Kozak序列对金黄色葡萄球菌黏附因子FnBPA-A DNA疫苗诱导小鼠免疫应答的影响

黏附因子FnbpA(纤连蛋白结合蛋白A,Fibronectin binding protein A)是金黄色葡萄球菌表面的蛋白质成分,是该菌感染早期最重要的致病因子,可促进其对寄主组织的侵入,也是一个有潜力的免疫靶标。将牛乳源金黄色葡萄球菌中FnBPA基因的A功能区克隆至真核表达载体中,分别构建含Kozak序列和不含Kozak序列的FnBPA-A基因真核表达载体,重组质粒经鉴定测序正确后,免疫C57BL/6小鼠检测其抗体水平和淋巴细胞增殖情况,并对各组小鼠进行攻毒实验。检测的结果表明Kozak修饰的重组DNA在血清抗体效价(P<0.05)和免疫保护率方面均优于不含Kozak序列的重组DNA,在刺激淋巴细胞增殖方面Kozak修饰的重组DNA的刺激效果虽然也高于不经修饰的重组DNA,但是差异不显著(P>0.05)。根据总体的免疫效果来看,Kozak序列对增强FnBPA的重组DNA疫苗诱导的免疫应答起了不容忽视的作用。     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.     

携带HBV包膜大蛋白DNA疫苗的减毒沙门菌在小鼠诱导免疫应答

探索一种简便、有效的乙型肝炎病毒DNA疫苗免疫方法。将编码绿色荧光蛋白的真核表达质粒pEGFPN1转化到减毒鼠伤寒沙门菌SL7207,灌胃饲服BALB/c小鼠,流式细胞术检测出小鼠脾细胞内表达的绿色荧光蛋白;构建编码HBV包膜大蛋白的DNA疫苗pCIS1S2S,分别以SL7207为载体的口服途径或直接肌肉注射途径免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,结果表明两种免疫途径均能在小鼠体内诱生细胞和体液免疫应答,但口服途径诱导免疫应答的强度明显强于肌肉注射途径。口服携带HBV DNA疫苗的减毒伤寒沙门菌可能代表一种简便、有效的治疗乙型肝炎的新方法。      

The activation of guinea pig T lymphocytes by anti-beta 2-microglobulin serum.

In order to study further the role of beta 2-m in the regulation of the immune response, we have examined the effects of a goat anti-guinea pig beta 2-m serum on a number of T lymphocyte functions in vitro. Anti-beta 2-m serum produced a marked inhibition of the response of peritoneal exudate T cells to antigen and mitogen stimulation. Surprisingly, a marked activation of lymph node T lymphocyte proliferation was observed in the absence of antigen or mitogen stimulation. This stimulatory effect of anti-beta 2-m serum was shown to be specific for beta 2-m and required the presence of macrophages. The T cell proliferative response induced by anti-beta 2-m could not be blocked by antisera to the antigens of the guinea pig MHC. These studies suggest that beta2-m may play some critical role in the immune response at the level of T cell activation.     

免疫共刺激分子OX40L对乙型肝炎核酸疫苗的免疫佐剂作用

[目的]为了进一步增强HBV DNA疫苗的免疫反应,本研究将共刺激分子OX40L 作为HBV DNA疫苗的分子佐剂免疫小鼠,旨在探讨共刺激分子OX40L对HBV DNA疫苗诱导体液和细胞免疫应答的影响.[方法]我们将HBV DNA疫苗(pcDS2)单独或联合共刺激分子质粒pOX40L免疫C57BL/6小鼠;分别在第0,2,4周进行免疫,在第6周检测抗-HBs IgG、IgG1和IgG2a,T淋巴细胞增殖指数,细胞因子表达水平和体内细胞毒性T淋巴细胞杀伤作用(CTL)等免疫学指标.[结果]pceDS2联合pOX40L免疫组小鼠的抗-HBs水平显著提高,抗-HBs IgG亚类以IgG2a占优;免疫小鼠的T淋巴细胞体外经乙型肝炎表面抗原(HBsAg)刺激后,联合免疫组刺激指数(SI)明显高于pcDS2组;联合免疫组CD4 + T淋巴细胞的IL-4和IFN-γ表达水平及CD8 + T淋巴细胞的IFN-γ表达水平显著升高;DNA疫苗免疫的各组小鼠,HBsAg特异性体内CTL高于对照组,其中联合免疫组小鼠的体内CTL杀伤作用最强.[结论]共刺激分子OX40L不仅能增强HBV DNA疫苗诱导特异性体液免疫应答,还能增强特异性细胞免疫反应,尤其增强体内CTL的杀伤活性,为HBV DNA疫苗的研究奠定了基础.     

Enhancement of the DNA synthetic response of antigen-primed lymph node cells by splenic promoter cells: characterization of the splenic promoter cells.

Rabbits were immunized in the footpad with diphtheria toxoid in complete Freund's adjuvant. At various times after immunization, cells harvested from the spleen, the draining (immune) and the opposing (control) lymph nodes (LN) were assayed for their proliferative response to incorporate tritiated thymidine upon exposure to the priming antigen. Although the immune LN cells responded by a substantial incorporation of thymidine, cells from the control LN and the spleen either did not respond or responded feebly. An enhancement in the response of the immune LN cells was observed when they were cultured in the presence of nonresponsive spleen cells. Pretreatment of spleen cells with mitomycin C did not abolish the enhancement. This suggests that the target cells which respond to the antigen are derived from the immune LN whereas the promoter cells which enhance the response are present in the spleen. The removal of adhering cells by glass wool columns and of Ig-bearing cells by anti-Ig immunoabsorbent columns from the spleen did not reduce the enhancing capacity of the nonadhering cells. Conversely, the killing of splenic T cells by specific heterologous antiserum directed against rabbit thymus lymphocyte antigen abolished the enhancement. Thus, the promoter cell which facilitates the enhancement has been characterized as a nonadherent, splenic T cell.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

猪细小病毒核酸疫苗的构建及其对小鼠免疫原性的研究

将猪细小病毒VP2基因克隆至pCI-neo真核表达载体中,构建了pCIneo-VP2重组质粒,转染至PK-15细胞中,利用免疫荧光方法检测在体外表达情况;并以小鼠为动物模型,将pCIneo-VP2、pCI-neo重组质粒、猪细小病毒活疫苗和对照组通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-VP2质粒 1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-VP2能够有效诱导机体产生体液免疫和细胞免疫,为研制出高效、新型猪细小病毒疫苗提供了科学依据和试验依据。     

A HER-2/neu peptide admixed with PLA microspheres induces a Th1-biased immune response in mice

The elimination of cancer cells requires strong cellular immune responses, and these responses are induced by the activation of Th1 lymphocytes. In this work, the possibility of inducing a Th1 type of immune response in vivo by mixing a HER-2/neu synthetic CTL (cytotoxic T lymphocyte) peptide [HER-2/neu (789-797)], with poly-lactide (PLA) microspheres was investigated. Various formulations of the peptide were administered to HLA-A2.1 transgenic (HHD) mice. Cellular experiments, assessing proliferation and cytokine determination in splenocyte culture supernatants, were carried out in order to evaluate the type of immune response to the antigen. The in vivo administration of the peptide antigen admixed with the PLA microspheres induced a potent immune response which was comparable to that induced by the combination of the antigen in complete Freund's adjuvant (CFA). Furthermore, the cytokine profile produced by the T lymphocytes of the immunized animals indicated that the combination of the peptide antigen with the PLA microspheres induced a strong Th1 biased immune response to the antigen. The time of peptide incubation with the microspheres prior to administration did not affect the immune response, which further simplifies the preparation of this type of vaccine. The results justify further investigation of the possibility of inducing effective cellular immune responses against cancer cells overexpressing HER-2/neu molecules by simply mixing appropriate HER-2/neu peptide antigens with PLA microspheres.     

Generation of helper T cells that recognize a cross-reactive idiotype through a network mechanism.

T cells that recognize the cross-reactive idiotype expressed on the heavy (H) chain of M104E (IgM, lambda 1) were induced in BALB/c mice by immunization with Dextran B-1355. T cells derived from mice immunized with 1 mg of Dextran B-1355 showed a marked proliferative response against M104E, whereas T cells from mice immunized with Ficoll or lesser amounts of Dextran B-1355 did not. BCL1Id, which had an immunoglobulin isotype identical to M104E, did not induce proliferation of the T cells. These T cells also proliferated against J558 (IgA, lambda 1) which shared the cross-reactive idiotype of the anti-alpha (1----3) glucosidic linkage antibody with M104E on the H chain. The T cells proliferated more efficiently against F(ab')2-104E, Fab-104E and H104E, the H chain of M104E, than against intact M104E. The T cell proliferative response against the idiotype on M104E or even H104E required macrophages as antigen-presenting cells (APC) and the response was inhibited when APC were treated with NH4Cl or chloroquine, inhibitors of antigen processing. Moreover, anti-CD4 antibody or anti-Ia antibody inhibited the proliferative response. These results indicated that anti-idiotypic T cells of the helper type, which recognized a cross-reactive idiotype associated with Ia molecules in processed form, could be induced physiologically through a network mechanism.     

IL-12增强HBV融合抗原DNA疫苗在小鼠诱导的细胞免疫应答

乙型肝炎病毒(hepatitis B virus,HBV)极易形成慢性感染,主要机制在于感染者不能产生强有力的细胞免疫应答以清除病毒[1].慢性HBV感染者体内虽然存在HBV抗原特异性T淋巴细胞,但对HBV抗原的反应性较低.研究发现,增强这类T淋巴细胞的反应性,可以促进HBV的清除[2].     

Measles virus nucleocapsid protein protects rats from encephalitis.

Lewis rats immunized with recombinant vaccinia virus expressing the nucleocapsid (N) protein of measles virus were protected from encephalitis when subsequently challenged by intracerebral infection with neurotropic measles virus. Immunized rats revealed polyvalent antibodies to the N protein of measles virus in the absence of any neutralizing antibodies as well as an N protein-specific proliferative lymphocyte response. Depletion of CD8+ T lymphocytes did not abrogate the protective potential of the N protein-specific cell-mediated immune response in rats, while protection could be adoptively transferred with N protein-specific CD4+ T lymphocytes. These results indicate that a CD4+ cell-mediated immune response specific for the N protein of measles virus is sufficient to control measles virus infections of the central nervous system.     

Chicken T lymphocyte clones with specificity for Eimeria tenella. I. Generation and functional characterization

T lymphocyte clones reacting specifically with the antigenic components of Eimeria tenella were generated from splenic lymphocytes of immunized chickens and were maintained for 12 to 14 wk in vitro. These T cell growth factor-dependent T lymphocyte clones from bursectomized and normal chickens proliferated in vitro when stimulated with antigens from different developmental stages of homologous but not heterologous species of the parasite. Specific proliferative responses of the cloned T cells showed an absolute requirement for antigen presentation by histocompatible antigen-presenting cells. Some of the T cell clones exhibited functionally discrete interactions with syngeneic primed B cells; 25% of the T cell clones from immunized normal chickens and 7% of those obtained from immunized bursectomized chickens showed antigen-dependent helper activity and induced specific antibody production by syngeneic primed B cells. Of the T cell clones from immunized normal chickens, 19% showed suppression of in vitro antibody production in comparison to 7% of those isolated from immunized bursectomized chickens. The frequency of cloned T cells with ability to induce cytotoxic activity in macrophages against the sporozoites of E. tenella was much higher in those isolated from bursectomized chickens (80%) than in those isolated from normal chickens. Because both bursectomized and normal chickens can be immunized by repeated infections, differences in the distribution among cloned T cells suggest different effector mechanisms of immunity against coccidiosis in these chickens. Lack of B cells seem to affect the development of T cell immunity as reflected by slower development of immunity and enhanced activation of cytotoxic T cell function.     

甲状腺素对大鼠实验性自体免疫心肌炎的作用

实验选用清洁级F344/NSIc大鼠,腹腔内注射甲状腺素（10μg/100g体重）,观察甲状腺素对肌球蛋白诱导的实验性自体免疫心肌炎的作用,并用免疫荧光技术结合流式细胞仪分析甲状腺素对免疫反应的影响。结果显示,甲状腺素对肌球蛋白免疫所致的心肌坏死,炎性细胞浸润和纤维化没有明显的改善作用;甲状腺素可诱导外周血B淋巴细胞比例升高。但对辅助T细胞和抑制T细胞比例。辅助T细胞/抑制T细胞比率没有影响。     

猪繁殖与呼吸综合征病毒GP5蛋白实验性核酸疫苗的构建及其免疫原性的初步研究

扩增了猪繁殖与呼吸综合征病毒(PRRSV)长春分离株ORF5基因,并将其克隆至真核表达载体pVAX1、pVIR-IL-18的CMV启动子下游,构建成真核表达质粒。通过Western blot以及IFA检测方法确认,所构建的2个重组DNA疫苗质粒在真核细胞能进行有效的转录,并表达了目的蛋白(GP5)。同时辅以不同佐剂进行了BALB/c小鼠免疫试验,检测了免疫后的ELISA抗体水平和脾T淋巴细胞亚群数量。结果表明:共表达猪IL-18基因的核酸疫苗pVIR-IL-18-ORF5免疫后的ELISA抗体水平和脾T淋巴细胞亚群数量均优于pVAX1-ORF5,而且加佐剂实验组优于未加佐剂的实验组。     

Altered T Lymphocyte Proliferation upon Lipopolysaccharide Challenge Ex Vivo

Context

Sepsis is characterized by the development of adaptive immune cell alterations, which intensity and duration are associated with increased risk of health-care associated infections and mortality. However, pathophysiological mechanisms leading to such lymphocyte dysfunctions are not completely understood, although both intrinsic lymphocyte alterations and antigen-presenting cells (APCs) dysfunctions are most likely involved.

Study

The aim of the current study was to evaluate whether lipopolysaccharide (LPS, mimicking initial Gram negative bacterial challenge) could directly impact lymphocyte function after sepsis. Therefore, we explored ex-vivo the effect of LPS priming on human T lymphocyte proliferation induced by different stimuli.

Results

We showed that LPS priming of PBMCs reduced T cell proliferative response and altered IFNγ secretion after stimulation with OKT3 but not with phytohaemagglutinin or anti-CD2/CD3/CD28-coated beads stimulations. Interestingly only LPS priming of monocytes led to decreased T cell proliferative response as opposed to LPS priming of lymphocytes. Importantly, LPS priming was associated with reduced expression of HLA-DR, CD86 and CD64 on monocytes but not with the modification of CD3, CTLA4, PD-1 and CD28 expressions on lymphocytes. Finally, IFNγ stimulation restored monocytes accessory functions and T cell proliferative response to OKT3.

Conclusion

We conclude that LPS priming does not directly impact lymphocyte functions but reduces APC’s capacity to activate T cells. This recapitulates ex vivo indirect mechanisms participating in sepsis-induced lymphocyte alterations and suggests that monocyte-targeting immunoadjuvant therapies in sepsis may also help to improve adaptive immune dysfunctions. Direct mechanisms impacting lymphocytes being also at play during sepsis, the respective parts of direct versus indirect sepsis-induced lymphocyte alterations remain to be evaluated in clinic.     

Improved cellular immune response elicited by a ubiquitin-fused DNA vaccine against Mycobacterium tuberculosis

This study evaluated the immune response elicited by a ubiquitin (Ub)-fused MPT64 DNA vaccine against Mycobacterium tuberculosis. BALB/c mice were vaccinated with plasmid DNA encoding MPT64 protein, Ub-fused MPT64 DNA vaccine (UbGR-MPT64), and negative DNA vaccines, respectively. MPT64 DNA vaccine immunization induced a Thl-polarized immune response. The production of Thl-type cytokine (interferon-gamma [IFN-γ]) and proliferative T cell responses were enhanced significantly in mice immunized with UbGR-MPT64 fusion DNA vaccine, compared with nonfusion DNA vaccine. Moreover, this fusion DNA vaccine also resulted in an increased relative ratio of IgG2a to IgGl and the cytotoxicity of T cells. IFN-γ intracellular staining of splenocytes indicated that UbGR-mpt64 fusion DNA vaccine activated CD4+ and CD8+ T cells, particularly CD8+ T cells. Thus, this study demonstrated that the UbGR-MPT64 fusion DNA vaccine inoculation could improve antigen-specific cellular immune responses, which is helpful for protection against TB.     

重组质粒与重组蛋白共免疫诱导HBsAg特异性T细胞免疫抑制

摘要：【目的】为了探索治疗急性乙型肝炎和爆发性乙型肝的新策略,本研究将HBV DNA疫苗和相应抗原的蛋白质分子联合免疫小鼠,旨在探讨联合免疫对小鼠抗原特异性T细胞增殖反应的影响。【方法】我们将HBV DNA疫苗（pcDS2）和相应抗原蛋白质分子(HBsAg)联合免疫BALB/c小鼠;分别在第0、2和4周进行免疫,在第6周用ELISA方法检测抗-HBs IgG效价,MTT和流式细胞仪检测T细胞增殖反应,及流式细胞仪检测细胞因子表达水平。【结果】pcDS2和HBsAg联合免疫组小鼠的抗-HBs水平显著提高;免疫小鼠的T细胞体外经HBsAg刺激后, 联合免疫组刺激指数(SI)明显降低;经流式细胞仪检测进一步证实联合免疫组T细胞增殖反应被显著抑制;联合免疫组T细胞表达IL-10和Foxp3水平显著升高。【结论】pcDS2和HBsAg联合免疫能诱导产生特异性体液免疫应答,但不能诱导产生抗原特异性T细胞增殖反应;T细胞增殖反应被显著抑制可能与T细胞表达IL-10和Foxp3上调有关;本研究为急性乙型肝炎和爆发性乙型肝炎治疗及HBV疫苗的研究奠定了基础。     

Major-histocompatibility-complex-class-II-positive cells and interleukin-2-dependent proliferation of immune T cells are required to reject carcinoma cells in the guinea pig

Summary Tumor immunity induced by bacillus Calmette-Guérin was studied in the line 10 hepatocellular carcinoma (line 10) in the strain-2 guinea pig. Line 10 immunity was investigatedin vitro with a lymphocyte proliferation assay using line 10 tumor protein extracted with 3 M KCl andin vivo by adoptive transfer of line-10-immune spleen cells. Monoclonal antibodies against guinea pig leucocyte markers were used to block functional properties of the immune cells in order to determine which cell types or cell markers are involved in the immune response to the line 10 tumor.In vitro cells from the spleen, peripheral blood and regional lymph node of immune animals reacted with a proliferative response to line 10 protein. This antigen-specific response was caused by T cells and was regulated by major histocompatibility complex (MHC) class II molecules. In blocking experiments it was found that CT5 (anti-PanT), or MSgp4 [anti-(MHC class I antigen)] monoclonal antibodies did not block but some-times stimulated the proliferative response. The effect of H159 (anti-PanT) was irregular, while H155 [anti-(T helper)], and 5C3 [anti-(IL-2 receptor)] monoclonal antibodies blocked the response almost completely. We studied the relevance of the resultsin vitro obtained and found that mAb 5C3 [anti-(IL-2 receptor)] inhibited the adoptive transfer of line 10 immunity, suggesting that the rejection of line 10 cells is caused by a mechanism that is interleukin-2 (IL-2)-dependent. Moreover, complement lysis of MHC-class-II-antigen-positive immune spleen cells inhibited completely the rejection of the line 10 tumor cell challenge in the adoptive-transfer experiments. In conclusion, our data show that MHC class II molecules or cells possessing these molecules are involved in immunity against line 10 tumor cells, as (a) monoclonal antibodies against MHC class II antigens inhibited thein vitro proliferative response of T cells to tumor antigens and (b) removal of MHC-class-II-positive immune spleen cells abrogated the antitumor effect in the adoptive-transfer experiments. Interleukin-2-dependent proliferation of immune T cells is required for the rejection of line 10 tumor cells.