双价痢疾菌角结膜免疫豚鼠后泪液中特异性抗体的检测

目的：研究痢疾杆菌侵袭蛋白与局部免疫反应的关系,为痢疾菌苗的研制提供理论依据。方法：用表达和不表达侵袭蛋白（Ipa^ /Ipa^-）的两株福氏、宋内氏双价痢疾菌苗经角结膜免疫豚鼠,并分别用福氏、宋内氏毒株攻击,BA－ELISA方法检测豚鼠泪液中特异性抗体升高情况。结果：发现角结膜免疫后豚鼠泪液中特异性双价抗体水平较对照显著升高,差别具有统计学意义,Ipa^ 菌苗免疫组内氏抗体水平较Ipa^-免疫组升高,差别具有统计学意义;攻击后,两免疫组局部抗体呈二次反应变化。结论：侵袭蛋白的表达可加强痢疾菌苗诱导的局部抗体水平。两株双价痢疾菌苗均有较好的免疫原性。     

双价痢疾菌苗免疫小鼠后抗体记忆反应的观测

目的：FSM－2117和FS－5416是我室构建的具有不同生物表型的福氏、宋内氏双价痢疾菌苗株,前者不表达侵袭蛋白,无侵袭力,而后者表达侵袭蛋白（Ipa）,具有侵袭力。拟观测两株菌苗FSM－2117（Ipa^-）、FS－5416（Ipa^ ）免疫后所引起的肠粘膜局部和系统中的免疫记忆反应,为研制痢疾菌苗提供理论依据。方法：以小鼠灌胃免疫为模型,3次免疫后,间隔3个月再次免疫,第7天取血清和小肠冲洗液,应用ELISA方法检测福氏、宋内氏特异性抗体。结果：两菌苗株免疫组,血清中局部抗原特异性sIgA、IgG抗体明显升高。两株菌苗与对照组相比,都具有显著差异。结论：两株双价痢疾菌苗免疫小鼠后,可产生一定的记忆反应。     

侵袭蛋白对双价志贺菌苗免疫保护影响的研究

为了研究侵袭蛋白与免疫保护的关系 ,为志贺菌苗的研制提供理论依据。经志贺菌苗角结膜免疫豚鼠 ,毒株攻击 ,观察侵袭蛋白对免疫保护的影响 ,观察到表达侵袭蛋白的菌苗较不表达侵袭蛋白的菌苗对毒株攻击有较高的保护率 ;用BA ELISA方法检测免疫后豚鼠泪液中特异性抗体水平 ,其结果较对照显著升高。说明侵袭蛋白的表达有一定的增加菌苗的免疫保护作用     

双价痢疾菌苗不同途径免疫小鼠对脾细胞及GALT中ASC影响的实验研究

目的观察侵袭蛋白表达阴性(FSM-2117)和阳性(FS-5416)的两株双价痢疾菌苗经不同途径免疫小鼠后,脾细胞和粘膜相关淋巴组织(gut associated lymphoid tissues,GALT)中抗体分泌细胞(antibody secreting cell,ASC)的变化,以探讨免疫途径及侵袭蛋白表达与否对痢疾菌苗免疫效果的影响.方法BALB/c小鼠随机分为3组,每组20只,FSM-2117或FS-54164×107CFU分别经滴鼻、灌胃及肠内注射3种途径免疫小鼠,间隔2w,第3次免疫后7d活杀分离脾、小肠PP及MLN淋巴细胞,采用BA-ELISASPOT法检测其中特异性抗福氏、宋内氏LPSIgA和IgG分泌细胞.结果滴鼻途径及小肠内免疫都能够诱生脾细胞特异性IgA-ASC、IgG-ASC显著升高(P＜0.01);仅滴鼻免疫组Ipa+株较Ipa-株升高显著(P＜0.01).滴鼻和小肠内免疫同时诱生肠GALT中派伊尔小结(Payer'spatch,PP)和肠系膜淋巴结(mesenteric lymph     

两株双价痢疾菌苗免疫小鼠后GALT中CD4+、CD8+ T细胞亚群的反应

目的 FSM-2117和FS-5416是两株具有不同生物表型的福氏、宋内氏双价痢疾菌苗株,FSM-2117无侵袭表型,无溶血活性,不表达侵袭蛋白抗原(Ipa-),FS-5416则为Ipa+,本实验是为了观测两株菌苗(Ipa-,Ipa+)免疫后引起的肠粘膜相关淋巴组织(GALT)中的细胞免疫反应,以探明痢疾菌苗在肠粘膜的免疫保护机制.方法 以BALB/c小鼠随机分成两组,每组25只,灌胃免疫FSM-2117及FS-5416,8×108CFU/只,分别在1,4,7,10,13天随机取各组小鼠5只分离GALT淋巴细胞;同时设一组PBS对照组,以间接免疫荧光法检测GALT中诱导部位[派伊尔小结(PP)、肠系膜淋巴结(MLN)]及效应部位[上皮间淋巴细胞(IEL)、粘膜固有层(LPL)]T细胞亚群的变化,荧光显微镜计数200个细胞计算阳性率.结果 用SAS软件,单因素方差分析,表明两株双价菌苗株均可引起肠粘膜GALT中CD4+、CD8+淋巴细胞亚群改变,不同粘膜部位免疫反应不同.诱导部位(派氏小结、肠系膜淋巴结)CD4+细胞亚群明显升高,末次免疫后第7天达峰值,而后迅速下降;在效应部位,粘膜固有层表现为CD4+细胞亚群明显升高,而IEL主要表现为CD8+细胞亚群的升高,均于第7天达峰值.两株菌苗与对照组相比,差异都具有显著性(FSM-2117 P≤0.01,FS-5416 P＜0.01),但两菌苗株之间差别无统计学意义(P＞0.05).结论 两株双价痢疾菌苗均可在小鼠GALT引起免疫反应,说明其均有较好的免疫原性.     

不同表型的双价痢疾菌苗的滴鼻免疫研究

为了探讨滴鼻免疫不同表型的双价痢疾菌苗的免疫效果 ,应用FSM 2 117(Ipa )及FS 5 40 2 (Ipa+)、FS 5 411(Ipa+)、FS 5 414(Ipa+)、FS 5 416 (Ipa+) 5株菌苗株分别滴鼻免疫小鼠 ,4× 10 7CFU/只 ,间隔 14d ,3次免疫后第 7天收集血清和小肠、鼻咽、肺、阴道冲洗液 ,ELISA法检测其中特异性福氏、宋内LPSIgA和IgG抗体水平 ,发现滴鼻免疫能够诱生血清中的双价IgA、IgG抗体显著升高 ,Ipa+株较Ipa 株升高显著 (P <0 0 1)。同时诱生鼻咽、肺、小肠内特异性抗体升高 ,尤其是生殖道冲冼液内抗体升高极为明显。表明鼻粘膜免疫不仅诱导多个粘膜部位 (特别是生殖道 )的抗体反应 ,且能诱导系统免疫反应 ,是一个安全有效的免疫途径。研究中发现侵袭蛋白表达在鼻粘膜部位能够显著加强系统特异性抗体生成。     

两株双价痢疾工程菌苗不同粘膜免疫途径的免疫原性

目的探讨免疫途径、接种剂量及侵袭蛋白表达对两株双价痢疾工程菌苗免疫效果的影响。方法小鼠分为滴鼻、灌胃和对照组 ,分别以不同剂量免疫 3次 ,间隔 2周 ,3次免疫后 7d收集血清、小肠、鼻咽、肺、阴道冲洗液 ,EL ISA法检测其中特异性福氏、宋内氏 L PS- Ig A和 Ig G。结果 4× 10 7CFU菌苗经鼻途径免疫即可诱导多个粘膜部位以及血清双价特异性抗体显著增高 ;菌苗剂量增加 2 0、2 0 0倍时经灌胃免疫诱发较为局限的粘膜特异性抗体增高。结论鼻粘膜免疫不仅诱导多个粘膜部位 (特别是生殖道 )以及系统免疫的抗体反应 ,是一个安全有效的免疫途径。     

不同表型双价痢疾菌苗滴鼻免疫小鼠后黏膜和系统记忆反应观测

目的：观察5株2类不同表型的双价痢疾菌苗滴鼻免疫后所引起的黏膜和系统的记忆反应，为痢疾菌苗的构建提供理论依据。方法：以小鼠滴鼻免疫为模型，3次免疫（间隔2周）后，20周再次免疫，分别于3次免疫及20周加强免疫后第7天，收取鼻咽、肺、小肠及阴道冲洗液和血清，应用ELISA方法检测福氏、宋内氏特异性抗体。结果：2类双价菌苗株滴鼻免疫3次后，多个黏膜部位及血清均产生了抗原特异性sIgA、IgG，较PBS对照组有明显升高；20周后再次加强免疫，仍可诱导黏膜及系统的免疫反应，但较3次免疫后水平明显降低。结论：2株双价痢疾菌苗滴鼻免疫小鼠后可诱导多个黏膜及系统的特异性抗体产生，并可产生一定时间的记忆反应，侵袭蛋白与免疫记忆有关。     

双价志贺菌苗灌胃免疫小鼠后肠粘膜的免疫应答

为了观察两株福氏、宋内双价志贺菌苗 (Ipa ,Ipa+ )免疫后所引起的肠粘膜诱导部位的免疫应答及为研制有效腹泻疫苗提供理论基础。应用小鼠灌胃免疫为模型 ,以间接免疫荧光法和BA ELISPOT方法 ,检测了肠粘膜相关淋巴组织 (gutasso ciatedlymphoidtissues ,GALT )中诱导部位派伊尔小结 (Payer’spatches ,PP )、肠系膜淋巴结 (mesentericlymphoidnode ,MLN )T淋巴细胞亚群和特异性抗体分泌细胞的变化 (ASC )及小肠局部和系统的特异性抗体变化。发现两株双价菌苗株均可引起肠粘膜GALT诱导部位CD4+ 淋巴细胞亚群、抗体分泌细胞明显升高 ;同时局部及血清中特异性抗体明显升高。说明双价志贺菌苗灌胃免疫小鼠后 ,诱导部位的免疫应答早期以体液免疫为主 ,灌胃不仅可诱导局部特异性抗体免疫应答也可诱导系统的抗体免疫应答。     

免疫途径及侵袭蛋白表达对痢疾疫苗免疫效果影响的实验研究

目的探讨免疫途径及侵袭蛋白表达对2株痢疾疫苗免疫效果的影响.方法FSM-2117或FS-5416以4×107CFU/只分别经滴鼻、灌胃、肠内注射3种途径免疫小鼠,三免后7?d收集血清、小肠、鼻咽、肺、阴道冲洗液,ELISA法检测其中特异性福氏、宋内LPSIgA和IgG抗体(Ab)水平.结果滴鼻和肠内免疫都能够诱生血清中双价IgA、IgGAb显著升高,但仅滴鼻免疫组Ipa+株较Ipa-株升高显著(P＜0.01).滴鼻免疫组同时诱生鼻咽、肺、小肠内特异性抗体升高,尤其是生殖道冲洗液内抗体升高极为明显;而肠内免疫组诱生小肠、肺冲洗液内特异性抗体升高,但鼻咽及生殖道冲洗液内抗体未见明显升高.结论鼻粘膜免疫不仅诱导多个粘膜部位(特别是生殖道)的抗体反应,且能诱导系统免疫反应.是一个安全有效的免疫途径.研究中发现侵袭蛋白表达在鼻粘膜部位能够显著加强系统特异性抗体生成,但在胃肠粘膜却无此作用.     

Prospective study of systemic and mucosal immune responses in dysenteric patients to specific Shigella invasion plasmid antigens and lipopolysaccharides.

Shigellosis is a major cause of infant morbidity and mortality in developing countries. To find immunological correlates of specific protection against shigellosis, we examined chronological samples of sera, stool extracts, duodenal aspirates, and saliva samples from 39 adults and 22 children with shigellosis from Peru for the presence of specific antibody to invasion plasmid antigens (Ipa) common to all virulent Shigella strains, by using both a whole-organism enzyme-linked immunosorbent assay (ELISA) and a Western blot (immunoblot) assay. Antibody responses to lipopolysaccharide (LPS) from Shigella serotypes both homologous and heterologous to the infecting strain were also determined by ELISA. ELISAs showed that the highest serum immunoglobulin G (IgG) antibody titers to Shigella whole organisms both with and without surface Ipa were found in adults and malnourished children, the two groups with the shortest and longest durations of disease, respectively. Mucosal IgA antibody titers to Shigella strains decreased over time to a much greater extent than serum IgG titers, and IgA to Ipa in mucosal secretions was found in adults and well-nourished children but not in malnourished children. The presence of mucosal antibody to Ipa may limit the spread and severity of the infection, as indicated by the prolonged illness observed in malnourished children who have no significant mucosal antibody to Shigella Ipa. Serum antibody titers to the Ipa antigens were high relative to anti-Shigella LPS antibody titers, especially in pediatric patients. In contrast to the anti-Ipa responses observed, no differences in antibody responses to LPS in children compared by nutritional status were found. High levels of serum and mucosal cross-reacting antibody to heterologous serotype LPS were found between Shigella flexneri serotypes 1a and 2a. Different patterns of immune response to Ipa proteins and LPS that may aid in the definition of Shigella antigens important in host protection were observed in adults, well-nourished children, and malnourished children.     

Humoral response in mice immunized with outer membrane proteins of Shigella flexneri

Intraperitoneal immunization of mice with outer membrane proteins (OMP) of Sh. flexneri induced in the animals a synthesis of specific antibodies. Their level determined by ELISA test was found to be relatively low in the sera of animals immunized with a single dose (10 micrograms) of OMP; it was markedly higher in mice immunized with two doses of OMP, and very high after three fold immunization. The specific antibodies maintained in the animals for 8-16 weeks after immunization. Anti-OMP sera given to normal mice by intraperitoneal route protected them not only against challenge with homologous Shigella but also against Proteus and Escherichia.     

双价志贺菌苗滴鼻免疫对小鼠NALT、GALT和脾淋巴细胞的影响

本文是探讨滴鼻免疫对NALT、GALT和脾淋巴细胞中特异性抗体分泌细胞及淋巴细胞表型变化的影响。将Balb/c小鼠随机分为 3组 ,每组 2 0只。FSM 2 117或FS 5 416 4× 10 7CFU/只经滴鼻途径免疫小鼠 ,间隔 2周 ,3次免疫后 7d活杀 ,分离NALT、鼻通道、脾、小肠PP及MLN淋巴细胞 ,采用BA ELISPOT法检测其中特异性抗福氏、宋内LPSIgA和IgG分泌细胞 ;采用流式细胞术检测其淋巴细胞表型的变化。结果是两株菌苗经鼻内免疫都诱发了NALT、NP、GALT内 (PP、MLN、LPL )以及代表系统免疫反应的脾淋巴细胞中特异性抗福氏、宋内LPSIgA、IgG ASC的显著增加 (P <0 0 1) ;NALT、NP和PP淋巴细胞中CD3+T细胞显著增加 ,其中以CD4+T细胞增加为主 ;FSM 2 117免疫组的脾细胞中B2 2 0 +细胞显著增加 (P <0 0 1) ,而FS 5 416免疫组的脾细胞中CD3+T细胞显著增加 (P <0 0 1)。说明两株菌苗经鼻粘膜免疫均可诱导鼻粘膜局部、GALT及系统免疫反应的发生 ,是一个安全有效的免疫途径     

Mucosal adjuvant properties of the Shigella invasin complex

The Shigella invasin complex (Invaplex) is an effective mucosal vaccine capable of protecting against Shigella challenge in animal models. The major antigenic constituents of Invaplex are the Ipa proteins and lipopolysaccharide. The cell-binding capacity of the Ipa proteins prompted the investigation into the adjuvanticity of Invaplex. Using ovalbumin (OVA) as a model antigen, intranasal immunization with OVA combined with Invaplex was found to enhance anti-OVA serum immunoglobulin G (IgG) and IgA responses and induce OVA-specific mucosal antibody responses at sites located both proximal and distal to the immunization site. The immune responses induced with OVA and Invaplex were comparable in both magnitude and duration to the immune responses induced after immunization with OVA and cholera toxin. The OVA-specific immune response was characterized by high levels of serum IgG1 and increased production of interleukin-4 (IL-4), IL-5, or IL-10 from lymphoid cells of immunized animals, suggesting a Th2 response. In addition to enhancing the immunogenicity of OVA, Invaplex-specific immune responses were also induced, indicating the potential for the development of a combination vaccine consisting of Invaplex and other immunogens. Preexisting Invaplex-specific immunity did not interfere with the capacity to enhance the immunogenicity of a second, unrelated vaccine antigen, suggesting that Invaplex could be used as a mucosal adjuvant in multiple vaccine regimens.     

Antibody responses in the lungs of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strains expressing the filamentous hemagglutinin of Bordetella pertussis.

The filamentous hemagglutinin (FHA) of Bordetella pertussis was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, and in a strain of Escherichia coli harboring Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness for epithelial cells. Expression of FHA in these strains did not interfere with their ability to invade Henle cells. Immunoglobulins A and G specific for FHA were detected in lung washes of mice following oral immunization with the live recombinant organisms; antibody levels were significantly higher than those in mice immunized with killed bacteria administered orally or intraperitoneally. Live oral vaccines carrying protective antigens of B. pertussis may be an important alternative to new-generation component vaccines against whooping cough.