecular weight and antigenicity of the released protein from NPs. Administration of LTB-entrapped NPs stimulated antibody responses in immunized mice. Immunization induced protection against LT toxin of ETEC in ileal loops and inhibits enterotoxin binding to GM1-gangliosides. Alginate NPs are also appropriate vehicle for antigen delivery purpose. Moreover because of their astonishing properties, they have the potential to serve as an adjuvant.

Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells.

Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein-coupled receptor (GPCR) leading to PLCß activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome.

Protective immunity of a Multivalent Vaccine Candidate against piglet diarrhea caused by enterotoxigenic Escherichia coli (ETEC) in a pig model.

As one of the most challenging problems in swine industry, piglet diarrhea has caused huge economic loss globally. Currently, vaccination is the most effective way of controlling enterotoxigenic Escherichia coli (ETEC) diarrhea. However, existing commercial vaccines could not provide broad protection against different types of ETEC. In this study, we mixed a enterotoxin fusion protein SLS (STa-LTB-STb) with the main fimbrial F4ac and F5 antigens as a novel multivalent vaccine candidate. Then an overall evaluation of this vaccine candidate against ETEC was carried out in a pig model. We found that the IgG titers in serum as well as colostrum in all the vaccinated sows were significantly higher than that in the control group (P < 0.05). By using a sensory evaluation method, we demonstrated that piglets in the vaccinated group exhibited significantly healthier status than the unimmunized group. Moreover, in response to F41 + ETEC challenge, none of the piglets with the vaccine candidate experienced diarrhea, whereas 30% of the piglets suffered without vaccination. In conclusion, these results showed that the candidate vaccine could elicit multiple high-titer antibodies against all the main virulence factors and provide a broad and effective protection against ETEC diarrhea.

Dietary Animal Plasma Proteins Improve the Intestinal Immune Response in Senescent Mice.

Increased life expectancy has promoted research on healthy aging. Aging is accompanied by increased non-specific immune activation (inflammaging) which favors the appearance of several disorders. Here, we study whether dietary supplementation with spray-dried animal plasma (SDP), which has been shown to reduce the activation of gut-associated lymphoid tissue (GALT) in rodents challenged by S. aureus enterotoxin B (SEB), and can also prevent the effects of aging on immune system homeostasis. We first characterized GALT in a mouse model of accelerated senescence (SAMP8) at different ages (compared to mice resistant to accelerated senescence; SAMR1). Second, we analyzed the SDP effects on GALT response to an SEB challenge in SAMP8 mice. In GALT characterization, aging increased the cell number and the percentage of activated Th lymphocytes in mesenteric lymph nodes and Peyer's patches (all, p < 0.05), as well as the expression of IL-6 and TNF-α in intestinal mucosa (both, p < 0.05). With respect to GALT response to the SEB challenge, young mice showed increased expression of intestinal IL-6 and TNF-α, as well as lymphocyte recruitment and activation (all, p < 0.05). However, the immune response of senescent mice to the SEB challenge was weak, since SEB did not change cell recruitment or the percentage of activated Th lymphocytes. Mice supplemented with SDP showed improved capacity to respond to the SEB challenge, similar to the response of the young mice. These results indicate that senescent mice have an impaired mucosal immune response characterized by unspecific GALT activation and a weak specific immune response. SDP supplementation reduces non-specific basal immune activation, allowing for the generation of specific responses.

Oral vaccination of broiler chickens against necrotic enteritis using a non-virulent NetB positive strain of Clostridium perfringens type A.

Necrotic enteritis (NE) is a severe disease of chickens and turkeys caused by some strains of Clostridium perfringens type A. The disease is well controlled by the use of in-feed antibiotic growth promoters (AGPs). However, due to worldwide public and regulatory pressure to reduce the use of AGPs inter alia, there is an urgent need to develop non-antibiotic based preventative measures. Vaccination would be a suitable control measure, but currently there is no commercial vaccine. NetB (necrotic enteritis toxin B-like) is a pore-forming toxin produced by C. perfringens that has been reported as an important virulence factor in the pathogenesis of NE. The present study tests a non-virulent NetB producing strain of C. perfringens (nvNetB+), with or without adjuvants, as an orally administered live vaccine. Adjuvants used were Gel 01™, Cholera toxin (CT), Escherichia coli wild type heat-labile holotoxin (LT) and mutant E. coli LT (dmLT) (R192G/L211A). Several vaccine administration regimes were tested. All vaccination regimes elicited serum and mucosal antibody responses to alpha toxin and to secreted proteins of both nvNetB+ and a very virulent NetB positive (vvNetB+) strain (p<0.0001 to p<0.05). In some vaccinated groups, there was milder intestinal pathology upon disease challenge. 55% of birds vaccinated orally at days 2, 12 with nvNetB+ adjuvanted with CT did not develop any lesions of NE by 6 days post challenge, compared to a 100% incidence of NE lesions in the unvaccinated disease challenged group.

Bioactive Immune Components of Anti-Diarrheagenic Enterotoxigenic Escherichia coli Hyperimmune Bovine Colostrum Products.

Diarrhea is a common illness among travelers to resource-limited countries, the most prevalent attributable agent being enterotoxigenic Escherichia coli (ETEC). At this time, there are no vaccines licensed specifically for the prevention of ETEC-induced traveler's diarrhea (TD), and this has propelled investigation of alternative preventive methods. Colostrum, the first milk expressed after birthing, is rich in immunoglobulins and innate immune components for protection of newborns against infectious agents. Hyperimmune bovine colostrum (HBC) produced by immunization of cows during gestation (and containing high levels of specific antibodies) is a practical and effective prophylactic tool against gastrointestinal illnesses. A commercial HBC product, Travelan, is available for prevention of ETEC-induced diarrhea. Despite its demonstrated clinical efficacy, the underlying immune components and antimicrobial activity that contribute to protection remain undefined. We investigated innate and adaptive immune components of several commercial HBC products formulated to reduce the risk of ETEC-induced diarrhea, including Travelan and IMM-124E, a newer product that has broader gastrointestinal health benefits. The immune components measured included total and ETEC-specific IgG, total IgA, cytokines, growth factors, and lactoferrin. HBC products contained high levels of IgG specific for multiple ETEC antigens, including O-polysaccharide 78 and colonization factor antigen I (CFA/I) present in the administered vaccines. Antimicrobial activity was measured in vitro using novel functional assays. HBC greatly reduced ETEC motility in soft agar and exhibited bactericidal activity in the presence of complement. We have identified immune components and antimicrobial activity potentially involved in the prevention of ETEC infection by HBC in vivo.

Preparation and preclinical evaluation of a freeze-dried formulation of a novel combined multivalent whole-cell/B-subunit oral vaccine against enterotoxigenic Escherichia coli diarrhea.

A promising liquid killed multivalent whole-cell plus enterotoxin B-subunit oral vaccine against enterotoxigenic Escherichia coli (ETEC), the primary cause of diarrhea among children in low-income countries and travelers to these areas, has recently been developed and tested in preclinical and phase-I and phase-II clinical studies. The vaccine contains killed E. coli bacteria over-expressing the main ETEC colonization factors (CFs) CFA/I, CS3, C5 and C6, and a recombinant enterotoxin B subunit protein (LCTBA) given together with a recently developed enterotoxin-derived adjuvant, dmLT. A dry-powder vaccine formulation should be advantageous especially for use in low-income countries. Here we describe a method to produce a dry-powder formulation by freeze-drying of the vaccine using inulin as stabilizer. Although not completely preventing aggregation of bacteria during freeze-drying, the stabilizer provided both improved overall bacterial morphology and almost complete recovery of the CF and B subunit antigens. Most importantly, oral-intragastric immunization of mice with the freeze-dried vaccine together with dmLT adjuvant elicited strong intestinal mucosal and serum antibody responses against all vaccine antigens, which were comparable to those achieved with the liquid vaccine. Our results indicate the feasibility to use freeze-drying with inulin as stabilizer for preparing a dry-powder formulation of the novel ETEC vaccine with retained oral-mucosal immunogenicity compared to the liquid formulation.

Analysis and application of a neutralizing linear epitope on liable toxin B of enterotoxin Escherichia coli.

Heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) is one of the major virulence factors for causing diarrhea in piglets, and LT is a strong immunogen. Thus, LT represents an important target for development of vaccines and diagnostic tests. In this study, bioinformatic tools were used to predict six antigenic B cell epitopes in the B subunit of LT protein (LTB) of ETEC strains. Then, seven antigenic B cell epitopes of LTB were identified by polyclonal antisera (polyclonal antibody (PAb)) using a set of LTB-derived peptides expressed as maltose-binding protein (MBP) fusion protein. In addition, one LTB-specific monoclonal antibody (MAb) was generated and defined its corresponding epitope as mentioned above. This MAb was able to specifically bind with native LT toxin and has no cross-reaction with LT-II (type II heat-labile enterotoxin), Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), STa (heat-stable enterotoxin I), and STb (heat-stable enterotoxin II) toxins. Further, this MAb was able to interrupt LT toxin specific binding to GM1 receptor, indicating that the corresponding epitope is the specific binding region to GM1 receptor. Moreover, in vitro and in vivo assay showed that the MAb was able to neutralize the native LT toxin. Diarrheal suckling pigs challenged with LT-positive ETEC strain recovered when an enema with this purified MAb was administered. This study will provide the foundation for further studies about the interaction between LT toxin and GM1 receptor and about the developing of epitope-based vaccines and specific therapeutic agent.

Exploring effects of a natural combination medicine on exercise-induced inflammatory immune response: A double-blind RCT.

Traumeel (Tr14) is a natural, combination drug, which has been shown to modulate inflammation at the cytokine level. This study aimed to investigate potential effects of Tr14 on the exercise-induced immune response. In a double-blind, randomized, controlled trial, healthy, untrained male subjects received either Tr14 (n = 40) or placebo (n = 40) for 24 h after a strenuous experimental exercise trial on a bicycle (60 min at 80%VO2 max). A range of antigen-stimulated cytokines (in vitro), white blood cell count, lymphocyte activation and apoptosis markers, and indicators of muscle damage were assessed up to 24 h following exercise. The area under the curve with respect to the increase (AUCI ) was compared between both groups. The Tr14 group showed a reduced exercise-induced leukocytosis and neutrocytosis (P < 0.01 for both), a higher AUCI score of antigen-stimulated IL-1ß and IL-1α (absolute and per monocyte, all P < 0.05), a lower AUCI score of antigen-stimulated GM-CSF (P < 0.05) and by trend a lower AUCI score of antigen-stimulated IL-2 and IL-4 as well as a higher AUCI score of antigen-stimulated IL-6 (all P < 0.1). Tr14 might promote differentiated effects on the exercise-induced immune response by (a) decreasing the inflammatory response of the innate immune system; and (b) augmenting the pro-inflammatory cytokine response.

Effect of maternal supplementation with seaweed extracts on growth performance and aspects of gastrointestinal health of newly weaned piglets after challenge with enterotoxigenic Escherichia coli K88.

In the present study, a 2 × 2 factorial arrangement was conducted to investigate the effect of maternal supplementation with seaweed extracts ( - SWE v. +SWE, n 20) from day 83 of gestation until weaning (day 28) on post-weaning (PW) growth performance, faecal score, faecal enterotoxigenic Escherichia coli (ETEC) toxin quantification, intestinal histology and cytokine mRNA of unchallenged and ETEC-challenged pigs. Pigs were ETEC challenged on day 9 PW. There was a maternal treatment × challenge (SWE × ETEC) interaction effect on growth performance and faecal score (P< 0.05). Pigs from SWE-supplemented sows and ETEC-challenged (SE) had higher average daily gain (ADG) during 0-13 d PW and reduced faecal score during 0-72 h post-challenge than those from basal-fed sows and ETEC-challenged (BE) (P< 0.05). However, there was no difference between unchallenged pigs from the SWE-supplemented sows (SC) and basal-fed sows (BC) (P>0.10). Pigs from the SWE-supplemented sows had reduced heat-labile enterotoxin gene copy numbers than those from the basal-fed sows (P< 0.05). Maternal SWE supplementation increased the villus height in the ileum of pigs (P< 0.05). There was a SWE × ETEC interaction effect (P< 0.05) on IL-6 mRNA and a SWE × gastrointestinal (GI) region interaction effect (P< 0.05) on transforming growth factor-ß1 (TGF-ß1) and TNF-α mRNA. IL-6 mRNA was down-regulated in SC pigs than BC pigs (P< 0.05). However, there was no difference in IL-6 mRNA between SE and BE pigs. The mRNA of TGF-ß1 and TNF-α was down-regulated in the colon of pigs from the SWE-supplemented sows compared with those from the basal-fed sows (P< 0.05). However, there was no difference in TGF-ß1 and TNF-α mRNA in the ileum between the pigs from the SWE-supplemented sows and basal-fed sows. In conclusion, maternal SWE supplementation improves ADG and the aspects of GI health of weaned pigs following an ETEC challenge.

Exogenous phosphatidylcholine supplementation improves intestinal barrier defense against Clostridium difficile toxin.

BACKGROUND: The incidence and severity of Clostridium difficile colitis have increased dramatically in the last decade. Disease severity is related to C. difficile virulence factors, including toxins A and B, as well as the patient's immune status. The intestinal mucus is an important component of innate barrier function in the intestine. Phosphatidylcholine (PC) is a key constituent of the intestinal mucus barrier, and exogenous PC administration has had therapeutic efficacy in patients with ulcerative colitis. We studied the protective function of exogenous PC on C. difficile toxin effects on the intestinal barrier in vitro. METHODS: Mucus-producing (HT29-MTX strain) and non-mucus-producing (HT29 strain) intestinal epithelial monolayers were cocultured with PC and C. difficile toxin A added to the apical media. Basal chamber culture supernatants were subsequently obtained, and tumor necrosis factor and interleukin 6 were quantitated by enzyme-linked immunosorbent assay. In other experiments, HT29 toxin A uptake, intestinal monolayer permeability, necrosis, and actin microfilament disruption were determined. RESULTS: There was a threefold to fourfold decrease in tumor necrosis factor and interleukin 6 levels and similar decreases in toxin A uptake and permeability changes in intestinal epithelial cells with mucus or PC versus control. Intestinal epithelial cell necrosis was reduced by more than 50% with either mucus or PC versus control. The integrity of HT29 cell cytoskeleton was demonstrated by both the mucus layer of the HT29-MTX strain and by exogenous PC administration by phalloidin staining of actin microfilaments. CONCLUSION: PC supplementation was effective in improving intestinal barrier defense against C. difficile toxin A challenge. PC administration may be a useful therapeutic adjunct in severe cases of C. difficile colitis or in patients who do not improve with conventional treatment.

A legume product fermented by Saccharomyces cerevisiae modulates cutaneous atopic dermatitis-like inflammation in mice.

BACKGROUND: Isoflavone-containing soy products modulate allergic inflammation in mice. In our previously study, IFN-γ and IL-10 production increased in mice fed with Saccharomyces cerevisiae legume fermented product (SCLFP), demonstrating that SCLFP had immunomodulatory activity. In this study, we tested the anti-inflammatory effects of SCLFP in a mouse model of cutaneous atopic dermatitis inflammation induced by epicutaneous sensitization. METHODS: Epicutaneous exposure to protein allergens plus Staphylococcal enterotoxin B induced a T helper (Th)-2-dominant immune response as well as cutaneous atopic dermatitis-like inflammation in BALB/c mice. The thickness of the skin epithelium, eosinophil migration, and T helper responses were determined in patched skin and draining lymph nodes of mice fed with and without SCLFP. RESULTS: Epicutaneous exposure to protein allergens plus Staphylococcal enterotoxin B induced a T helper (Th)-2-dominant immune response as well as cutaneous atopic dermatitis-like inflammation in BALB/c mice. SCLFP feeding attenuated this cutaneous Th2 response, as evidenced by decreased thickening of the epidermis, less eosinophil infiltration, and lower levels of IL-5, IL-13, and CXCL11 expression compared to controls. Oral administration of SCLFP also modulated Th1 responses in draining lymph nodes, with lower levels of IFN-γ, IL-4, and IL-17 expression. CONCLUSION: Oral intake of SCLFP modulated the induced Th2 inflammatory responses in skin and might have potential applications for the prevention and treatment of atopic dermatitis.

Hyperimmune bovine colostrum for treatment of GI infections: a review and update on Clostridium difficile.

Hyperimmune bovine colostrum (HBC), produced by vaccination of a cow during gestation, is rich in targeted immunoglobulins, and can be used to treat a variety of diseases. The published history of HBC use for treating gastrointestinal infections in humans has developed over the past several decades and demonstrates the promise of this type of therapeutic for GI infectious disease. HBC, or purified derivative products, have been used successfully for treatment or prevention of cryptosporidiosis, shigellosis, rotavirus, enterotoxigenic E. coli, and C. difficile infection (CDI). Given the positive results of previous studies using HBC for treatment of CDI, we have produced HBC with antibodies against the two most important virulence factors of C. difficile, TcdA and TcdB, using a novel recombinant vaccine. Our preliminary results demonstrate efficacy of the HBC product for treatment of CDI in the gnotobiotic piglet model, and warrant more thorough investigation. HBC may provide an effective treatment alternative to antibiotics, which can spare the normal gut microflora, and reduce rates of recurrence and antibiotic resistance.

Immunisation of mares with binding domains of toxins A and B of Clostridium difficile elicits serum and colostral antibodies that block toxin binding.

REASONS FOR PERFORMING STUDY: Enterocolitis caused by Clostridium difficile (C. difficile) is a serious, sometimes fatal, disease of neonatal foals and older horses. Toxins A and B (TcdA and B) produced by C. difficile are important virulence factors. Immunisation of mares with receptor binding domains of toxins may prevent or reduce the severity of C. difficile colitis in foals. OBJECTIVES: To determine whether antibodies generated in the pregnant mare to the binding regions of TcdA and B will neutralise TcdA and B toxicity. METHODS: Sequences encoding the binding domains of each toxin were isolated by PCR amplification from C. difficile JF09, a foal isolate, and cloned and expressed into pET15b. Thirteen mares were immunised twice 2 weeks apart with 200 µg of each recombinant protein with Quil A 2 months prior to foaling. Antibodies were assayed in the sera and colostrum by ELISA and for ability to block the cytopathic activity of each of toxin for equine endothelial cells. RESULTS: All mares produced strong serum antibody responses to the binding domain of each toxin. A high level of toxin-specific antibodies was also detected in colostrum and in most foal sera 2 days after suckling. Diluted sera and colostrum premixed with either TcdA or B had no effect on the morphology of equine endothelial cells. Application of the same concentration of toxins alone or premixed with nonimmune mare/foal serum or colostrum led to an unambiguous cytopathic effect that ranged from complete degradation to varying degrees of cell rounding. CONCLUSIONS: Immunisation of pregnant mares with recombinant binding domains of TcdA and B of C. difficile resulted in the production of specific antibodies in serum and colostrum that blocked the cytopathic activity of toxins. POTENTIAL RELEVANCE: Results of studies support the feasibility of a prepartum vaccine against C. difficile enterocolitis in foals.

Specific IgE against Staphylococcus aureus enterotoxins: an independent risk factor for asthma.

BACKGROUND: The role of IgE in patients with severe asthma is not fully understood. OBJECTIVE: We sought to investigate whether IgE to Staphylococcus aureus enterotoxins might be relevant to disease severity in adult asthmatic patients. METHODS: Specific IgE antibody concentrations in serum against enterotoxins, grass pollen (GP), and house dust mite allergens and total IgE levels were measured in adult cohorts of 69 control subjects, 152 patients with nonsevere asthma, and 166 patients with severe asthma. Severe asthma was defined as inadequately controlled disease despite high-dose inhaled corticosteroids plus at least 2 other controller therapies, including oral steroids. RESULTS: Enterotoxin IgE positivity was significantly greater in patients with severe asthma (59.6%) than in healthy control subjects (13%, P< .001). Twenty-one percent of patients with severe asthma with enterotoxin IgE were considered nonatopic. Logistic regression analyses demonstrated significantly increased risks for enterotoxin IgE-positive subjects to have any asthma (OR, 7.25; 95% CI, 2.7-19.1) or severe asthma (OR, 11.09; 95% CI, 4.1-29.6) versus enterotoxin IgE-negative subjects. The presence of GP or house dust mite IgE antibodies was not associated with either significantly increased risk for asthma or severity. Oral steroid use and hospitalizations were significantly increased in patients with enterotoxin IgE and nonatopic asthma. GP IgE was associated with a higher FEV(1) percent predicted value, and enterotoxin IgE was associated with a lower FEV(1) percent predicted value. CONCLUSIONS: Staphylococcal enterotoxin IgE antibodies, but not IgE against inhalant allergens, are risk factors for asthma severity. We hypothesize that the presence of enterotoxin IgE in serum indicates the involvement of staphylococcal superantigens in the pathophysiology of patients with severe asthma.

Expression and immunogenicity of enterotoxigenic Escherichia coli heat-labile toxin B subunit in transgenic rice callus.

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.