Expression of the Nontypeable Haemophilus influenzae Type IV Pilus Is Stimulated by Coculture with Host Respiratory Tract Epithelial Cells.

The type IV pilus (Tfp) of nontypeable Haemophilus influenzae (NTHI) mediates adherence, colonization, motility, and biofilm formation, and the major protein subunit, PilA, is a promising vaccine candidate. Thus, it is crucial to understand how Tfp expression is regulated within the microenvironments of the human nasopharynx, which NTHI colonizes asymptomatically, and the more distal regions of the respiratory tract where NTHI-induced diseases occur. Here, we examined the effects of coculture of NTHI with human airway epithelial cells and heme availability on Tfp expression at temperatures typical of the human nasopharynx (34°C) or warmer anatomical sites during infection (37°C). Tfp expression was estimated by pilA promoter activity, pilA gene expression, and relative abundances of PilA and pilin protein. The results revealed that at both temperatures, NTHI cocultured with airway epithelial cells demonstrated significantly greater expression of pilA, PilA/pilin protein, and likely, fully assembled Tfp than NTHI cultured on an abiotic surface. Because NTHI is a heme auxotroph, we hypothesized that availability of heme from host cells might be a signal for Tfp expression. Thereby, we cultured NTHI in iron-limited medium, and we observed that supplementation with heme significantly increased pilA promoter activity. Collectively, our data suggested that NTHI Tfp expression was stimulated by soluble factor(s) released by epithelial cells, which are present in all microenvironments of the respiratory tract. The expression of this target antigen under conditions that mimic the human airway strongly supports the rationale for the use of PilA as a vaccine immunogen to prevent NTHI-induced diseases of the respiratory tract.

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.

Prophylactic Efficacy of Hyperimmune Bovine Colostral Antiadhesin Antibodies Against Enterotoxigenic Escherichia coli Diarrhea: A Randomized, Double-Blind, Placebo-Controlled, Phase 1 Trial.

Background: Tip-localized adhesive proteins of bacterial fimbriae from diverse pathogens confer protection in animal models, but efficacy in humans has not been reported. Enterotoxigenic Escherichia coli (ETEC) commonly elaborate colonization factors comprising a minor tip adhesin and major stalk-forming subunit. We assessed the efficacy of antiadhesin bovine colostral IgG (bIgG) antibodies against ETEC challenge in volunteers. Methods: Adults were randomly assigned (1:1:1) to take oral hyperimmune bIgG raised against CFA/I minor pilin subunit (CfaE) tip adhesin or colonization factor I (CFA/I) fimbraie (positive control) or placebo. Two days before challenge, volunteers began a thrice-daily, 7-day course of investigational product administered in sodium bicarbonate 15 minutes after each meal. On day 3, subjects drank 1 × 109 colony-forming units of colonization factor I (CFA/I)-ETEC strain H10407 with buffer. The primary efficacy endpoint was diarrhea within 120 hours of challenge. Results: After enrollment and randomization, 31 volunteers received product, underwent ETEC challenge, and were included in the per protocol efficacy analysis. Nine of 11 placebos developed diarrhea, 7 experiencing moderate to severe disease. Protective efficacy of 63% (P = .03) and 88% (P = .002) was observed in the antiadhesin bIgG and positive control groups, respectively. Conclusions: Oral administration of anti-CFA/I minor pilin subunit (CfaE) antibodies conferred significant protection against ETEC, providing the first clinical evidence that fimbrial tip adhesins function as protective antigens.

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.

Development of a novel live vaccine delivering enterotoxigenic Escherichia coli fimbrial antigens to prevent post-weaning diarrhea in piglets.

The efficacy of a novel, live delivery vaccine was examined for protection against post-weaning diarrhea in pigs. An expression/secretion plasmid harboring genes encoding enterotoxigenic Escherichia coli K88ab, K88ac, FedA and FedF fimbriae was constructed and harbored in an attenuated Salmonella, which was used as the vaccine candidate. Groups A (n=3) and B (n=3) sows were orally immunized with the candidate vaccine and PBS as a control, respectively, at 8 and 11 weeks of pregnancy. All group piglets were challenged with two challenge strains at 5-week-old. All immunized sows had significantly increased IgG and IgA levels in both serum and colostrum to individual adhesins compared to the control (p ≤ 0.05). Immune response in Group A piglets were significantly increased (p ≤ 0.05). Furthermore, no clinical signs were observed in Group A piglets after the challenge and no challenge strains were detected in rectal swabs, while diarrhea was observed in 47.8% control piglets and challenge strains were isolated from all the diarrheic piglets. These results show that immune response of sucking piglets can maintain at higher levels through the milk of the immunized sows and vaccination of sows with the candidate may protect colibacillosis in weaned piglets.

Pseudomonas aeruginosa: mannose sensitive hemagglutinin inhibits the growth of human hepatocarcinoma cells via mannose-mediated apoptosis.

A vaccine derived from the outer membrane proteins of the Gram-negative bacterium Pseudomonas aeruginosa has been shown to have immune modulatory properties. An inactivated mutant strain of P. aeruginosa with mannose sensitive hemagglutinin fimbria (PA-MSHA) has been used for adjuvant therapy for malignant cancer. In this study, the growth of human hepatocellular carcinoma Hep G2 and BEL-7402 cells is inhibited by PA-MSHA, but not by mannose-cleaved PA-MSHA. PA-MSHA-treated cells arrested in the S phase of the cell cycle and underwent apoptosis. We hypothesize that apoptosis induced by treatment of Hep G2 and BEL-7402 cells with PA-MSHA is mediated by the mannose residues of PA-MSHA and is propagated through the extrinsic apoptosis pathway directly through caspase-8. These data provide mechanistic details for the potential application of PA-MSHA-based treatment of hepatocellular carcinoma.

Immune response, ciprofloxacin activity, and gender differences after human experimental challenge by two strains of enterotoxigenic Escherichia coli.

In order to test vaccines against enterotoxigenic Escherichia coli (ETEC)-induced diarrhea, challenge models are needed. In this study we compared clinical and immunological responses after North American volunteers were orally challenged by two ETEC strains. Groups of approximately eight volunteers received 10(9) or 10(10) CFU of E. coli B7A (LT+ ST+ CS6+) or 10(8) or 10(9) CFU of E. coli H10407 (LT+ ST+ CFA/I+). About 75% of the volunteers developed diarrhea after challenge with 10(10) CFU B7A or either dose of H10407. B7A had a shorter incubation period than H10407 (P = 0.001) and caused milder illness; the mean diarrheal output after H10407 challenge was nearly twice that after B7A challenge (P = 0.01). Females had more abdominal complaints, and males had a higher incidence of fever. Ciprofloxacin generally diminished or stopped symptoms and shedding by the second day of antibiotic treatment, but four subjects shed for one to four additional days. The immune responses to colonization factors CS6 and colonization factor antigen I (CFA/I) and to heat-labile toxin (LT) were measured. The responses to CFA/I were the most robust responses; all volunteers who received H10407 had serum immunoglobulin A (IgA) and IgG responses, and all but one volunteer had antibody-secreting cell (ASC) responses. One-half the volunteers who received B7A had an ASC response to CS6, and about one-third had serum IgA or IgG responses. Despite the differences in clinical illness and immune responses to colonization factors, the immune responses to LT were similar in all groups and were intermediate between the CFA/I and CS6 responses. These results provide standards for immune responses after ETEC vaccination.

Human colostrum contains IgA antibodies reactive to colonization factors I and II of enterotoxigenic Escherichia coli.

Diarrhea is an important cause of morbidity and mortality amongst infants of low socio-economic levels in developing countries and in travelers who visit such areas. Enterotoxigenic E. coli strains express two sets of virulence-associated factors: enterotoxins (heat-stable toxins or heat-labile toxins) and colonization factors. Studies have shown that breast-feeding protects infants against infectious diseases, such as diarrhea, as it presents a great variety of immunological components. The aim of this study was to analyze the reactivity of immunoglobulin A from human colostrum to colonization factor antigens I and II. The colostrum ability in preventing enterotoxigenic E. coli adhesion to Caco-2 cells was also evaluated. Colostrum samples were collected from 32 healthy women, and a human colostrum pool was prepared. Enterotoxigenic E. coli strains expressing colonization factor antigens I and II were utilized. The colostrum pool and individual samples showed variable antienterotoxigenic E. coli immunoglobulin A titers, that were reactive with colonization factor antigen I and CS1/CS3 (colonization factor antigen II). The human colostrum pool and individual samples inhibited enterotoxigenic E. coli colonization factor antigen I and II adhesion to Caco-2 cells, at variable levels, and this ability was a result of immunoglobulin A antibodies reactive to these colonization factors. The immunoglobulin A-depleted pool lost this inhibitory ability. As bacterial adhesion is the initial mechanism of enterotoxigenic E. coli infection, breast-feeding could protect the offspring against diarrhea caused by this agent.

Intranasal immunization with a recombinant truncated FimH adhesin adjuvanted with CpG oligodeoxynucleotides protects mice against uropathogenic Escherichia coli challenge.

In this work, we assessed the efficacy of an experimental intranasal vaccine against urinary-tract infections. The vaccine contained a recombinant truncated FimH (rFimHt) adhesin plus CpG oligodeoxynucleotides. The efficacy of the vaccine was compared with that of an intramuscular vaccine that was formulated with the same immunogen plus Freund's adjuvant. Our results show that serum immunoglobulin G titers of vaccinated animals were similarly enhanced in both cases. However, the intranasal vaccine elicited higher vaginal-wash-specific immunoglobulin A titers against rFimHt than the intramuscular route. Both vaccines reduced the in vivo colonization of the bladder by uropathogenic Escherichia coli more than 100-fold in a murine cystitis model. Our results indicate that a recombinant truncated FimH adhesin plus CpG oligodeoxynucleotides is a suitable immunogenic combination that can contribute to the development of a highly efficacious urinary tract infection vaccine.

Protection against neonatal Escherichia coli diarrhoea in pigs by vaccination of sows with a new vaccine that contains purified enterotoxic E. coli virulence factors F4ac, F4ab, F5 and F6 fimbrial antigens and heat-labile E. coli enterotoxin (LT) toxoid.

The efficacy of a new vaccine against neonatal Escherichia coli diarrhoea in piglets containing purified F4ab, F4ac, F5 and F6 fimbriae and detoxified heat-labile toxin (LT) was tested in challenge experiments by the method described by the European Pharmacopoeia (3rd edn, EDQM, Council of Europe, Strasbourg, France). A group of 11 young sows from a herd without E. coli problems was vaccinated 6-8 and 2-4 weeks prior to expected farrowing and another group of nine young sows were non-vaccinated controls. Escherichia coli antibody titres were determined in serum samples taken from the sows before first vaccination and before farrowing and in colostrum samples. The newborn piglets were allowed to suckle colostrum from their mother immediately after birth. The piglets were marked with individually numbered ear tags. Approximately 12 h after birth, 118 piglets from vaccinated sows and 79 piglets from non-vaccinated control sows were challenged by oral instillation of 5 ml of a freshly prepared culture of one of the challenge strains [O8:K87:F4ab (LT+) or O149:K91:F4ac (LT+) or O9:K30:F5 or O9:K103:F6 respectively]. The challenge cultures contained as a mean 6.8x10(9) CFU/ml. After challenge the piglets were observed for 7 days and mortality and morbidity were recorded. Vaccinated sows developed significant levels of antibody titres in colostrum and serum. Control sows stayed at a low/seronegative level. The protective efficacy was excellent because 66.7-87.5% of the piglets from vaccinated sows remained without clinical signs after challenge. Only 0.0-28.0% of the piglets from non-vaccinated sows remained healthy and more than 47.1% of the piglets in this group died after challenge. It is concluded that the new vaccine is very effective in protection of piglets against neonatal E. coli diarrhoea.

Partially assembled K99 fimbriae are required for protection.

Antibodies to K99 fimbriae afford protection to F5+ bovine enterotoxigenic Escherichia coli (ETEC). Previous studies show that murine dams immunized with Salmonella vaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion of fanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion of fanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion of fanGH is sufficient to confer protection.

Prevalent transfer of human colostral IgA antibody activity for the enteropathogenic Escherichia coli bundle-forming pilus structural repeating subunit A in neonates.

The frequency of IgA antibody activity to the structural protein subunit BfpA of the enteropathogenic Escherichia coli bundle-forming pilus was determined in 40 mother-infant pairs by immunoblot analysis using affinity purified recombinant BfpA to monitor for IgA in maternal colostrum and in feces of the neonates. Fecal samples were collected from exclusively breastfed term infants < 24-h after the first breastmilk feeding and colostral samples from their mothers. Infants were monitored prospectively with monthly visits to ascertain dietary practices and diarrheal illnesses. The percentage of colostral anti-BfpA IgA positive patients that were also coproantibody positive was 67.5%. The median duration of lactation was 108 days and the incidence of infantile diarrheal disease was 7.5%. Thus, colostral anti-BfpA IgA antibody activity survives passage through the gut of breastfed neonates, persisting in their feces. It is suggested that oral passive immunotherapy may be used to prevent and/or treat typical EPEC infection during infancy.

Oral immunization of sows: anti-K88 antibodies in serum and milk of the sow and in serum of the piglets.

Pregnant sows were immunized by oral application of live E. coli. The effect of immunization was demonstrated by measuring the titers of anti-K88 antibodies in sow serum, colostrum and milk as well as in piglet sera using enzyme-linked immunosorbent assays. The isotype involved in anti-K88 reactivity was found to be IgA. By comparing IgA-titers in colostrum and milk, the local production of this Ig-class in the mammary gland is suggested.