重组减毒细菌运送CD8+T细胞表位的效应分析

目的:分析重组减毒菌体外运送CD8 T细胞表位的效应。方法:以表达卵清蛋白(OVA)和淋巴细胞脉络丛脑膜炎病毒(LCMV)CD8 T细胞表位的重组菌13A(ptG2F)、25A(ptG2F)和SL7207(ptG2F)感染抗原提呈细胞(APC)LKb、LLd和骨髓源树突状细胞(BMDC),应用体外抗原提呈试验检测APC对重组菌运送的CD8 T细胞表位的提呈效应。结果:感染试验证实,减毒菌13A、25A和SL7207对LKb细胞或LLd细胞均具有良好的侵袭能力,BMDC对重组菌具有很好的摄取功能。抗原提呈试验结果显示,在感染的早期(2h),LKb、LLd细胞和BMDC均可提呈重组菌13A(ptG2F)或SL7207(ptG2F)运送的T细胞表位;在感染的晚期(48h),LKb细胞对OVA257-264CD8 T细胞表位的提呈效应降低,LLd细胞对LCMV118-132CD8 T细胞表位的提呈效应增强。3种APC均不能提呈25A(ptG2F)运送的T细胞表位。另外,在同样的作用条件下,BMDC对减毒菌运送的抗原表位的提呈效应要强于LKb和LLd细胞。结论:重组菌能运送CD8 T细胞表位,为基于减毒细菌的新型基因工程疫苗的分子设计提供了有益借鉴。     

重组减毒大肠杆菌对真核表达的CD8+ T细胞表位的运送研究

目的：分析体外、体内重组减毒大肠杆菌运送真核表达的CD8^＋T细胞表位的效应。方法：将携带融合表达绿色荧光蛋白（GFP）与OVACD8^＋T细胞表位基因真核表达质粒的重组大肠杆菌13A（pG2F）感染抗原提呈细胞（APC）IX^b和骨髓源树突状细胞（BMDC），应用体外抗原提呈试验检测APE对重组菌运送的CD8^＋T细胞表位的提呈效应。同时，重组菌13A（pG2F）以静脉注射方式免疫C57BL／6小鼠，应用ELISPOT法检测特异性IFN-γ分泌细胞。结果：感染试验表明，大肠杆菌13A能向APE中运送真核表达质粒，并且外源GFP基因获得了表达。体外抗原提呈试验结果显示，在感染的早期（2小时），LK^b和BMI）C均可提呈重组菌13A（pG2F）运送的T细胞表位；在感染的晚期（48小时），LK^b细胞对CD8^＋T细胞表位的提呈效应增强。在同样的作用条件下，BMI）C对减毒菌运送的抗原表位的提呈效应要强于LK^b细胞。体内结果显示，大肠杆菌可以有效运送真核表达的CD8^＋T细胞表位并诱导小鼠产生特异性细胞免疫应答。结论：重组减毒大肠杆菌在体外和体内均能有效运送真核表达的CD8^＋T细胞表位，为基于减毒细菌的新型基因工程疫苗的分子设计提供了有益借鉴。     

Th9细胞与自身免疫性疾病

Th1、Th2、Th17和调节性T细胞（Treg）亚群是CD4＋T细胞亚群中的重要成员,其参与了人类及动物自身免疫性疾病的发病过程.既往认为,IL-9是由CD4＋Th2细胞分泌的细胞因子,是机体免疫应答中重要的调节因子.最近研究表明,机体内可能存在着一群新型的具有分泌IL-9和IL-10能力的CD4＋Th细胞亚群,称之为＂Th9＂细胞.该细胞亚群与自身免疫性疾病的相关性尚不清楚.     

以减毒沙门菌作为DNA疫苗载体的研究

目的 以真核表达质粒pCMVβ为报告基因，研究用芳香族氨基酸合成缺陷的沙门菌SL7207为载体以提高DNA疫苗免疫应答的可行性。方法 携带质粒pCMVβ的SL7207体外感染小鼠腹腔巨噬细胞后，用X-gal染色方法和RT-PCR方法检测巨噬细胞内β-gal的表达。小鼠口服免疫SL7207(pCMVβ)后，用RT-PCR方法检测β-gaⅠ基因在淋巴组织中的转录产物；用ELISA方法检测体液免疫；用^3H-TdR掺入法检测脾淋巴细胞增殖；用JAM试验检测杀伤性T淋巴细胞反应。结果 SL7207能有效地将质粒DNA传递到体外培养的巨噬细胞中并进行表达；小鼠口服携带有pCMVβ的SL7207后，能在脾脏、派伊尔结、肠系膜淋巴结中检测到目的基因的转录，并可诱导小鼠产生特异性体液免疫和细胞免疫。与肌内注射pCMVβ相比较，口服SL7207(pCMVβ)能更有效地诱导出细胞免疫应答。结论 减毒沙门菌SL7207作为DNA疫苗的载体，可经口服途径进行免疫，并可将质粒直接传递给抗原递呈细胞，诱导出以细胞免疫为主的免疫应答。     

Th9细胞及相关因子的研究进展

CD4+辅助性T(Th)细胞参与宿主防御反应,维持免疫细胞动态平衡。初始CD4+T细胞分化为不同细胞亚群由不同细胞因子决定。与亚群相关的相对特异的典型细胞因子不仅能够诱导初始CD4+T细胞分化,亦可作为感受器启动该细胞亚群发挥细胞免疫作用,还可以直接参与免疫应答反应。Th9细胞是一种CD4+辅助T细胞亚群,分泌白细胞介素9,已经发现多种细胞因子与Th9细胞有关,Th9细胞对自身免疫性疾病的作用越来越受到重视。本文就Th9细胞的生物学特性、分化相关信号分子及对自身免疫性疾病的作用综述如下。     

不同Th1/Th2细胞免疫应答支气管肺泡灌洗液中细胞学的变化

目的 探讨不同Th细胞优势应答下支气管肺泡灌洗液（BALF）中的细胞学变化，了解Th1/Th2细胞免疫应答的细胞和分子机制。方法 用鸡卵清蛋白（OVA）致敏Wistar大鼠（每组10只），制作致敏大鼠哮喘模型；用“冻干BCG”皮内注射制作BCG免疫大鼠模型。收集BALF并做HE染色，进行细胞分类计数。采用流式细胞术，测定BALF中，CD2^ ,CD28^ 及γδTCR^ T细胞占总淋巴细胞的百分率及平均荧光密度（MIF）。用原位杂交法，检测肺组织中IL－4mRNA的IFN－γmRNA的表达。用ELISA法检测血清IL－4和IFN－γ的浓度。结果 哮喘组BALF中淋巴细胞，嗜酸性粒细胞（EOS），浆细胞和中性粒细胞的总数，均显著多于正常组（P＜0．01）；BCG免疫组BALF中，淋巴细胞和巨噬细胞的总数也显著高于正常组（P＜0．001）。哮喘组BALF中，CD2^ T 细胞的明显增加。但哮喘组CD2^ T细胞的F1显著高于正常组及BCG组（P＜0．05）；BCG组BALF中，CD^2 T细胞的百分率与正常组相比产无显著差异（P＞0．05），其CD2^ T细胞的MFI显著高于正常组(P＜0．05）。哮喘组和BCG组BALF中，CD28^ 细胞占淋巴细胞的百分率显著多于正常组（P＜0．01）；BCG组CD28^ 细胞的MFI高于哮喘组（P＜0．01）。两组的CD28^ 细胞的MF1均显著多于正常组（P＜0．05）。哮喘组和BCG组BALF中，γδTCR^ 细胞占淋巴细胞的百分率显著高于正常组（P＜0．01）。结论 支气管哮喘患者Th2细胞的优势应答，与BALF中的B细胞，EOS，浆细胞和中性粒细胞等APC数的增加及T细胞上CD2的高表达有关；而BCG免疫组中的Th1细胞的优势应答与BALF中巨噬细胞，T细胞增加及T细胞上CD28的高表达有关。γδT细胞可能存在Th1/Th2细胞免疫模式，既参与哮喘免疫也参与BCG免疫过程，可能是调节Th0细胞分化的重要始动细胞。     

环孢霉素A对OVA免疫TCR转基因DO11.10小鼠CD4+CD25+T细胞的影响

目的：研究免疫抑制剂Csh对处于免疫应答状态的小鼠脾脏调节性CD4^＋CD25^＋T细胞影响。方法：采用流式细胞术检测卵白蛋白（OVA）免疫的DO11．10小鼠脾脏CD4^＋CD25^＋T细胞及其中Foxp 3^＋T细胞的变化。结果：OVA免疫后脾脏CD4^＋CD25^＋T细胞占CD4^＋T细胞百分数及Foxp 3^＋T细胞占CD4^＋CD25^＋T细胞百分数均增加，显示CsA可明显减少正常及免疫应答状态下小鼠脾脏CD4^＋CD25^＋T细胞及CD4^＋CD25^＋Foxp3^＋T细胞百分数。结论：CsA在抑制免疫应答的同时也可能抑制了免疫耐受的诱导。     

NKT细胞在CFA促进的Th1型免疫应答中的作用

为探索CFA促进小鼠对蛋白质抗原产生Th1介导的特异性免疫应答的机制 ,我们用OVA +CFA和OVA +IFA免疫C5 7BL/6小鼠 ,并于第 3天、第 8天后用MACS +FACS法分离引流淋巴结中NKT细胞 ,在体外经CD3单抗刺激 2d后测定其分泌的细胞因子数量及格局。同时于免疫后第 8天分离引流淋巴结抗原特异性T细胞 ,测定其分泌细胞因子的格局 ;并于再次免疫后 2周检测小鼠血清抗体类型。ELISA结果显示 ,与OVA +IFA组相比 ,OVA +CFA免疫小鼠引流淋巴结NKT细胞和抗原特异性T细胞分泌IFN γ的能力均显著升高 (P <0 0 5 ) ,且血清抗体类型以IgG2a为主。表明CFA促进抗原特异性Th0细胞极化为Th1细胞可能是通过激活NKT细胞使之在免疫应答局部产生大量的IFN γ而实现的     

Th细胞及其分化调节

幼稚CD4^＋T细胞可分化为Th1和Th2细胞，Th1主要产生IL-2、IFN-γ、TNF，增强吞噬细胞介导的抗感染机制，促进细胞免疫，也在器官特异性自身免疫疾病中起作用；Th2细胞主要产生IL-4、IL-5、IL-10、IL-13，促进B细胞增殖、分化和产生抗体，增强B细胞介导的体液免疫应答，在变态反应和机体抗寄生虫免疫中发挥作用。Th细胞分化主要由局部环境中的细胞因子及细胞内关键转录因子调控。转录因子STAT1、STAT4、IRF1和T—bet促使Th1细胞分化；转录因子STAT6、IRF4和GATA-3促使Th2细胞分化。     

颗粒性HBV多CTL表位基因诱导BALB/c小鼠的免疫应答研究

目的：研究含HBV多CTL表位的杂合HBc基因免疫BALB/c小鼠所诱导的免疫应答.方法：构建以HBV多CTL表位取代MIR区基因的杂合HBc基因疫苗（pHBcMep）并以肌肉注射方式免疫BALB/c小鼠,ELISA、流式细胞术、LDH释放法等分别检测血清特异性抗体与淋巴细胞分泌的IFN-γ水平、CD4^＋/CD8^＋T细胞比例变化及特异性CTLs活性等免疫应答指标.结果：颗粒性杂合HBc基因疫苗pHBcMep免疫BALB/c小鼠诱导明显抗体应答,末次免疫后2周,特异性抗preS2抗体阳性率达100%（12/12）,最高效价1∶1 000,同时诱导淋巴细胞分泌IFN-γ能力增强和刺激CTLs活化,其杀伤活性达16 IU30,CD4^＋/CD8^＋T细胞比例明显升高,且诱导明显回忆反应.结论：颗粒性HBV多CTL表位基因疫苗pHBcMep具有良好免疫原性,能迅速诱导高活性体液和细胞免疫应答及回忆反应.     

Efficient induction of mouse immune responses to hepatitis C virus by viral core protein-carrying attenuated Salmonella typhimurium

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for inclusion in a protective vaccine. However, this protein may attenuate the development of systemic immune responses because of its immunomodulatory properties. In this study, a eukaryotic expression plasmid, pCI-C, and an in vivo-inducible prokaryotic expression plasmid, pZW-C, for HCV core protein were constructed and transformed into attenuated Salmonella typhimurium aroA strain SL7207. The recombinant expression plasmids SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and immune responses specific to core protein were assessed. Immunization with bacteria SL7207/pCI-C led to a persistent decrease in the percentage of CD3(+)CD4(+) T cells and triggered weak anti-core IgG production. Splenocytes from SL7207/pCIC-immunized mice developed relatively weak proliferation response, low interferon-gamma secretion, and inferior cytotoxic activity compared with those from mice immunized with SL7207/pZW-C. Boost immunization with SL7207/pCI-C yielded limited improvement in the immune response, whereas boost with bacteria SL7207/pZW-C enhanced immune responses significantly. These results suggest that de novo host synthesis of native HCV core protein may cut down the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.     

Augmented induction of CD8+ cytotoxic T-cell response and antitumour resistance by T helper type 1-inducing peptide

The effector CD8(+) T cells recognize major histocompatibility complex (MHC) class I binding altered self-peptides expressed in tumour cells. Although the requirement for CD4(+) T helper type 1 (Th1) cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumour responses remain unclear. We examined whether a potent immunogenic peptide of Mycobacterium tuberculosis eliciting Th1 immunity contributes to the generation of CD8(+) T cells and to protective antitumour immune responses to unrelated tumour-specific antigens. Peptide-25, a major Th epitope of Ag85B from M. tuberculosis preferentially induced CD4(+) Th1 cells in C57BL/6 mice and had an augmenting effect on Th1 generation for coimmunized unrelated antigenic peptides. Coimmunization of mice with Peptide-25 and ovalbumin (OVA) or Peptide-25 and B16 melanoma peptide [tyrosinase-related protein-2 (TRP-2)] for MHC class I led to a profound increase in CD8(+) T cells specific for OVA and TRP-2 peptides, respectively. This heightened response depended on Peptide-25-specific CD4(+) T cells and interferon-gamma-producing T cells. In tumour protection assays, immunization with Peptide-25 and OVA resulted in the enhancement of CD8(+) cytotoxic cell generation specific for OVA and the growth inhibition of EL-4 thymoma expressing OVA peptide leading to the tumour rejection. These phenomena were not achieved by immunization with OVA alone. Peptide-25-reactive Th1 cells counteractivated dendritic cells in the presence of Peptide-25 leading them to activate and present OVA peptide to CD8(+) cytotoxic T cells.     

Construction and study of antigenic characteristics of recombinant salmonella strain producing TBI protein

A recombinant strain producing TBI protein (artificial protein containing HIV-1 B and T cell epitopes) was constructed on the base of attenuated Salmonella typhimurium SL7207 strain and BALB/c mice were immunized with this strain in a dose of 10(9) live cells orally or rectally. A single immunization with the recombinant salmonella strain induced humoral and cellular immune response to HIV-1; hence, this strain is a promising candidate for vaccine against HIV.     

Pathogenicity island 2 mutants of Salmonella typhimurium are efficient carriers for heterologous antigens and enable modulation of immune responses

The potential use as vaccine delivery system of Salmonella typhimurium strains harboring defined mutations in the sseC (HH104) and sseD (MvP101) genes, which encode putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2, was evaluated and compared with that of the well-characterized aroA mutant strain SL7207 by using beta-galactosidase (beta-Gal) as a model antigen. When orally administered to immune-competent or gamma interferon-deficient (IFN-gamma-/-) BALB/c mice, both mutants were found to be highly attenuated (50% lethal dose, >10(9) bacteria). Both strains were also able to efficiently colonize and persist in Peyer's patches. Immunization with HH104 and MvP101 triggered beta-Gal-specific serum and mucosal antibody responses equivalent to or stronger than those observed in SL7207-immunized mice. Although immunoglobulin G2 (IgG2) serum antibodies were dominant in all groups, IgG1 was also significantly increased in mice vaccinated with MvP101 and SL7207. Comparable beta-Gal-specific IgA and IgG antibodies were detected in intestinal lavages from mice immunized with the different strains. Antigen-specific CD4(+) T-helper cells were generated after vaccination with all vaccine prototypes; however, responses were significantly more efficient when HH104 and MvP101 were used (P < 0.05). Significantly higher levels of IFN-gamma were produced by restimulated spleen cells from mice immunized with HH104 than from those vaccinated with the MvP101 or SL7207 derivatives (P 相似文献   

Rescue of memory CD8+ T cell reactivity in peptide/TLR9 ligand immunization by codelivery of cytokines or CD40 ligation

The capability of cellular immune components to rapidly recall upon challenge in most situations decides the efficacy of a vaccine. Here, we show that immunization of mice with SSIEFARL peptide (immunodominant epitope in glycoprotein B of herpes simplex virus type 1, aa498-505) combined with TLR9 ligand in the absence of helper CD4(+) T cell activation generates a functionally impaired CD8(+) T cell memory response. Codelivery of IL-12, IL-15, or anti-CD40 together with MHC class-I-restricted peptide combined with TLR9 ligand at inception of immunization resulted in generation of memory CD8(+) T cells that were several fold less compromised than immunization with peptide alone. Furthermore, administration of either plasmid DNA encoding IL-15 or anti-CD40 mAb but not rIL-12 during the memory phase restored the reactivity of memory CD8(+) T cells. Moreover, the rescued CD8(+) T cells preserved their cytotoxic capability and were able to clear a recombinant vaccinia virus encoding glycoprotein B of HSV. Our results indicate that good memory CD8(+) T cell response to peptide immunization can be achieved by using costimulatory procedures at the time of priming or recall immunization.     

Attenuated Salmonella typhimurium reduces ovalbumin-induced airway inflammation and T-helper type 2 responses in mice

Cytokines produced by Th2 cells are responsible for the pathogenesis of asthma. Th1-biased immune responses caused by attenuated salmonella have the potential to relieve asthmatic symptoms. We evaluated whether oral administration of attenuated salmonella could modulate allergic responses in a chicken ovalbumin (OVA)-induced asthmatic murine model. Mice were fed with attenuated salmonella SL7207 one dose before and three doses during the induction of an allergic response. Lung histology, percentages of eosinophil in bronchoalveolar lavage fluid, serum levels of OVA-specific antibodies and cytokine production by OVA-activated splenocytes were evaluated in mice with or without the administration of SL7207. A significant reduction in pulmonary eosinophilic infiltration was observed in mice receiving attenuated salmonella. Lower levels of OVA-specific IgG1 but higher titres of OVA-IgG2a in serum were also detected in this group. Splenocytes from salmonella-fed mice produced lower levels of Th2 cytokines upon OVA stimulation. The administration of attenuated salmonella significantly suppressed immunopathological symptoms in OVA-sensitized mice. Inhibition of Th2 responses might explain the potential mechanisms. This study provides some evidence for the feasibility of attenuated salmonella as an effective vaccine for allergic diseases.     

Induction of Cell-Mediated Immunity against Mycobacterium tuberculosis Using DNA Vaccines Encoding Cytotoxic and Helper T-Cell Epitopes of the 38-Kilodalton Protein

Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed DNA vaccines encoding cytotoxic T lymphocytes (CTL) and T helper cell (Th) epitopes of the 38-kDa lipoglycoprotein of Mycobacterium tuberculosis and analyzed and compared their immunogenicities with that of pXJ38, a DNA vaccine encoding the entire 38-kDa protein (X. Zhu, N. Venkataprasad, H. S. Thangaraj, M. Hill, M. Singh, J. Ivanyi, and H. M. Vordermeier, J. Immunol. 158:5921-5926, 1997). Plasmid DNAs encoding a CTL epitope, P3 (pP3), a Th epitope (vTh), or both the Th and the P3 epitopes (pThP3) were prepared and tested in C57BL6/J (H-2(b)) mice. Our results confirmed that DNA immunization with pXJ38 induces strong CD8(+) CTL and Th1 responses (high gamma interferon [IFN-gamma], low interleukin-4 [IL-4]). Coadministration of plasmid DNAs encoding a Th epitope with those encoding a CTL epitope (vTh+pP3) elicited both antigen-specific CD8(+) CTL and Th1 responses. High levels of IFN-gamma were secreted by spleen cells from all plasmid DNA-vaccinated mice after in vitro stimulation with the recombinant 38-kDa protein. Small or undetectable amounts of IL-4 were observed, which indicates the induction of a Th1-like response. Multiple-epitope vaccination by vTh+pP3 or pThP3 resulted in a broader Th1 response to peptide or epitopes than the single-epitope plasmid DNAs. Antigen-specific immunoglobulin G2a was only detected in sera from mice immunized with the plasmid pXJ38, and not in mice immunized with the epitope-based DNA vaccines. Thus, the absence of an antibody response after immunization with epitope plasmid DNAs and their ability to trigger only a specific cellular immune response may prove to be important advantages for a vaccine against tuberculosis.     

Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for T cells against HIV

An immunization strategy using attenuated bacteria to deliver DNA vaccine plasmids to mucosal sites may induce protective T cell responses against sexual HIV transmission. In a murine intranasal (i.n.) immunization model, we demonstrate that transiently persistent Deltaasd Shigella flexneri strain 15D harboring DNA vaccines induces HIV- and SIV-specific gamma interferon (IFN-gamma) producing CD8+ T cells among splenocytes more efficiently than either a longer persisting DeltaaroD Salmonella typhimurium strain SL7207 or transiently persistent S. typhi strain Ty21a harboring DNA vaccines. Also, the frequency of antigen-specific gamma interferon (IFN-gamma) producing cells induced by Shigella 15D harboring a DNA vaccine were comparable to that induced by intramuscular (i.m.) immunization with purified DNA vaccine. Moreover, the magnitude of mucosal and systemic antigen-specific IgA and IgG responses after immunization were dependent upon the route (i.m. vs. i.n.) of inoculation, with i.n. Shigella 15D DNA vaccines generating higher levels of HIV-specific IgA in vaginal washings than i.m. purified DNA vaccine. Deltaasd S. flexneri is a promising vector for mucosal DNA vaccine immunization against HIV.