共表达ETEC CFA/Ⅰ、CS6福氏痢疾减毒株与单独表达CFA/Ⅰ及CS6混合疫苗株的免疫原性比较

目的　以减毒志贺菌为载体 ,研制肠毒素大肠杆菌 (enterotoxigenicEscherichiacoli,ETEC)多价疫苗可以有效防治细菌性腹泻。方法　以共表达肠毒素大肠杆菌定居因子CFA/Ⅰ和CS6的重组减毒福氏痢疾疫苗株FWL0 1(pZCF16 )和两种混合的表达CFA/Ⅰ的FWL0 1(pZHY2 1)和表达CS6的FWL0 1(pZLG6 )减毒福氏痢疾疫苗株 ,分别以同等剂量口服免疫BALB/c小鼠 ,比较二者肠毒素大肠杆菌定居因子的免疫效果。结果　免疫BALB/c小鼠全部产生了抗CFA/Ⅰ和CS6的特异性血清抗体IgG和分泌型抗体sIgA ,共表达CFA/Ⅰ、CS6的减毒福氏痢疾菌FWL0 1(pZCF16 )的免疫原性与混合的FWL0 1(pZHY2 1)和FWL0 1(pZLG6 )相似或稍高。结论　在同一菌株可以表达多种菌毛 ,构建ETEC多价疫苗有效可行     

共表达ETEC CFA／I、CS6福氏痢疾减毒株与单独表达CFA／I及CS6混合疫苗株的免疫原性比较

目的以减毒志贺菌为载体，研制肠毒素大肠杆菌(enterotoxigenic Escherichia coli，ETEC)多价疫苗可以有效防治细菌性腹泻。方法以共表达肠毒素大肠杆菌定居因子CFA/I和CS6的重组减毒福氏痢疾疫苗株FWL01(pZCF16)和两种混合的表达CFA/I的FWL01(pZHY21)和表达CS5的FWL01(pZLG6)减毒福氏痢疾疫苗株，分别以同等剂量口服免疫BMLB／c小鼠，比较二者肠毒素大肠杆菌定居因子的免疫效果。结果免疫BMLB／c小鼠全部产生了抗CFA/I和CS6的特异性血清抗体IgG和分泌型抗体sIgA，共表达CFA/I、CS6的减毒福氏痢疾菌FWL01(pZCF16)的免疫原性与混合的FWL01(pZHY21)和FWL01(pZLG6)相似或稍高。结论在同一菌株可以表达多种菌毛，构建。ETEC多价疫苗有效可行。     

双价志贺疫苗滴鼻免疫小鼠后粘膜免疫与系统免疫应答持续时间的观察

本文探讨双价志贺疫苗滴鼻免疫小鼠一段时间后,粘膜免疫和系统免疫应答的变化。将BALB/c小鼠随机分为三组,每组30只。PBS、FSM-2117和FS-5416(菌量为5×10~6、1×10~7、4×10~7和4×10~7CFU/只/次)经滴鼻途径免疫小鼠。间隔2周,4次免疫后7、30和90d活杀,收集鼻咽、肺、肠、生殖道冲洗液和血清。采用ELISA法检测其中特异性抗福氏、宋内LPSIgA和IgG。结果是两株疫苗经鼻内免疫后,诱发鼻咽、肺、胃肠道和生殖道等不同粘膜部位及血清中特异性抗福氏、宋内LPSIgA、IgG的显著增加(P<0.01)。特异性抗体水平虽然在免疫后30、90d明显下降,但仍明显高于PBS对照组水平。故认为两株双价志贺疫苗滴鼻免疫小鼠后能有效诱导粘膜免疫和系统免疫应答,并持续较长时间。     

痢疾杆菌脂多糖抗独特型(AId)单克隆抗体的制备和鉴定

以抗宋内志贺氏痢疾杆菌(Shigella sonnei)脂多糖单克隆抗体(Ab1)为抗原,免疫同系BALB/c小鼠和异系DBA小鼠,以其脾细胞与SP2/0骨髓瘤细胞株融合,得到16株能稳定分泌抗独特型单克隆抗体(Ab2)的杂交瘤细胞株,对其中5株抗体进行了血清学、免疫化原和免疫生物学鉴定,证明其中2株(AC1和BH8)为内影像抗独特型单克隆抗体(Ab2β)。它们能够模拟宋内菌脂、多糖抗原位点,使动物产生功能类似Ab1的抗体(Ab3),为进一步研究其作为疫苗的可行性打下了基础。     

HIV-1中国流行株gag与hIL-2在重组痘苗病毒中的共表达及其与核酸疫苗的实验免疫研究

目的：将HIV-1中国流行株B亚型gag基因、gag和hIL-2基因在天坛株痘苗病毒中进行共表达，以期获得重组痘苗病毒，观察细胞因子的佐剂作用，与核酸疫苗混合免疫，评价免疫效果，为新型艾滋病疫苗研制开发打下基础。方法：将HIV-1中国流行株 gag基因、gag和hIL-2基因片段插入到 pJ38载体启动子下游，经同源重组和血凝素阴性空斑筛选重组痘苗病毒，SDS-PAGE、Western blot检测目的蛋白。以重组病毒和核酸疫苗免疫Balb/c小鼠，进行淋巴细胞转化实验、CTL、CD4 、CD8 T细胞数目以及血清抗体的细胞免疫和体液免疫指标检测。结果：获得了重组痘苗病毒 vJ38gag和 vJ38gag-IL-2。与 vJ38-gag相比，vJ38gag-IL-2，具有更好的免疫原性，重组痘苗病毒免疫3次的效果好于重组病毒免疫2次，以2rVV-DNA混合免疫效果最好。结论：重组痘苗病毒vJ38gag和vJ38gag-IL-2能够表达外源蛋白并诱导机体产生细胞免疫和体液免疫。细胞因子IL-2发挥了免疫佐剂的作用。     

表达HIV-1 B亚型env基因的质粒DNA及腺病毒载体疫苗的免疫原性研究

目的 构建表达中国B亚型HIV-1流行株env基因的DNA及重组腺病毒载体疫苗,将其用于预防或治疗HIV感染.方法 构建质粒DNA疫苗pVR-gp160及重组腺病毒载体疫苗rAdV-gp160.将这两种疫苗以不同的方式免疫BALB/c小鼠,分别采用ELISPOT方法 和ELISA方法 检测免疫小鼠中HIV-1 Gp120特异性细胞免疫反应及抗体反应.结果 DNA疫苗单独免疫及DNA疫苗初免/腺病毒疫苗加强免疫的联合免疫方案皆可诱导较高水平的Gp120特异性细胞免疫反应;而在体液免疫方面,各实验组产生的Gp120特异性抗体水平都较低.结论 所构建的DNA疫苗及rAdV疫苗能有效表达Gp160蛋白,并可有效激活机体的细胞免疫反应.     

志贺氏菌双价疫苗候选株DOM3的残余毒力、免疫原性和保护力

目的 研制多价、安全、高效和无抗生素抗性的志贺氏菌疫苗. 方法 利用动物模型,评价志贺氏菌双价(S.flexneri 2a -S.dysenteriae I)疫苗候选株DOM3的残余毒力、免疫原性和保护力.结果 DOM3的残余毒力分别比野生型毒株S.flexneri 2a 301和S.dysenteriae I 112低120倍和57倍(昆明系小鼠),873和195倍(SPF BALB/c小鼠).DOM3丧失了侵袭上皮细胞的能力.DOM3具有和S.flexneri 2a 301几乎相同的免疫原性,但较S.dysenteriae I 112 的免疫原性略差.皮下免疫小鼠血清中特异性抗体IgA、IgG滴度峰值出现在最后一次皮下注射的第2周, 而IgM滴度峰值出现在第1周, 然后滴度逐步下降, 在6周后趋于稳定.血清中同时具有抗S.flexneri 2a 菌和抗S.dysenteriae I 菌的抗体, 且其滴度变化的趋势基本一致.使用昆明系小鼠,以30 LD50(6.13×107CFU) 的野生型毒株S.flexneri 2a 301和21 LD50(6.26×107 CFU) 野生型毒株S.dysenteriae I 112 攻击,保护率分别为90%和70%; 使用SPF BALB/c小鼠,以20 LD50(1.38×106 CFU)的 S.flexneri 2a 301和23 LD50( 6.95×106 CFU )的S.dysenteriae I 112攻击,保护率分别为100%和80%;与生理盐水和E.coli LE392对照比较,差异均具有显著性(P＜0.05). 结论 志贺菌双价疫苗候选株DOM3是一株有希望的疫苗候选株,为今后DOM3的安全性和免疫原性志愿者试验及疫苗改进提供了依据.     

含密码子优化的SIV gag基因的DNA疫苗与rAd5疫苗构建及免疫原性评价

目的 构建含有SIVgag基因的DNA疫苗和重组腺病毒疫苗,为后期在SIV感染的猴模型中进行多载体疫苗联合免疫策略的治疗效果评价奠定基础.方法 将SIV gag基因按照哺乳动物偏嗜密码子进行优化并构建至pVR载体,作为DNA疫苗.以Western Blot方法比较优化前后gag基因表达水平.将优化后的gag基因插入重组腺病毒载体,构建rAd5-SIVgag疫苗.在BALB/c小鼠中分别比较DNA疫苗及rAd5-SIV.gag疫苗单独及联合免疫的效果.结果 密码子优化的SIVgag基因的表达水平远高于野生型SIVgag基因.重组腺病毒疫苗免疫一次或两次诱导的细胞免疫反水平分别高于DNA疫苗免疫一次或两次.两种疫苗联合免疫的反应水平与腺病毒疫苗免疫两次的水平相当,高于DNA疫苗单独免疫及腺病毒疫苗单独免疫一次的结果.结论 成功优化了SIV gag基因,使其不依赖Rev高水平表达;成功构建了表达优化后SIV gag基因的DNA疫苗和rAd5疫苗,可以在小鼠体内诱导较强的gag基因特异性CTL应答.     

多房棘球绦虫混合重组BCG-EmⅡ/3和BCG-Em14-3-3疫苗诱导小鼠脾细胞因子的变化

目的:探讨多房棘球绦虫混合重组BCG-EmⅡ/3和BCG-Em14-3-3疫苗免疫后再以Em原头节攻击后小鼠脾细胞因子的变化。方法:将疫苗采用皮下注射和鼻腔内接种分别免疫BALB/c小鼠后8周,用多房棘球绦虫原头节进行攻击感染。感染后18周杀鼠取脾,分离脾细胞,用EmAg或ConA刺激培养,并收集脾细胞培养上清液,用试剂盒检测脾细胞培养上清液中IL-2、IFN-γ、TNF-α和IL-4的水平,同时设有空载体、BCG和PBS对照。结果:疫苗接种组的IFN-γ和TNF-α水平升高,IL-4水平降低;皮下注射组的TNF-α水平高于鼻腔内接种组。结论:多房棘球绦虫混合重组BCG-EmⅡ/3和BCG-Em14-3-3疫苗可诱导小鼠产生Th1型细胞应答,抵抗Em原头节的攻击感染。疫苗皮下注射途径优于鼻腔内接种。     

狂犬病毒CTN株糖蛋白重组鸡痘病毒与核酸疫苗的联合免疫

目的 构建狂犬病毒糖蛋白重组鸡痘病毒，研究联合运用重组病毒与同一抗原核酸疫苗对小鼠的免疫效果，为狂犬病疫苗的研究提供基础。方法 以中国鸡痘病毒疫苗株282E4为基础构建的表达载体pUTA-2的ATI-P7．5复合启动子下游，插入狂犬病毒CTN-18l株糖蛋白基因，构建了重组转移载体pUTA-RgC。以脂质体转染法将pUTA-RgC转染至已感染鸡痘病毒282E4株(FPV)2～3h的鸡胚成纤维(CEF)细胞中。待细胞出现明显病变后收获病毒，用BrdU进行连续3次加压筛选，挑取蚀斑毒株、纯化、扩增，间接免疫荧光鉴定。用重组鸡痘病毒vUTA-RgC单独及与核酸疫苗联合免疫小鼠，在细胞及体液免疫，攻毒保护水平评价所构建的重组病毒及联合免疫效果。结果 间接免疫荧光结果表明已成功构建重组鸡痘病毒，免疫后能诱导机体产生细胞与体液免疫，并能抵抗标准攻毒株的攻击，联合免疫效果较为理想。结论 获得一株能够表达狂犬病毒糖蛋白的vUTA．RgC，并证明有一定的免疫原性，与核酸疫苗联合应用能够克服各自的缺点。     

Attenuated Shigella flexneri 2a Delta guaBA strain CVD 1204 expressing enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 fimbriae as a live mucosal vaccine against Shigella and ETEC infection

To construct a prototype hybrid vaccine against Shigella and enterotoxigenic Escherichia coli (ETEC), the genes encoding the production of ETEC CS2 and CS3 fimbriae were isolated and expressed in attenuated Shigella flexneri 2a guaBA strain CVD 1204. The CS2 cotA to -D genes, isolated from ETEC strain C91F, and the CS3 cstA to -H genes, subcloned from plasmid pCS100, were cloned into ~15-copy-number-stabilized pGA1 behind the osmotically regulated ompC promoter, resulting in high expression of both fimbriae. Under nonselective in vitro growth conditions, pGA1-CS2 and pGA1-CS3 were stable in CVD 1204, exhibiting a plasmid loss of only approximately 1% per duplication. Expression of CS2 and CS3 reduced the invasiveness of Shigella for HeLa cells and slowed the intracellular growth rate. Guinea pigs immunized intranasally with CVD 1204(pGA1-CS2) or CVD 1204(pGA1-CS3), or with a mixture of these strains, developed secretory immunoglobulin A (IgA) in tears and serum IgG antibodies against Shigella lipopolysaccharide, CS2, and CS3 antigens. Moreover, the animals were protected against keratoconjunctivitis following conjunctival challenge with virulent S. flexneri 2a strain 2457T. Animals immunized with Shigella expressing CS2 or CS3 developed serum antibodies that agglutinated Shigella as well as an ETEC strain bearing the homologous fimbriae, whereas animals immunized with combined CVD 1204(pGA1-CS2) and CVD 1204(pGA1-CS3) developed antibodies that agglutinated all three test strains. These observations support the feasibility of a multivalent vaccine against shigellosis and ETEC diarrhea consisting of multiple Shigella live vectors expressing relevant ETEC antigens.     

A combination vaccine consisting of three live attenuated enterotoxigenic Escherichia coli strains expressing a range of colonization factors and heat-labile toxin subunit B is well tolerated and immunogenic in a placebo-controlled double-blind phase I trial in healthy adults

Immune responses against colonization factors (CFs) and the nontoxic B component of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LTB) are considered to be important for immunity against diarrhea caused by ETEC. Individual live attenuated ETEC derivatives that have had their toxin genes removed and whose aroC, ompC, and ompF genes are deleted have shown promise as vaccines against ETEC. The development of such strains has culminated in the testing of a three-strain-combination live attenuated vaccine known as ACE527, comprised of strains ACAM2025 expressing colonization factor antigen I (CFA/I) and LTB; ACAM2022, expressing CS5, CS6, and LTB; and ACAM2027, expressing CS1, CS2, CS3, and LTB. The recombinant CF and LTB genes expressed in the three strains were inserted into the bacterial chromosome to ensure their stable inheritance and expression without the requirement for any selection. ACE527 has been tested in a randomized placebo-controlled, double-blind, phase I safety and immunogenicity study in healthy adult volunteers and proved to be well tolerated and immunogenic at dose levels of 10(10) and 10(11) total CFU. There was no indication of strain interference on the basis of fecal shedding patterns, with all three being detected in the feces of 50% and 83% of low- and high-dose vaccine recipients, respectively. Similarly, strong immune responses to LTB and to CFs expressed on all three constituent strains were induced, with at least 50% of subjects in the high-dose group responding to LTB, CFA/I, CS3, and CS6.     

Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain

The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA, -B, -C, -E, and -D', were isolated from strain E11881A. The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD'). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The csaA, -B, -C, -E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter. pGA2-CS4 directs production of CS4 fimbriae in both E. coli DH5alpha and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine.     

Vaccines against enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among children less than 3 years of age in developing countries and in travelers to these areas. The key pathogenic mechanisms that contribute to the pathogenesis of ETEC are the production of colonization factors (CFs) and a heat-labile enterotoxin (LT) and/or a heat-stable enterotoxin. To provide broad-spectrum protection, an ETEC vaccine should, most likely, contain the most prevalent fimbrial antigens, that is, CF antigen I and CS1-CS6, and/or a LT toxoid. Different strategies have been taken to deliver ETEC fimbriae and toxin antigens to the human immune system to elicit strong mucosal, in particular, intestinal immune responses that are considered to be of prime importance for protection against ETEC disease. There has been some promise when testing different ETEC candidate vaccines for protection against diarrhea in adult travelers. However, no ETEC candidate vaccine has been shown to be effective in the most important target group, which is infants and young children in endemic areas. Against this background, intense efforts are in progress to try to improve the immunogenicity of different available candidate vaccines, as well as to develop new types of ETEC vaccines.     

Dose-dependent circulating immunoglobulin A antibody-secreting cell and serum antibody responses in Swedish volunteers to an oral inactivated enterotoxigenic Escherichia coli vaccine

The immunogenicity of different preparations of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine was evaluated in Swedish volunteers previously unexposed to ETEC infection. The vaccine preparations consisted of recombinant cholera toxin B subunit (CTB) and various amounts of formalin-killed whole bacteria expressing the most prevalent colonization factor antigens (CFAs). Significant immunoglobulin A (IgA) antibody-secreting cell (ASC) responses against CTB and the various CFA components were seen in a majority of volunteers after two doses of ETEC vaccine independent of the vaccine lot given. The IgA ASC responses against CTB were significantly higher after the second than after the first immunization, whereas the CFA-specific IgA ASC responses were almost comparable after the first and second doses of ETEC vaccine. Two immunizations with one-third of a full dose of CFA-ETEC bacteria induced lower frequencies of IgA ASC responses against all the different CFAs than two full vaccine doses, i.e., 63 versus 80% for CFA/I, 56 versus 70% for CS1, 31 versus 65% for CS2, and 56 versus 75% for CS4. The proportion of vaccinees responding with rises in the titer of serum IgA antibody against the various CFA antigens was also lower after immunization with the reduced dose of CFA-ETEC bacteria. These findings suggest that measurements of circulating IgA ASCs can be used not only for qualitative but also for quantitative assessments of the immunogenicity of individual fimbrial antigens in various preparations of ETEC vaccine.     

Further characterization of delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for delivering antigens of enterotoxigenic Escherichia coli.

The use of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a live vector for enterotoxigenic Escherichia coli (ETEC) antigens is reported. CVD 1203 alone or expressing colonization factor antigen fimbriae and CS3 fibrillae of ETEC was given to guinea pigs and mice, orogastrically (o.g.) or intranasally (i.n.). CVD 1203 given i.n. elicited high titers of antilipopolysaccharide (anti-LPS) immunoglobulin A (IgA) and was protective in guinea pigs against a homologous conjunctival challenge. Whereas a strong IgA response against colonization factor antigen CS3, and Shigella LPS was detected in tears and serum of guinea pigs after o.g. or i.n. immunization, the i.n. route elicited significantly higher antibody titers. A strong serum IgG response was also observed against the ETEC antigens, although no serum anti-LPS IgG response was detected. The immune response in mice followed a pattern similar to that in guinea pigs, and the difference between the responses after o.g. and i.n. administration was even more remarkable.     

Assessment by electron-microscopy of recombinant Vibrio cholerae and Salmonella vaccine strains expressing enterotoxigenic Escherichia coli-specific surface antigens.

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) requires adhesion of microorganisms to enterocytes. Hence, a promising approach to immunoprophylaxis is to elicit antibodies against colonisation factor antigens (CFAs). Genes encoding the most prevalent ETEC-specific surface antigens were cloned into Vibrio cholerae and Salmonella vaccine strains. Expression of surface antigens was assessed by electron-microscopy. Whereas negative staining was effective in revealing CFA/I and CS3, but not CS6, immunolabelling allowed identification of all surface antigens examined. The V. cholerae vaccine strain CVD103 did not express ETEC-specific colonisation factors, whereas CVD103-HgR expressed CS3 only. However, expression of both CFA/I and CS3 was demonstrated in Salmonella Ty21a.     

Vaccines inf the prevention of fecal hazards

In addition to the imperative improvement of hygiene standards, vaccines can also be used for the prevention of diseases transmitted by the feco-oral route. Vaccination is essential to protect against poliomyelitis, the eradication of which is targeted through vaccination campaigns using the live, attenuated vaccine in countries where the disease is still endemic and the inactivated vaccine or a combination of both vaccines (mixed schedule) in countries where the disease is under control. The introduction of a specific routine vaccination program against hepatitis A in endemic countries is now starting to be considered. Travellers to these countries must be protected. Among bacterial diseases, only typhoid fever can be prevented by means of an effective vaccine. The difficulties encountered in improving hygiene standards in numerous countries have prompted the WHO to encourage search for new vaccines for the prevention of the diseases transmitted by the feco-oral route. A vaccine against rotavirus that has just been licensed in the USA should permit the global reduction of a significant number of deaths attributable to these viruses. Vaccine prospects for the prevention of hepatitis E are more distant. Several vaccines against Shigella (injectable polysaccharide conjugate vaccine or oral, live, attenuated vaccines obtained by construction of mutant strains) and enterogenic Escherichia coli (oral, inactivated vaccine containing several strains of ETEC and sub-unit B of the cholera toxin), not to mention candidate vaccines against cholera.     

Dose-Dependent Circulating Immunoglobulin A Antibody-Secreting Cell and Serum Antibody Responses in Swedish Volunteers to an Oral Inactivated Enterotoxigenic Escherichia coli Vaccine

The immunogenicity of different preparations of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine was evaluated in Swedish volunteers previously unexposed to ETEC infection. The vaccine preparations consisted of recombinant cholera toxin B subunit (CTB) and various amounts of formalin-killed whole bacteria expressing the most prevalent colonization factor antigens (CFAs). Significant immunoglobulin A (IgA) antibody-secreting cell (ASC) responses against CTB and the various CFA components were seen in a majority of volunteers after two doses of ETEC vaccine independent of the vaccine lot given. The IgA ASC responses against CTB were significantly higher after the second than after the first immunization, whereas the CFA-specific IgA ASC responses were almost comparable after the first and second doses of ETEC vaccine. Two immunizations with one-third of a full dose of CFA-ETEC bacteria induced lower frequencies of IgA ASC responses against all the different CFAs than two full vaccine doses, i.e., 63 versus 80% for CFA/I, 56 versus 70% for CS1, 31 versus 65% for CS2, and 56 versus 75% for CS4. The proportion of vaccinees responding with rises in the titer of serum IgA antibody against the various CFA antigens was also lower after immunization with the reduced dose of CFA-ETEC bacteria. These findings suggest that measurements of circulating IgA ASCs can be used not only for qualitative but also for quantitative assessments of the immunogenicity of individual fimbrial antigens in various preparations of ETEC vaccine.     

Attenuated Shigella flexneri 2a vaccine strain CVD 1204 expressing colonization factor antigen I and mutant heat-labile enterotoxin of enterotoxigenic Escherichia coli

A multivalent live oral vaccine against both Shigella spp. and enterotoxigenic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution-serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into S. flexneri 2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM(1) binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-S. flexneri 2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type S. flexneri 2a. This prototype Shigella-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses against both the heterologous antigens and vector strain.