Over-expression of major colonization factors of enterotoxigenic Escherichia coli, alone or together,on non-toxigenic E. coli bacteria

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveller's diarrhea. Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. We have recently described the development of recombinant strains over-expressing CFA/I; the most prevalent CF among human clinical ETEC isolates. Here, non-toxigenic recombinant E. coli strains over-expressing Coli surface antigen 2 (CS2), CS4, CS5, and CS6, either alone, or each in combination with CFA/I were constructed by cloning the genes required for expression and assembly of each CF into expression vectors harboring a strong promoter. Immunological assays showed that recombinant strains expressing single CFs produced those in significantly larger amounts than did corresponding naturally high producing reference strains. Recombinant strains co-expressing CFA/I together with another CF also expressed significantly larger amounts of both CFs compared with the corresponding references strains. Further, when tested in mice, oral immunization with formalin-killed recombinant bacteria co-expressing one such double-expression CF pair, CFA/I + CS2, induced specific serum IgG + IgM and fecal IgA antibody responses against both CFs exceeding the responses induced by immunizations with natural reference strains expressing CFA/I and CS2, respectively. We conclude that the described type of recombinant bacteria over-expressing major CFs of ETEC, alone or in combination, may be useful as candidate strains for use in an oral whole-cell CF-ETEC vaccine.     

Surveys of human enterotoxigenic Escherichia coli from three different geographical areas for possible colonization factors.

Enterotoxigenic Escherichia coli (ETEC) from Burma, central Africa (Rwanda and Zaire) and Peru, were screened by enzyme-linked immunoassays for the colonization factor antigens (CFAs) and putative colonization factors (PCFs): CFA/I, CFA/II, which consists of three coli surface-associated (CS) antigens, CS1, CS2 and CS3, CFA/III, CFA/IV (CS4, CS5, CS6), CS7, PCFO9, PCFO159. H4, PCFO166, and CS17. The highest proportion of ETEC with identifiable colonization factors (71%) were found in the strains from Burma, which were mainly positive for CFA/I (38%), but strains producing CFA/II (4%), CFA/IV (11%), CS7 (10%), CS17 (4%), PCFO159, H4 (2%) and PCFO166 (2%) were also found. Sixty-nine percent of the ETEC from central Africa were positive for known colonization factors. While CFA/I positive strains were important (12%), a higher number of ETEC producing CFA/IV (33%) and CS17 (24%) were found. Fifty-two percent of the Peruvian strains produced identifiable colonization factors. The largest group of strains produced antigens of the CFA/IV complex (17%), while ETEC producing CFA/II (6%), CFA/III and CS6 (2%), CS7 (6%), PCFO9 (6%), PCFO166 (8%) and CS17 (7%) were also found. These surveys show that there is a considerable variation in the proportions and types of colonization factor found in different geographical areas. From 29 to 48% of the ETEC did not possess an identifiable colonization factor. These were particularly of the LT only producing type. These results have important implications for vaccine formulation.     

Analysis of efficacy of CVD 103-HgR live oral cholera vaccine against all-cause travellers' diarrhoea in a randomised, double-blind, placebo-controlled study

Enterotoxigenic Escherichia coli (ETEC), which produces heat labile toxin (LT) and/or heat stable toxin (ST), is considered to be the most common known cause of travellers' diarrhoea (TD). Owing to the antigenic similarity between cholera toxin and LT, immunization with inactivated oral B-subunit/whole-cell cholera vaccine (BS-WC) offers short term (3 months) but significant (>67%) protection against TD caused by LT-related ETEC. Since it expresses the cholera toxin B (CTB) subunit, the live attenuated oral cholera vaccine strain CVD 103-HgR, may induce similar protection. A trial was performed to determine if CVD 103-HgR live oral cholera vaccine would provide a protective efficacy of at least 50% against TD. In addition, the protective efficacy of the vaccine against TD specifically due to LT-ETEC and LT/ST-ETEC was determined. Volunteers (n=134) travelling to Indonesia, India, Thailand or West-Africa were randomised to receive either a placebo (n=65) or the vaccine (n=69). In the placebo group, 46% reported an episode of diarrhoea, compared to 52% in the vaccine group. No significant group differences were found with regard to incidence, duration or severity of all caused TD or ETEC-associated TD. However, ETEC-associated TD occurred earlier in the placebo group (median 5 days), compared to the vaccine group (median 15 days). In conclusion, CVD 103-HgR live oral cholera vaccine failed to provide a 50% protection against TD. This study does not exclude that the vaccine may offer a short-lived protection against ETEC-associated TD. However, the power of the study was limited by the unexpected low incidence of LT-ETEC-associated diarrhoea (9% of all TD) compared to ST-associated TD (24% of all TD).     

Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae

To express high quantities of colonization factor antigen I (CFA/I) derived from enterotoxigenic Escherichia coli (ETEC) for use in ETEC vaccines, the entire CFA/I operon consisting of four genes (cfa-A, -B, -C, -E) was cloned into plasmid expression vectors that could be maintained either with or without antibiotic selection. Expression from the powerful tac promoter was under the control of the lacIq repressor present on the plasmids. Fimbriae were expressed on the surface of both a non-toxigenic E. coli K12 strain and a non-toxigenic strain of Vibrio cholerae following induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). It was found that the recombinant E. coli strains expressed up to 16-fold higher levels of CFA/I fimbriae compared to a reference strain which had previously been shown to be among the highest natural producers of the CFA/I fimbriae among tested wild type ETEC strains. Oral immunization with formalin-killed recombinant E. coli bacteria over-expressing CFA/I induced significantly higher serum IgA and IgG+M antibodies responses compared to the reference strain. Oral immunization with formalin-killed recombinant V. cholerae bacteria also induce strong CFA/I-specific serum IgA and IgG+M responses. We conclude that our constructs may be useful as candidate strains in an oral killed CF-ETEC vaccine.     

Immune responses elicited against multiple enterotoxigenic Escherichia coli fimbriae and mutant LT expressed in attenuated Shigella vaccine strains

Shigella and enterotoxigenic Escherichia coli (ETEC) continue to be important causes of diarrheal disease in infants and young children in developing countries and are major etiologic agents of traveler's diarrhea. Since attenuated strains of Shigella have been developed as live oral vaccines against shigellosis, we have adapted these attenuated Shigella strains to serve as carriers of ETEC antigens, thereby constituting a hybrid vaccine. Since protective immunity against ETEC is largely directed against fimbrial antigens (of which there are multiple antigenic types), we have individually expressed four different ETEC fimbriae, including CFA/I, CS2, CS3, and CS4, using deltaguaBA attenuated Shigella vaccine strain CVD 1204 as a prototype live vector. Following mucosal (intranasal) immunization of guinea pigs, serum IgG and mucosal IgA responses were elicited against each fimbrial type. An additional strain was constructed expressing a detoxified version of the human ETEC variant of heat labile toxin (LThK63). Following mucosal immunization of guinea pigs with a mixed inoculum containing five Shigella strains each expressing a different ETEC antigen, immune responses were observed against each ETEC antigen plus the Shigella vector.     

Efficacy trial of single-dose live oral cholera vaccine CVD 103-HgR in North Jakarta, Indonesia, a cholera-endemic area

A randomized, double-blind, placebo-controlled efficacy trial of one dose of CVD 103-HgR live oral cholera vaccine was performed in Indonesia from 1993 to 1997. 67,508 persons aged 2-41 years ingested vaccine or placebo and were followed for four years, detecting cholera cases using hospital-based surveillance. A nested reactogenicity study (538 vaccinees, 535 controls) revealed no vaccine-attributable side effects. A nested immunogenicity study (N=657) showed vibriocidal seroresponses in 64-70% of vaccinees vs 1-2% of controls. Cholera incidence was lower than expected. 103 cases of Vibrio cholerae O1 El Tor diarrhea were detected, 93 evaluable for vaccine efficacy (43 vaccine, 50 placebo; efficacy=14%). A suggestion of protection was observed among persons with blood group O [P=0.12]. Only seven cases occurred within six months of vaccination, precluding assessment of short-term efficacy. In Jakarta, single-dose CVD 103-HgR did not confer long-term protection. Short-term protection from a single-dose and long-term protection from two doses have yet to be studied.     

Randomized double-blind placebo controlled trial to evaluate the safety and immunogenicity of the live oral cholera vaccine strain CVD 103-HgR in Swiss adults

A randomized, double-blind, placebo controlled trial was conducted in 50 healthy Swiss adults to assess the safety and immunogenicity of the live oral attenuated cholera vaccine candidate strain Vibrio cholerae CVD 103-HgR (classical, Inaba). A single dose of 5 × 10⁸ viable CVD 103-HgR organisms, administered in a buffered liquid formulation, was well tolerated as compared with individuals who received an equivalent amount of heat-killed Escherichia coli K-12 placebo. Eighty-eight percent of subjects receiving CVD 103-HgR mounted a significant (>fourfold) rise in Inaba vibriocidal titre while 68% did so for the heterologous Ogawa serotype. The magnitude of the vibriocidal antibody response (as measured by peak geometric mean titre and by fold-rise in titre over baseline) was greater for the homologous Inaba serotype. Nineteen out of 25 volunteers (76%) responded with a significant (p < 0.05) rise in serum antitoxin levels. No vaccine who received the E. coli K-12 placebo mounted a significant rise in either vibriocidal or antitoxin antibody levels. These results corrobrate the safety and immunogenicity of CVD 103-HgR in healthy adult volunteers.     

口服霍乱疫苗研究现状

[目的]了解目前国内外口服霍乱疫苗的研究现状.[方法]对近年来国内外医学期刊上发表的有关口服霍乱疫苗研究的论文进行综述.[结果]有3种疫苗具有良好的免疫原性、安全性,能有效预防霍乱流行.即灭活霍乱弧菌全菌体(WC)疫苗或霍乱毒素B亚单位-灭活霍乱弧菌全菌体(BS-WC)疫苗、重组霍乱毒素B亚单位-灭活霍乱弧菌全菌体(rBS-WC)疫苗、由活的基因修饰的O1群霍乱弧菌减毒株(CVD103-HgR)组成的减毒活菌苗.[结论]口服霍乱疫苗尚处在大规模的实验和论证中,仅在较小范围人群中被建议服用,未被作为大范围人群预防霍乱的公共卫生措施.     

Mucosal immunization of BALB/c mice using enterotoxigenic Escherichia coli colonization factors CFA/I and CS6 administered with and without a mutant heat-labile enterotoxin

Mice (BALB/c) were intranasally (IN) and intragastrically (IG) administered the ETEC colonization factors (CF), CFA/I and CS6, with and without the R192G mutant heat-labile enterotoxin (mLT), and immunogenicity and efficacy measured. The IN administration of CFA/I to mice induced strong serum and fecal IgG and IgA responses. The IG administration of CFA/I to mice induced serum IgG and fecal IgA responses, but only when mLT was co-administered with CFA/I were serum IgA titers detected. The IN administration of CS6 to mice induced serum IgG antibodies, and mLT, when co-administered with CS6, enhanced the serum IgG response. Only when the mLT was co-administered with CS6, were serum and fecal IgA responses detected. The IG administration of CS6 plus mLT induced serum IgG and fecal IgA responses. Partial protection against lethal challenge with ETEC strain H10407 was seen in the mice IN administered the CFA/I plus mLT (P<0.01), and H10407 was cleared from the lungs of CFA/I plus mLT-immunized mice at a significantly greater rate than from the control mice (P<0.05). CFA/I and CS6 administered IN and IG induced mixed Th1/Th2 immune responses with the Th2 type being predominant as evidenced by IgG1>IgG2a. The administration of colonization factors to mice, particularly by the IN route, potentially serves as a useful way to measure the serum and mucosal immune responses to these antigens prior to their use in volunteers.     

Safety and immunogenicity of an oral,inactivated enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Bangladeshi children 18-36 months of age

A phase II safety and immunogenicity study of an oral-formalin inactivated enterotoxigenic Escherichia coli (ETEC) vaccine containing six colonization factors (CFA/I, CS1, CS2, CS3, CS4, CS5) and 1mg of recombinant cholera toxin B subunit (the CF-BS-ETEC vaccine) was carried out in an urban slum of Dhaka city in Bangladesh. The study was carried out in a double blinded, placebo controlled design in 158 children, 18-36 months of age. Children were given two doses of the CF-BS-ETEC vaccine or the placebo which consisted of E. coli K12. The vaccine was well tolerated.The immune response was studied in 60 children (30 each in the placebo and vaccine group). Significant vaccine specific IgA antibody-secreting cell (ASC) responses were seen 7 days after ingestion of the first and second dose of the vaccine. The responses to CFA/I (P相似文献   

Construction of a non-toxigenic Escherichia coli oral vaccine strain expressing large amounts of CS6 and inducing strong intestinal and serum anti-CS6 antibody responses in mice

Coli surface antigen 6 (CS6) is one of the most prevalent non-fimbrial colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) bacteria, which are the most common cause of diarrhea among infants and children in developing countries. Since immune protection against ETEC is mainly mediated by locally produced IgA antibodies in the gut, much effort is focused on the development of an oral CF-based vaccine. Previous work has described the preparation of candidate E. coli vaccine strains expressing immunogenic amounts of fimbrial CF antigens such as CFA/I and CS2, which are retained after formalin treatment. However, attempts to generate E. coli expressing immunogenic amounts of CS6 and to preserve the immunological activity of the CS6 protein in a killed whole-cell vaccine have failed until now. Here we describe the construction of a recombinant non-toxigenic E. coli strain, with thyA as a non-antibiotic-based selection, which expresses large amounts of CS6 antigen on the bacterial surface, and show that phenol inactivation of the bacteria does not destroy the CS6 antigen properties. Oral immunization of mice with such phenol-killed CS6 over-expressing E. coli bacteria induced strong fecal and intestinal IgA and serum IgG + IgM antibody responses to CS6 that exceeded the responses induced by an ETEC reference strain naturally expressing CS6 and previously used as a vaccine strain. Our data indicate that the described phenol-inactivated non-toxigenic and CS6 over-expressing E. coli strain may be a useful component in an oral ETEC vaccine.     

Experimental evaluation of antitoxic protective effect of new cholera vaccines in mice.

Intraperitoneal immunization of mice and subsequent challenge with purified cholera toxin (CT) were employed to evaluate the anti-cholera toxin protective effect of two new oral cholera vaccines, live CVD 103-HgR and killed B subunit-whole cell (BS-WC). CVD 103-HgR vaccine demonstrated 100% protection of mice against 2.25 LD50 and 70% against 3 LD50 of CT. Mice immunized with BS-WC vaccine were protected against 2.25 and 3 LD50 of CT in 88 and 62% of cases, respectively. All three killed parenteral vaccines failed to protect against CT. We suggest this mouse system for preliminary evaluation of the antitoxic protective activity of cholera vaccines.     

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.     

Antibodies derived from an enterotoxigenic Escherichia coli (ETEC) adhesin tip MEFA (multiepitope fusion antigen) against adherence of nine ETEC adhesins: CFA/I,CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA

Diarrhea continues to be a leading cause of death in children younger than 5 years in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of children's diarrhea and travelers’ diarrhea. ETEC bacteria initiate diarrheal disease by attaching to host receptors at epithelial cells and colonizing in small intestine. Therefore, preventing ETEC attachment has been considered the first line of defense against ETEC diarrhea. However, developing vaccines effectively against ETEC bacterial attachment encounters challenge because ETEC strains produce over 23 immunologically heterogeneous adhesins. In this study, we applied MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA adhesins and to construct an adhesin tip MEFA peptide. We then examined immunogenicity of this tip MEFA in mouse immunization, and assessed potential application of this tip MEFA for ETEC vaccine development. Data showed that mice intraperitoneally immunized with this adhesin tip MEFA developed IgG antibody responses to all nine ETEC adhesins. Moreover, ETEC and E. coli bacteria expressing these nine adhesins, after incubation with serum of the immunized mice, exhibited significant reduction in attachment to Caco-2 cells. These results indicated that anti-adhesin antibodies induced by this adhesin tip MEFA blocked adherence of the most important ETEC adhesins, suggesting this multivalent tip MEFA may be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea.     

Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein

We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine.     

Cross-reactivity and avidity of antibody responses induced in humans by the oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX

We investigated whether the oral inactivated, multivalent enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of four E. coli strains over-expressing the colonisation factors (CFs) CFA/I, CS3, CS5 and CS6, combined with the toxoid LCTBA, could induce cross-reactive antibodies to CFs related to the CFA/I and CS5 families. We also evaluated the avidity of vaccine induced antibodies against the toxoid and CFs.Cross-reactivity was analysed in mucosal (faecal and antibodies in lymphocyte supernatants, ALS) samples, and antibody avidity in serum and ALS samples, from two phase I trials: a primary vaccination study, where two oral doses of ETVAX were given ± the double mutant heat labile toxin (dmLT) adjuvant at a 2-week interval, and a booster vaccination study, where a single booster dose of ETVAX was given 13–23 months after primary vaccinations.We found that 65–90% of subjects who had responded to CFA/I in ALS or faecal specimens also developed cross-reactive antibodies to the related CFs tested, i.e. CS1, CS14 and CS17, and that approximately 80% of those responding to CS5 also responded to the closely related CS7. For subjects who had developed cross-reactive antibodies, the magnitudes of responses against vaccine CFs and related non-vaccine CFs were comparable. Using both a simple method of antibody avidity determination based on limiting antigen dilution, as well as a chaotropic ELISA method, we found that the avidity of serum and ALS antibodies to key vaccine antigens increased after a late booster dose compared to after primary vaccination.Our results suggest that the cross-reactive antibody responses against multiple CFs may result in expanded ETEC strain coverage of ETVAX and that repeated vaccinations induce vaccine-specific antibodies with increased binding capacity.     

Infection with colonization factor antigen I-expressing enterotoxigenic Escherichia coli boosts antibody responses against heterologous colonization factors in primed subjects.

Enterotoxigenic Escherichia coli (ETEC) adhere to the intestinal mucosa by a number of fimbrial colonization factors (CFs) that have been claimed to induce only type-specific immunity. However, adult Bangladeshi patients infected with CFA/I-expressing bacteria, developed significant plasma IgA antibody responses, as determined by enzyme-linked immunosorbent assay, not only against the homologous fimbriae but also against several heterologous CFs, i.e. CS1, CS2, CS4 and PCFO166 fimbriae. In contrast, North American volunteers, who had probably not been infected by ETEC previously, responded with serum IgA against CFA/I fimbriae but not against any other CFs after symptomatic infection with CFA/I-expressing ETEC. Thus, infection with CFA/I-expressing bacteria may boost immune responses against CFs with a related amino acid sequence in previously primed subjects.     

Reduced doses of oral killed enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine is safe and immunogenic in Bangladeshi infants 6-17 months of age: dosing studies in different age groups

The oral-formalin inactivated whole cell enterotoxigenic Escherichia coli (ETEC) vaccine needs to be further tested in developing countries in order to determine the dose at which it will be safe and immunogenic for infants who are the target population for the vaccine. To determine the immunogenicity of reduced doses, studies were first carried out in children, 2-12 years of age (n = 60). The full, half or a quarter doses of the vaccine were comparable in immunogenicity with similar frequency of responses seen to the different antigens (P = NS). Following this result, a pilot study carried out in infants, 6-17 months of age (n = 50), showed that the frequency of episodes of vomiting was lowest when a quarter of the full dose was used. The infants however showed comparable immune responses to the half and quarter dose of vaccine that was tested (P = NS). Based on these results in the infants, a randomized double blind placebo-controlled Phase II study was carried out in 158 children, 6-17 months of age, where a quarter dose of the ETEC vaccine was tested. Adverse events of mild vomiting were seen in only 4% of vaccinees and in 2.5% of placebo recipients. The IgA-antibody secreting cell (ASC) responses to CFA/I (GM: 28.1 ASC/10(7) PBMC) and BS (GM: 55.7 ASC/10(7) PBMC) were elevated compared to placebo recipients (CFA/I-2.0; BS-4.8 ASC/10(7) PBMC) (P = 0.01 to < 0.001). The plasma-IgA antibody titers in vaccinees were also significantly elevated to CFA/I (GM-93.00), CS1 (GM-62.0), CS2 (GM-55.0), CS4 (GM-66.0) and BS (1057.0) compared to preimmune levels or responses or levels in placebo recipients (P < or = 0.05-0.001). This study thus demonstrates that reduced doses of the ETEC vaccine is immunogenic in children and infants as well as safe in infants down to 6 months of age.     

2011—2013年上海西区哨点医院成人急性腹泻细菌感染的病原学分析

目的分析上海地区2015—2018年产毒性大肠埃希菌(ETEC)毒力基因及定植因子(CF)的携带特征,为相关腹泻疾病防控及研究提供依据。方法采用多重荧光PCR的方法,对上海地区2015—2018年分离到的300株ETEC所携带的毒力基因(est,elt)及部分主要定植因子(CFA/I,CS1/PCFO71,CS2-8,CS12,CS14,CS17-19,CS21)进行检测,统计分析其在人群中的分布特征等。结果目标菌株中,57.00%(171/300)携带est基因,30.33%(91/300)携带elt基因,12.67%(38/300)同时携带est与elt基因;68.33%(205/300)的菌株根据所检测的14组定植因子可分为18种型别,其中最常出现的3种型别为13型(CS6,85/300),18型(CS21,65/300)和7型(CS2+CS3+CS21,17/300);出现频次最多的定植因子为CS21与CS6,其携带者在年龄分布上的差异有统计学意义,CS21携带者多为40岁及以下患者,而CS6多见于40岁以上的患者。结论上海地区2015—2018年检出的ETEC中est毒力基因比例较大;虽然不同年份间ETEC菌株携带的毒力基因和定植因子在构成上存在较大差异,但大多菌株携带的定植因子种类相对集中;其主要携带的定植因子为CS6(以elt为主)与CS21(均为est),并呈现出一定的年龄分布特征。     

Comparison of the antibody in lymphocyte supernatant (ALS) and ELISPOT assays for detection of mucosal immune responses to antigens of enterotoxigenic Escherichia coli in challenged and vaccinated volunteers

In the present study we compared the ELISPOT and antibody in lymphocyte supernatants (ALS) assays as surrogate measures of mucosal immunity. In separate studies, 20 inpatient volunteers received oral doses of 6 x 10(8) or 4 x 10(9)cfu of ETEC strain E24377A (LT+, ST+, CS1+, CS3+) and 20 subjects received 1 (n = 9) or 2 (n = 11) oral doses of the attenuated ETEC vaccine, PTL-003 expressing CFA/II (CS1+ and CS3+) (2 x 10(9)cfu/dose). Peripheral blood mononuclear cells (PBMCs) from all subjects were assayed for anti-colonization factor or toxin-specific IgA antibody responses using the ALS and ELISPOT procedures. ALS responses were measured using a standard ELISA, as well as by time-resolved fluorescence (TRF). Following challenge with E24377A, significant anti-CS3, CS1 and LT ALS responses were detected in the lymphocyte supernatants of 75-95% of the subjects. A similar proportion (75%) of subjects mounted an ALS response to CFA/II antigen after vaccination with the PTL-003 vaccine. Inter-assay comparisons between ALS and ELISPOT methods also revealed a high degree of correlation in both immunization groups. ALS sensitivity versus the ELISPOT assay for LT, CS3 and CS1-specific responses following challenge were 95%, 94% and 78%, respectively and 83% for the ALS response to CFA/II antigen after vaccination with PTL-003. Correlation coefficients for the LT and CS3 antigens were 0.94 (p<0.001) and 0.82 (p<0.001), respectively after challenge and 0.78 (p<0.001) after vaccination. The association between ALS and ELISPOT for the CS1 antigen was however, significant only when ALS supernatants were tested by TRF (r = 0.91, p<0.001). These results demonstrate the value and flexibility of the ALS assay as an alternative to ELISPOT for the measurement of mucosal immune responses to ETEC antigens, particularly when the complexities of ELISPOT may make it impractical to perform.