Generation of high-titer of neutralizing polyclonal antibodies against heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli

In this study, polyclonal antibodies with high titer and avidity to native heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC) have been generated and evaluated for their neutralizing effect in STa-induced enterotoxic animal model. Native STa was purified to homogeneity and coupled to modified bovine serum albumin (MBSA) using dimethylformamide (DMF)-based conjugation protocol. STa conjugate was used for immunization of female New Zealand white rabbits. The humoral immune response of the rabbits against native STa was monitored and evaluated for its antibody binding and neutralization capacity by ELISA and suckling mouse assay, respectively. After three subsequent boosts by STa conjugate, the animals were capable of eliciting high levels of STa-antibody binding titer (10(6)) and STa-neutralizing antibody capacity (3×10(4) mouse units of STa/ml serum). STa antibody maturation (avidity) was improved dramatically after multiple boosters with the STa conjugate. Comparison of the avidity of STa antibodies demonstrated that the strength in the STa antibody avidity developed in time corresponding to the development of the STa-neutralizing and binding titers. High avid STa antibodies (48.21% avidity index) were demonstrated 24 weeks post immunization (PI). However, differences in the onset of STa antibody production were noticed among animals and may need further investigation.     

A totally synthetic lipopeptide-based self-adjuvanting vaccine induces neutralizing antibodies against heat-stable enterotoxin from enterotoxigenic Escherichia coli

ST-based lipopeptide vaccine candidates were constructed in which ST was chemically synthesized and folded into the correct conformation prior to ligation to a module containing a T-helper cell epitope (T(H)) and the Toll-like receptor 2 (TLR2) agonist, S-[2,3-bis(palmitoyloxy)propyl]cysteine (P2C). Two different chemistries, thioether-based and oxime-based, were then used to ligate ST to the lipidated T(H) epitope. The enterotoxic activity of synthetic ST and the ST-based lipopeptide vaccines was determined in mice followed by an evaluation of immunological efficacy. The importance of the fine detail in chemical composition used in vaccine design was demonstrated by the findings that (i) the oxime-based vaccine exhibited little or no toxicity but the thioether-based vaccine, exhibited residual toxicity in suckling mice, (ii) although each of the synthetic vaccines generated specific anti-ST antibodies, it was the low titer antibodies induced by the oxime-based vaccine that demonstrated better neutralizing activity suggesting that the chemical linkage also affects the specificity of antibodies, (iii) the geometric arrangement of ST within a vaccine can profoundly affect the specificity and biological function of the antibodies that are elicited, and (iv) the lipopeptide-based ST vaccine candidate assembled using oxime chemistry induced a better neutralizing antibody response to ST when administered by the mucosal (intranasal) route.     

Virus-like particle-display of the enterotoxigenic Escherichia coli heat-stable toxoid STh-A14T elicits neutralizing antibodies in mice

Enterotoxigenic Escherichia coli (ETEC) causes diarrhoea by secreting enterotoxins into the small intestine. Human ETEC strains may secrete any combination of three enterotoxins: the heat-labile toxin (LT) and the heat-stable toxins (ST), of which there are two variants, called human ST (STh) and porcine ST (STp). Strains expressing STh, either alone or in combination with LT and/or STp, are among the four most important diarrhoea-causing pathogens affecting children in low- and middle-income countries. ST is therefore an attractive target for ETEC vaccine development. To produce a safe ST-based vaccine, several challenges must be solved. ST must be rendered immunogenic and non-toxic, and antibodies elicited by an ST vaccine should neutralize ST but not cross-react with the endogenous ligands uroguanylin and guanylin. Virus-like particles (VLPs) tend to be highly immunogenic and are increasingly being used as carriers for presenting heterologous antigens in new vaccines. In this study, we have coupled native STh and the STh-A14T toxoid to the coat protein of Acinetobacter phage AP205 by using the SpyCatcher system and immunized mice with these VLPs without the use of adjuvants. We found that both STs were efficiently coupled to the VLP, that both the STh and STh-A14T VLPs were immunogenic in mice, and that the resulting serum antibodies could completely neutralize the toxic activities of native STh. The serum antibodies showed a high degree of immunological cross-reaction to STp, while there was little or no unwanted cross-reaction to uroguanylin and guanylin. Moreover, compared to native STh, the STh-A14T mutation did not seem to negatively impact the immunogenicity of the construct or the neutralizing ability of the resulting sera. Taken together, these findings demonstrate that VLPs are suitable carriers for making STs immunogenic, and that the STh-A14T-coupled AP205 VLP represents a promising ETEC vaccine candidate.     

Passive antibodies derived from intramuscularly immunized toxoid fusion 3xSTaN12S-dmLT protect against STa+ enterotoxigenic Escherichia coli (ETEC) diarrhea in a pig model

Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of children’s diarrhea and travelers’ diarrhea. Developing effective vaccines against ETEC associated diarrhea becomes a top priority. ETEC heat-labile toxin (LT) and heat-stable toxin (STa) toxoid fusion 3xSTa_N12S-dmLT was demonstrated recently to induce neutralizing antitoxin antibodies in intraperitoneally or subcutaneously immunized mice. However, whether antibodies derived from this toxoid fusion are protective against ETEC diarrhea has not been examined. In this study, we intramuscularly immunized pregnant gilts with toxoid fusion 3xSTa_N12S-dmLT, challenged suckling piglets with a STa-positive ETEC strain, and assessed protective efficacy of passive acquire antitoxin antibodies against ETEC diarrhea. Data showed all three immunized gilts developed anti-STa IgG and IgA antibodies, and piglets born to the immunized dams acquired anti-STa and anti-LT antibodies. When challenged with a STa+ ETEC strain, none of the piglets born to the immunized dams developed watery diarrhea, with 20 piglets remained normal and the other 8 piglets developed mild diarrhea indicated with stained butt. In contrast, the control dams and born piglets had no anti-STa or anti-LT antibodies detected, and 26 out 32 piglets developed watery diarrhea after challenge of the STa+ ETEC strain. These results indicated that passive acquired anti-STa antibodies are protective against ETEC diarrhea, and suggested potential application of toxoid fusion 3xSTa_N12S-dmLT in ETEC vaccine development.     

Microencapsulated enterotoxigenic Escherichia coli and detached fimbriae for peroral vaccination of pigs

The feasibility of peroral immunisation with microencapsulated Escherichia coli and detached fimbriae to prevent enterotoxigenic E. coli infections in pigs was examined. For this E. coli and fimbriae were microencapsulated into poly(lactide-co-glycolide) microspheres by spray-drying. Various microsphere formulations designed to deliver priming and booster doses were fed to new-born and weaned pigs. The pigs were challenged 19 days after the booster dose by peroral administration of an infective dose of the homologous E. coli. Serum IgA antibody titres and excretion of challenge E. coli, as indicators for colonisation, were determined. The data showed that no significant serum antibodies were induced, and E. coli colonisation was not reduced by the peroral administration of the various antigen-loaded microspheres. These results are in contradiction to some of the previously published experiments typically in rats or rabbits, where model antigens or unpractical immunisation procedures have frequently been used.     

Progress towards a vaccine against enterotoxigenic Escherichia coli

A variety of approaches are being investigated in the development of a vaccine against enterotoxigenic Escherichia coli (ETEC). These approaches include purified fimbriae vaccines, toxoid vaccines, live attenuated E. coli vaccine strains and ETEC antigens expressed in carrier organisms. Studies of the pathogenesis and immune response to ETEC indicate that development of a vaccine against human ETEC is a realistic goal but considerable work remains before this goal is realized.     

Chinese hamster ovary cells produce an enzyme that Nicks heat-labile enterotoxin from enterotoxigenic Escherichia coli

Chinese hamster ovary (CHO) cells were examined for production of an enzyme that nicked the polypeptide chain of the heat-labile enterotoxin from enterotoxigenic Escherichia coli between the A1 and A2 fragments of its A subunit. Serum-free culture medium prepared each day after CHO cell inoculation was concentrated 100 times and its proteolytic activity for formation of the A1 fragment was examined by Western blotting with anti-LT A antibody. The A subunit was detected in culture medium on day 6 after cell inoculation, although not in media on day 1 or 3, indicating that CHO cells produced a nicking enzyme. This nicking enzyme had an optimal pH of about 7.5 and an apparent Mr. of 120,000, as seen by Superose 12 TM gel filtration with an FPLC system. The activity of this enzyme was strongly inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by p-chloromercuribenzoic acid, EDTA or ethyleneglycol bis (beta-aminoethylether)-N,N-N',N'-tetraacetic acid, suggesting that this enzyme was a serine protease. The activity was not stimulated by plasminogen or fibrin. These findings suggest that the nicking enzyme was different from proteases such as elastase, collagenase and plasminogen activator, which are probably also secreted by fibroblast-like CHO cells.     

DNA immunisation against the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC)

The CFA/I fimbria promotes the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes. The generation of a protective immune response requires the induction of antibodies able to block the CFA/I-mediated binding of ETEC to receptors located on the small intestine epithelium or on the surface of human red blood cells, in hemagglutination tests. An eukaryotic expression plasmid, pBLCFA, encoding the CFA/I gene under the control of the human cytomegalovirus major immediate-early promoter was constructed as a prototype DNA vaccine against ETEC. pBLCFA-tranfected BHK-21 cells secreted a peptide cross-reacting with a monoclonal antibody raised against CFA/I subunits. BALB/c mice immunized intramuscularly with one or two doses of purified pBLCFA developed CFA/I-specific serum antibodies for at least 52 weeks, composed predominantly of the IgG1 subclass. pBLCFA-induced antibodies bind mainly to epitopes exposed on the surface of intact CFA/I fimbriae and do not react with immune recessive epitopes found in other ETEC fimbra sharing amino acid homologies with CFA/I. Furthermore, pBLCFA-induced antibodies were able to block the adhesive properties of the CFA/I fimbriae, as evaluated by the ability to inhibit the hemagglutination promoted by CFA/I-expressing ETEC cells. These results suggest that secretion of CFA/I encoded by pBLCFA preserves important conformational epitopes required for the generation of protective antibodies against the adhesive properties of the CFA/I fimbriae and open new perspectives for the development of DNA vaccines against enteric bacterial pathogens.     

SARS coronavirus spike polypeptide DNA vaccine priming with recombinant spike polypeptide from Escherichia coli as booster induces high titer of neutralizing antibody against SARS coronavirus

Different forms of SARS coronavirus (SARS-CoV) spike protein-based vaccines for generation of neutralizing antibody response against SARS-CoV were compared using a mouse model. High IgG levels were detected in mice immunized with intraperitoneal (i.p.) recombinant spike polypeptide generated by Escherichia coli (S-peptide), mice primed with intramuscular (i.m.) tPA-optimize800 DNA vaccine (tPA-S-DNA) and boosted with i.p. S-peptide, mice primed with i.m. CTLA4HingeSARS800 DNA vaccine (CTLA4-S-DNA) and boosted with i.p. S-peptide, mice primed with oral live-attenuated Salmonella typhimurium (Salmonella-S-DNA-control) and boosted with i.p. S-peptide, mice primed with oral live-attenuated S. typhimurium that contained tPA-optimize800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide, and mice primed with oral live-attenuated S. typhimurium that contained CTLA4HingeSARS800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide. No statistical significant difference was observed among the Th1/Th2 index among these six groups of mice with high IgG levels. Sera of all six mice immunized with i.p. S-peptide, i.m. DNA vaccine control and oral Salmonella-S-DNA-control showed no neutralizing antibody against SARS-CoV. Sera of the mice immunized with i.m. tPA-S-DNA, i.m. CTLA4-S-DNA, oral Salmonella-S-DNA-control boosted with i.p. S-peptide, oral Salmonella-tPA-S-DNA, oral Salmonella-tPA-S-DNA boosted with i.p S-peptide, oral Salmonella-CTLA4-S-DNA and oral Salmonella-CTLA4-S-DNA boosted with i.p. S-peptide showed neutralizing antibody titers of <1:20-1:160. Sera of all the mice immunized with i.m. tPA-S-DNA boosted with i.p. S-peptide and i.m. CTLA4-S-DNA boosted with i.p. S-peptide showed neutralizing antibody titers of >or=1:1280. The present observation may have major practical value, such as immunization of civet cats, since production of recombinant proteins from E. coli is far less expensive than production of recombinant proteins using eukaryotic systems.     

Oligosaccharides from human milk block binding and activity of the Escherichia coli heat-stable enterotoxin (STa) in T84 intestinal cells

Enterotoxin-producing Escherichia coli are major causes of pediatric diarrhea in developing countries. The heat-stable enterotoxin of Escherichia coli (STa) causes diarrhea by virtue of its ability to bind to and stimulate intestinal membrane-bound guanylate cyclase, generating cyclic GMP (cGMP). Previous work showed that a fucosylated oligosaccharide fraction of human milk was able to protect suckling mice from the secretory effects of STa, but the mechanism of the protection could not be determined. Oligosaccharide fractions from human milk were tested for their ability to block the biochemical effects of STa in T84 cells, a human colon carcinoma line responsive to the toxin. Total and fucosylated oligosaccharide fractions were found to inhibit STa-stimulated guanylate cyclase activity in T84 cell membranes and cGMP production in intact T84 cells by 60-80%. In addition, the total oligosaccharide fraction and the fucosylated oligosaccharide fraction inhibited 125I-STa binding significantly (17% and 27% inhibition, respectively). These findings demonstrate the protective activity of human milk oligosaccharides against STa in a human-derived cell line and show that the biochemical step blocked by oligosaccharides is STa-mediated stimulation of guanylate cyclase. This represents a novel mechanism by which human milk oligosaccharides protect against diarrhea.     

双重实时荧光PCR检测肠产毒大肠埃希菌肠毒素基因方法的建立

目的:建立一种快速、准确、灵敏的检测肠产毒大肠埃希菌肠毒素基因方法。方法:于GenBank中查找ETEC肠毒素基因lt和st序列并进行比对后设计引物及TaqMan-MGB探针。对引物、探针、Mg2+、TaqDNA聚合酶及dNTPs浓度进行优化,并对方法的灵敏度、特异性、重复性进行分析。结果:建立了双重实时荧光PCR法检测ETEC肠毒素基因lt和st方法。结论:建立了一种灵敏、特异的检测ETEC肠毒素基因的方法。该法在疾病控制相关和临床实验室中具有广泛的实际应用价值。     

Towards a live oral vaccine against enterotoxigenic Escherichia coli of swine

A live oral vaccine has been developed against scouring induced in piglets by enterotoxigenic Escherichia coli (ETEC). An attenuated strain of Salmonella typhimurium, G30, has been used as a vector for plasmids encoding the production of the fimbrial colonization factors of porcine ETEC. Initial studies with clones expressing K88 or K99 fimbriae have shown them to be well tolerated when administered orally in very high doses. The clones elicited serum, colostral and milk antibodies to the fimbrial antigens, and a challenge trial indicated that such responses were sufficient to ensure the passive transfer of protective immunity to suckling piglets. The possible advantages of this approach are discussed.     

Safety and immunogenicity of intramuscularly administered CS6 subunit vaccine with a modified heat-labile enterotoxin from enterotoxigenic Escherichia coli

IntroductionEnterotoxigenic Escherichia coli (ETEC) is a common cause of infectious diarrhoea and a leading cause of morbidity and mortality in children living in resource-limited settings. It is also the leading cause of travellers’ diarrhoea among civilian and military travellers. Its dual importance in global public health and travel medicine highlights the need for an effective vaccine. ETEC express colonization factors (CFs) that mediate adherence to the small intestine. An epidemiologically prevalent CF is coli surface antigen 6 (CS6). We assessed the safety and immunogenicity of a CS6-targeted candidate vaccine, CssBA, co-administered intramuscularly with the double-mutant heat-labile enterotoxin, dmLT [LT(R192G/L211A)].MethodsThis was an open-label trial. Fifty subjects received three intramuscular injections (Days 1, 22 and 43) of CssBA alone (5 µg), dmLT alone (0.1 µg) or CssBA (5, 15, 45 µg) + dmLT (0.1 and 0.5 µg). Subjects were actively monitored for adverse events for 28 days following the third vaccination. Antibody responses (IgG and IgA) were characterized in the serum and from lymphocyte supernatants (ALS) to CS6 and the native ETEC heat labile enterotoxin, LT.ResultsAcross all dose cohorts, the vaccine was safe and well-tolerated with no vaccine-related severe or serious adverse events. Among vaccine-related adverse events, a majority (98%) were mild with 79% being short-lived vaccine site reactions. Robust antibody responses were induced in a dose-dependent manner with a clear dmLT adjuvant effect. Response rates in subjects receiving 45 µg CssBA and 0.5 µg dmLT ranged from 50 to 100% across assays.ConclusionThis is the first study to demonstrate the safety and immunogenicity of CssBA and/or dmLT administered intramuscularly. Co-administration of the two components induced robust immune responses to CS6 and LT, paving the way for future studies to evaluate the efficacy of this vaccine target and development of a multivalent, subunit ETEC vaccine.     

Durability of neutralizing antibodies against yellow fever virus after vaccination in healthy adults

In accordance with the World Health Organization, one dose of yellow fever vaccine may guarantee protection lifelong in healthy adults. However, relatively little information is still available from ad hoc studies. We evaluated the persistence of neutralizing antibodies, which are considered to be an immune correlate of protection, in a large number of military personnel vaccinated up to 47 years before. Overall, 322 individuals were studied. The median time from vaccination to blood collection for neutralizing antibody evaluation was 9 years, ranging from <1 to 47 years. Of the 322 participants, 319 had neutralizing antibodies (99.1 %). The highest median PRNT₅₀ value was observed in those vaccinated ≤1 year before (median PRNT₅₀ = 320). In conclusion, our study confirms on a larger scale that, in healthy adults, neutralizing antibodies may persist as long as 47 years after a single yellow fever vaccines dose.     

Status of vaccine research and development for enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of diarrhea-associated morbidity and mortality, particularly among infants and young children in developing countries. Still, the true impact on child and traveler health is likely underestimated. There are currently no licensed vaccines for ETEC, but studies indicate high public health impact, cost-effectiveness, and feasibility of immune protection through vaccination. ETEC vaccine development remains a World Health Organization priority. Traditionally, ETEC vaccine development efforts have focused on inducing antitoxin and anticolonization antigen immunity, as studies indicate that antibodies against both antigen types can contribute to protection and thus have potential for vaccines. Leading cellular vaccine candidates are ETVAX (a mixture of four inactivated strains) and ACE527 (a mixture of three live attenuated strains), both of which have been found to be safe and immunogenic in Phase 1/2 trials. ETVAX is the furthest along in development with descending-age studies already underway in Bangladesh. Other ETEC vaccine candidates based on protein subunits, toxoids (both LT and ST), or novel, more broadly conserved ETEC antigens are also under development. Of these, a protein adhesin-based subunit approach is the most advanced. Impact and economic models suggest favorable vaccine cost-effectiveness, which may help expand market interest in ETEC vaccines. Combination vaccine formulations may help improve the economic case for development and use, and better point-of-care diagnostics will help to raise awareness of the true health burden of ETEC and highlight the potential public health benefit of ETEC vaccine introduction. Better diagnostics and vaccine demand forecasting will also improve vaccine development financing and support accelerated uptake once a licensed vaccine becomes available.     

Isolation of enterotoxigenic Escherichia coli from British troops in Saudi Arabia.

Specimens from 181 patients with diarrhoea were examined by a Military General Hospital in a 3-month period during deployment of troops to Saudi Arabia in 1990/1. DNA probes for heat labile (LT) and heat stable (ST) enterotoxin genes identified enterotoxigenic Escherichia coli (ETEC) in 47 of the specimens (26%) and 49 ETEC strains were isolated. The majority (55%) belonged to a novel ETEC serotype having the O-antigen 159 and a flagellar antigen designated as a provisional new type. They produced ST and the coli surface associated antigen (CS)6. Strains of serotype O6:H16 represented 22% of the ETEC examined. They produced ST, LT and CS3 together with either CS1 or CS2. The remaining ETEC belonged to seven O:H serotypes. Overall, ST was the only enterotoxin gene identified in 73% of the ETEC and 67% of the strains expressed CS6 in the absence of other colonization antigens. Resistance to three or more antibiotics was observed in 53% of the ETEC, including most of the O159 strains.     

Development of vaccines against cholera and diarrhoea due to enterotoxigenic Escherichia coli: memorandum from a WHO meeting.

This Memorandum summarizes current knowledge on the epidemiology of cholera and diarrhoea due to enterotoxigenic Escherichia coli (ETEC) and outlines the results of recent research to develop an effective oral vaccine against cholera. The meeting reviewed current research on the protective antigens of ETEC and made a number of recommendations with the aim of stimulating further efforts towards the development of vaccines against disease caused by ETEC.     

Demonstration of enterotoxigenic Escherichia coli in diarrheic broiler chicks

An investigation was made to survey the possible presence of enterotoxigenic Escherichia coli (ETEC) in the stools of diarrheal chicks.We analyzed two outbreaks of diarrhea in broiler chicks at two independent farms in the Philippines, from which no pathogens other than Escherichia coli were found. In one outbreak at Farm # 1, all 42 isolates produced heat-labile enterotoxin (LT), with 3 of these isolates also producing heat-stable enterotoxin (ST). The 0 serotypes of 15 strains tested randomly could not be identified as any known serotype (0-antigen; 1–170). In another outbreak at Farm #2, 7 out of 52 isolates produced only LT, their subtypes being identified as O-149 or O-8, common serotypes in pig ETEC. Strains from Farm #1 did not produce any pili usually found in human ETEC. We believe this to be the first isolation of ETEC from diarrheal chicks.Corresponding author.     

Immunogenicity and protective efficacy of a single-dose live non-pathogenic Escherichia coli oral vaccine against F4-positive enterotoxigenic Escherichia coli challenge in pigs

Enterotoxigenic Escherichia coli strains expressing F4 (K88) fimbriae (F4-ETEC) are one of the most important causes of post-weaning diarrhea (PWD) in pigs. F4, a major antigen, plays an important role in the early steps of the infection. Herein, the efficacy of a live oral vaccine consisting of a non-pathogenic E. coli strain expressing F4 for protection of pigs against PWD was evaluated. Three blinded, placebo-controlled, block design, parallel-group confirmatory experiments were conducted, using an F4-ETEC PWD challenge model, each with a different vaccination-challenge interval (3, 7, and 21 days). The pigs were vaccinated via the drinking water with a single dose of the Coliprotec® F4 vaccine one day post-weaning. Efficacy was assessed by evaluating diarrhea, clinical observations, intestinal fluid accumulation, weight gain, intestinal colonization and fecal shedding of F4-ETEC. The immune response was evaluated by measuring serum and intestinal F4-specific antibodies. The administration of the vaccine resulted in a significant reduction of the incidence of moderate to severe diarrhea, ileal colonization by F4-ETEC, and fecal shedding of F4-ETEC after the heterologous challenge at 7 and 21 days post-vaccination. The 7-day onset of protection was associated with an increase of serum anti-F4 IgM whereas the 21-day duration of protection was associated with an increase of both serum anti-F4 IgM and IgA. Significant correlations between levels of serum and intestinal secretory anti-F4 antibodies were detected. Maternally derived F4-specific serum antibodies did not interfere with the vaccine efficacy. The evaluation of protection following a challenge three days after vaccination showed a reduction of the severity and the duration of diarrhea and of fecal shedding of F4-ETEC. The 7-day onset and the 21-day duration of protection induced by Coliprotec® F4 vaccine administered once in drinking water to pigs of at least 18 days of age were confirmed by protection against F4-ETEC and induction of F4-specific protective immunity.