含CpG基序的寡脱氧核苷酸联合乙型肝炎表面抗原免疫转基因小鼠的实验研究

有研究表明,含CpG基序的寡脱氧核苷酸(CpG oli-godeoxynucleotides,CpG ODN)对天然和获得性免疫系统具有广泛的刺激作用,在动物体内可作为多种外源抗原,包括乙型肝炎表面抗原(HBsAg)的Th1型免疫佐剂,达到增强体液免疫及细胞免疫的作用,而Th1型免疫反应对乙型肝炎病毒(HBV)的清除是必要的,因此CpG ODN联合HBsAg可望成为慢性乙型肝炎的有效治疗性疫苗。为此,我们将人工合成硫     

CpG寡脱氧核苷酸在系统性红斑狼疮研究中的应用

CpG寡脱氧核苷酸(CpG oligodeoxynucleotides,CpG ODN)为含有胞嘧啶和鸟嘌呤二核苷酸的寡聚脱氧核苷酸单链,通过与Toll样受体9(TLR9)结合而发挥作用.天然的含有未甲基化CpG基序的脱氧寡核苷酸广泛存在于细菌DNA中,具有强大的免疫刺激效应,能够活化多种细胞并诱导产生多种细胞因子.人工合成的模拟细菌DNA的CpG ODN也具有免疫刺激效应,因具有抵抗核酶降解特性、稳定性好于天然DNA,而用于临床多领域的研究.CpG ODN不仅在机体过敏性疾病、免疫接种及诱导抗肿瘤免疫应答中发挥重要作用,近年来研究显示CpG ODN同样参与系统性红斑狼疮(systemic lupus erythematosus,SLE)的调控.本文就CpG ODN的生物学特征、活化机制及其在SLE研究中的应用作一综述.     

CpG寡核苷酸作为黏膜佐剂的研究进展

含未甲基化的CpG二核苷酸的寡聚脱氧核苷酸模体(CpG ODN)具有很强的黏膜佐剂活性,已成为近年来的研究热点之一。应用CpG ODN与各种病毒和细菌抗原进行了大量实验研究,所涉及的黏膜免疫途径包括滴鼻、口服和生殖道。CpG ODN可与传统的黏膜佐剂CT和LT产生协同效应,对人体无毒副作用,是一种极具应用前景的黏膜佐剂。目前认为,CpG ODN的佐剂效应发挥与TLR9识别和后续的一系列信号转导密切相关。     

白细胞介素2作为佐剂增强乙型肝炎病毒基因疫苗诱导的免疫应答

目的 :构建乙型肝炎病毒 (HBV)基因疫苗 pCR3.1 S ,观察重组人白细胞介素 2 (rhIL 2 )作为佐剂对其诱导BALB/c小鼠产生免疫应答的影响。方法 :以ELISA法检测免疫小鼠血清抗HBs抗体 ,另用3 H TdR掺入法测定淋巴细胞增殖活性 ,初步研究不同组的体液和细胞免疫应答。结果 :rhIL 2组免疫小鼠抗HBs抗体和淋巴细胞增殖活性与对照组比较差异有统计学意义 (P <0 .0 5 )。结论 :HBV基因疫苗可诱发BALB/c小鼠产生良好的免疫应答 ,rhIL 2作为佐剂可增强其免疫效应。     

应用含CpG基序的寡核苷酸增强乙型肝炎病毒基因疫苗诱导的细胞免疫应答

目的 探讨人工合成的含ＣｐＧ基序的寡核苷酸（ＯＤＮ）作为佐剂对乙型肝炎病毒（ＨＢＶ）基因疫苗诱导小鼠产生细胞免疫应答的影响。方法 构建编码ＨＢＶ基因疫苗,人工合成含ＣｐＧ基序（ｍｏｔｉｆ）的硫代磷酸寡核苷酸作为佐剂,以Ｂａｌｂ／ｃ．１－Ｓ小鼠作为实验动物进行免疫接种;采用＾３Ｈ－ＴｄＲ法、＾５１Ｃｒ４ｈ释放法等分别检测免疫小鼠的淋巴细胞增殖和杀伤功能。结果 与空载体对照组相比较,ＨＢＶ基因疫苗诱发     

CpG寡核苷酸可作为幽门螺杆菌疫苗的佐剂

目的　研究一种新型黏膜佐剂 (一段寡核苷酸 ,序列为 :非甲基化的 5′ …嘌呤 嘌呤 CpG 嘧啶 嘧啶 … 3′(CpG ODN)是否可作为幽门螺杆菌 (Hp)疫苗的佐剂成分。 方法　C5 7BL/6小鼠经口灌胃给予Hp整菌超声粉碎物 (WCS) /CpG ODN或WCS/霍乱毒素 (CT) ,或经鼻给予WCS/CpG ODN ,并设立相应对照组。免疫程序及途径为每周 1次 ,共 4次 ,最后 1次免疫后 1周 ,以 5× 10 8Hp活菌攻击小鼠 ,攻击后 2周和 8周时 ,处死小鼠 ,鉴定Hp感染情况。同时收集小鼠血清、唾液、胃液 ,ELISA法检测血清中IgG、IgG1、IgG2a及IgA水平和唾液、胃液中IgA水平。 结果　以WCS作为抗原 ,不同佐剂及接种途径免疫小鼠 ,保护率分别为 :口服CT组 75 % (9/12 ) ,口服CpG ODN组 0 % (0 /10 ) ,滴鼻CpG ODN组 70 % (7/10 )。第 2和第 8周滴鼻CpG ODN组小鼠血清IgG2a水平显著高于未免疫的对照组(P <0 .0 5 )。结论　通过鼻道免疫CpG ODN是一很有前景的Hp疫苗佐剂成分。     

STAg联合CpG ODN和CT鼻内免疫小鼠诱导的特异性抗体应答和脾淋巴细胞的特异性增殖反应

目的 观察可溶性速殖子抗原(STAg)联合CpG寡核苷酸(CpG ODN)和/或霍乱毒素(CT)鼻内免疫BALB/c小鼠后血清IgG、肠冲洗液IgA抗体水平及脾淋巴细胞体外特异性增殖反应,探讨CpG ODN和CT的佐剂效应. 方法 6周龄BALB/c小鼠60只,随机分为5组,分别用20 μl PBS(PBS组)、20 μg STAg(抗原组)、20 μg STAg 10 μg CpG ODN(CpG佐剂组)、20 μg STAg 1 μg CT(CT佐剂组)、20 μg STAg 1 μg CT 10 μg CpG ODN(联合佐剂组)滴鼻,间隔2周,免疫2次.末次免疫后第20 d颈椎脱臼处死小鼠,收集血清和肠冲洗液,ELISA法检测血清弓形虫特异性IgG和肠冲洗液IgA.分离脾淋巴细胞,用四甲基偶氮唑盐(MTT)法检测致敏脾淋巴细胞体外增殖活性. 结果 ELISA法测定PBS组、抗原组、CpG佐剂组、CT佐剂组及联合佐剂组小鼠血清IgG A值分别为0.025 6±0.012 1、0.054 2±0.024 4、0.109 4±0.007 5、0.110 1±0.009 1和0.124 6±0.013 7,肠冲洗液IgA A值分别为0.141 8±0.030 2、0.415 2±0.080 7、0.526 9±0.075 4、0.846 5±0.115 5和1.128 9±0.100 3,脾淋巴细胞刺激指数SI分别为1.159 0±0.275 5、1.364 0±0.349 8、1.582 0±0.375 3、1.928 0±0.278 9和2.371 0±0.646 9,差异均有统计学意义(P＜0.01);联合佐剂组肠冲洗液IgA水平和脾淋巴细胞的SI值与CpG佐剂组比较差异有统计学意义(P＜0.05). 结论 STAg联合CpG ODN和/或CT鼻内免疫小鼠可有效诱导机体免疫反应,两种佐剂联合免疫的效果优于单一抗原或抗原联合单一佐剂.     

日本血吸虫22.6 kDa抗原编码基因序列中抑制性片段的确定

目的探讨日本血吸虫22.6kDa（Sj22.6）抗原编码基因序列中是否存在抑制性片段。方法用人工合成的来自Sj22.6抗原编码基因序列中的不同寡脱氧核苷酸和自小鼠脾脏分离的单个核细胞共同孵育,以对小鼠具有刺激作用的免疫刺激序列CpG1826作为刺激物,淋巴细胞增殖试验检测待测定寡脱氧核苷酸对CpG1826诱导的增殖是否具有抑制作用及其作用特征。结果存在于Sj22.6抗原编码基因序列中的寡脱氧核苷酸F311能够抑制刺激性CpG1826诱导的淋巴细胞增殖作用（P〈0.05）;当寡脱氧核苷酸F311与CpG1826的物质的量之比为1∶10和1∶3时无明显抑制作用（P〉0.05）,当物质的量之比为1∶1和3∶1时,对CpG诱导的淋巴细胞增殖的抑制率分别为11%和58%;寡脱氧核苷酸F311在先于CpG1826孵育2h加入表现出最强抑制作用。结论Sj22.6抗原编码基因序列中存在具有抑制作用的片段,抑制作用与其浓度和作用时间呈正相关。     

CpG DNA在寄生虫感染免疫中的作用

近年研究发现，细菌胞嘧啶鸟嘌呤二核苷酸(CpG DNA)和合成的含CpG序列的寡脱氧核苷酸(CpG ODN)具有强烈的非特异性的免疫刺激作用，其共同特征为都含有一些具有免疫学活性的短核苷酸序列。美国依阿华大学医学院Krieg等将此类特定结构称为CpG基元，另外有人称其为CpG免疫刺激序列(ISS)。多年来，细菌DNA(bDNA)被认为是没有免疫作用的。     

CpG-ODN联合HBsAg对慢性乙型肝炎患者外周血树突状细胞表型和功能的影响

目的　研究含非甲基化CpG基序的免疫刺激寡核苷酸 (CpG- ODN)与重组HBsAg对慢性乙型肝炎患者 (CHB)外周血树突状细胞 (dendriticcell ,DC)表型和功能的影响。方法　以重组人GM CSF、IL 4自CHB患者和健康者外周血单个核细胞诱导扩增DC ;以CpG ODN和HBsAg单独或联合刺激DC ,并与TNF α比较 ,评价其对DC表达表面分子HLA DR、CD86、CD1a ,分泌IL 12p70以及刺激同种T细胞增殖能力的影响 ;同时检测血浆TGF- β、IFN- γ含量。 结果　与PBS组比 ,CpG- ODN单用或联合HBsAg均能明显提高CHB患者DC表面分子HLA DR的表达 ,使IL- 12分泌增加 ,刺激同种T细胞增殖的能力亦增强 ,CpG ODN联合HBsAg尚能明显提高CD1a的表达 ;CpG- ODN的上述刺激作用类似于TNF α ;CHB患者血浆TGF- β、IFN -γ含量明显高于正常对照。 结论　CpG -ODN与TNF α一样能够促进CHB患者外周血DC分化和成熟 ;CpG- ODN与HBsAg联合刺激能协同增强DC的特异性抗原递呈作用 ;CHB患者的细胞因子环境可能是DC功能沉默的重要原因。     

富含C、G碱基的核甘酸片段增强乙型肝炎表面抗原免疫反应

目的 研究如何高效率激发机体对HBsAg的体液和细胞免疫反应,寻求治疗乙型肝炎的有效方法。方法 合成全硫代修饰的富含C、G碱基的核甘酸(CpG ODN)片段作为免疫佐剂,与HBsAg按一定比例混合免疫小鼠,研究其增强HBsAg免疫反应的作用。实验分为单纯HBsAg组、HBsAg CpG ODN组、商用乙肝疫苗(与HBsAg亚型相同)组、商用乙肝疫苗 CpG ODN组4组。HBsAg及商用乙肝疫苗用量为1．67μg／次,CpG ODN为16．5μg／次。采用两剂免疫方案,间隔15d,末次免疫后15d,取血测抗-HBs;取脾细胞作细胞毒性T淋巴细胞(CTL)实验。结果 4组抗-HBs A值分别为0．109、0．435、0．422及0．575;CTL反应值分别为8．5％、37．0％、1．5％和28．0％,实验中未观察到明显的毒副作用。结论 (1)CpG ODN能明显增强HBsAg的体液免疫反应;(2)CpG ODN也能增强HBsAg的细胞免疫反应;(3)CpG ODN与商用乙肝疫苗中的铝剂有协同作用,两者联用可同时诱导高效价的抗体反应和有效的细胞免疫反应。进一步评价其安全性和有效性,可望用于临床治疗慢性乙肝病毒感染。     

Induction of humoral and cellular immune response to hepatitis B virus (HBV) vaccine can be upregulated by CpG oligonucleotides complexed with Dectin‐1 ligand

A persistent hepatitis B virus (HBV) infection is characterized by a lack of or a weak immune response to HBV, which may be reflective of tolerance to HBV. Efficient induction of HBV‐specific immune response leads to the clearance of HBV in patients with a chronic HBV infection. CpG oligodeoxynucleotides (ODN) has a powerful adjuvant effect in HBV vaccination. A recent report demonstrated that the immunization by B/K CpG ODN (K3) wrapped by the nonagonistic Dectin‐1 ligand, schizophyllan (SPG), namely K3‐SPG, was more effective in the induction of antigen‐specific immune response than that by K3. In this study, we examined the efficacy of K3‐SPG as a HBV vaccine adjuvant. Wild‐type (WT) mice and HBV transgenic (HBV‐Tg) mice were subcutaneously immunized with hepatitis B surface antigen (HBsAg) alone, HBsAg and K3, or HBsAg and K3‐SPG. The vaccination with HBsAg and K3‐SPG significantly enhanced humoral and cellular immune response to HBV antigen compared to the other vaccinations in WT and HBV‐Tg mice. K3‐SPG induced the accumulation of dendritic cells (DCs) into draining lymph node and the activation of DCs. The expression of cytokines and chemokines related to Th1 and Th2 responses was upregulated after the vaccination including with K3‐SPG. In conclusion, these results indicated that the vaccination using K3‐SPG may overcome tolerance even in patients with chronic HBV infection.     

Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization

Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund’s adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund’s adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund’s adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.     

CpG-containing ODN has a limited role in the protection against Toxoplasma gondii

Bacterial DNA containing immunostimulatory motifs (CpG) induces the development of a T(H1) immune response. Since protection against Toxoplasma gondii is correlated with this type of response, the aim of this work was to determine if a synthetic oligodeoxynucleotide (ODN) containing CpG sequences could be useful as adjuvant for the induction of a long-lasting protective immune response against T. gondii. BALB/c mice immunized with a total soluble antigen of T. gondii (TSA2) mixed with ODN-containing CpG sequences developed a typical TH1 response, as determined by antibody isotypes and interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by spleen cells. However, they did not resist a challenge with the virulent RH strain of the parasite. Absence of protection paralleled with lower levels of IFN-gamma, when compared with mice vaccinated with the live tachyzoites of the attenuated ts.4 strain of the parasite, which resisted this challenge. Intraperitoneal injection of ODN alone to mice induced a high degree of resistance to a lethal challenge inoculated by the same route. Nevertheless, this nonspecific protection was transient. Thus, the use of ODN containing CpG motifs as adjuvant is of limited value for the induction of a protective immune response against T. gondii.     

CpG DNA functions as an effective adjuvant for the induction of immune responses in aged mice.

The present studies demonstrate that the immunization of aged mice with Diphtheria toxoid in formulations containing unmethylated immunostimulatory CpG motifs, promotes the successful development of immune responses that are qualitatively and quantitatively comparable to those induced in young animals vaccinated in a similar manner. Aged mice given vaccines containing CpG oligodeoxynucleotides (ODNs) expressed primary and secondary systemic humoral immune responses having isotype profiles consistent with an enhancement in Th-1 type immunity. The ability to generate common mucosal immunity was also restored in aged animals given CpG ODN-containing vaccines. Dendritic cells (DCs) were determined to represent one of the cellular targets of CpG ODN activities in aged mice since restoration of immune function was observed when DCs from aged donors were pulsed with antigen and CpG ODNs, prior to injection into syngeneic young adult or aged recipients. Interestingly, antigen-pulsed DCs from young donors were fully capable of stimulating immune responses following their injection into syngeneic young adult or aged hosts, without a need for exposure to CpG ODNs. Although the mechanism(s) by which CpG DNA exerts its beneficial adjuvant effects on the aged immune system remains unclear, our findings suggest that the incorporation of CpG ODNs into vaccine formulations provided to the aged could prove useful in the development of more effective vaccines for the elderly.     

Unmethylated oligo-DNA containing CpG motifs aggravates collagen-induced arthritis in mice

OBJECTIVE: To investigate the effects of an intradermal injection of an unmethylated oligodeoxynucleotide (ODN) containing CpG motifs on the severity of collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 LacJ mice by immunization with bovine type II collagen (CII) in Freund's complete adjuvant followed 3 weeks later by immunization with CII in Freund's incomplete adjuvant (yielding CIA mice). Unmethylated ODN containing a CpG motif was injected intradermally into DBA/1 LacJ mice at a dosage of 20 microg (yielding CpG-CIA mice) 1 week prior to the first immunization with CII. Unmethylated ODN containing a GpC motif instead of a CpG motif and ODN containing a methylated CpG motif were used to produce controls (GpC-CIA mice and mCpG-CIA mice, respectively). After the second immunization with CII, arthritis scores were measured weekly up to the eighth week. At the eighth week, the mice were killed, histopathologic changes in the ankle joints were examined, and titers of interferon-gamma (IFNgamma) in the supernatants of splenocytes (1 x 10(7)) stimulated in culture by CII for 3 days were determined by enzyme-linked immunosorbent assay. RESULTS: CpG-CIA mice had significantly higher arthritis scores than CIA mice. CpG-CIA mice had more severe histopathologic changes than CIA mice and mCpG-CIA mice. Moreover, splenocytes in CpG-CIA mice produced higher IFNgamma titers in response to CII than did splenocytes in CIA mice and mCpG-CIA mice. CONCLUSION: Injection of unmethylated oligo-DNA containing CpG motifs aggravated CIA through activation of the Th1-type immune response, suggesting that microbial infection could be one of the mechanisms for aggravation or exacerbation of arthritis or, alternatively, that such infection could be an adjuvant in the induction of arthritis in rheumatoid arthritis.     

A Prophylactic Effect of an Oligodeoxynucleotide Containing a Cytidine–Guanosine Motif against Japanese Cedar Pollen-Induced T-Helper Type 2 Allergic Response

Background. Over 10% of entire population in Japan suffer from allergic diseases induced by Japanese cedar pollen (JCP) every spring. In terms of preventive medicine, it has become a matter of urgency to establish successful prophylactic and therapeutic strategies for controlling the disorders. The effect of an oligodeoxynucleotide containing a cytidine–guanosine motif (CpG ODN) on the regulation of immune responses induced by JCP was investigated in this study. Methods. BALB/c mice were inoculated with CpG ODN intraperitoneally before intranasal sensitization to JCP. Cellular infiltration in the lung of BALB/c mice after treatment with CpG ODN or JCP was performed by hematoxylin and eosin (H&E) staining. Antibody titers and cytokines levels were determined by ELISA. Results. Intranasal inoculation of BALB/c mice with JCP induced a T-helper type 2 (Th2-type) dominant immune response, as characterized by the production of interleukin (IL)-4 and IL-5 in the lung and of JCP-specific IgE antibody in serum. Prior intraperitoneal administration of CpG ODN to mice suppressed the subsequent JCP-induced antibody production and infiltration of inflammatory cells in the lung. The inhibitory mechanism of CpG ODN seemed to be attributable to a CpG ODN-induced Th1-type dominant environment, which down-regulated Th2-type response subsequently induced by JCP allergen sensitization. Furthermore, administration with CpG ODN decreased the production of JCP-induced IL-17, which has been found to play a pivotal role in several inflammatory diseases including allergic asthma. The decreased production of IL-17, together with reduced secretion of IL-4 and IL-5, may contribute to diminish the inflammation in the lung of JCP-sensitized mice. Conclusion. This work provides evidence that the CpG ODN has a prophylactic effect on the JCP-induced Th2-type allergic responses by establishing or restoring a Th1-type shift of immune environments.     

Enhancement of humoral immune responses to HBsAg by heat shock protein gp96 and its N-terminal fragment in mice

AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragment of gp96 could substitute native gp96 to stimulate the immune system. METHODS: gp96 isolated from livers of normal mice and its N-terminal fragment (amino acid 22-355) expressed in E coli were used for immunization of BALb/c mice. Eight groups of mice received one of the following regiments subcutaneously in 100 μL phosphate buffered saline (PBS) at an interval of 3 wk. Group 1: PBS only; group 2: gp96 only; group 3: N-terminal fragment only; group 4: HBsAg only; group 5: HBsAg+gp96; group 6: HBsAg+N-terminal fragment; group 7: HBsAg+incomplete Freud's adjuvant; group 8: HBsAg+N-terminal fragment (95℃ heated for 30 min). Serum anti-HBsAg antibody levels were assayed by ELISA. CTL responses in splenocytes were analyzed by ELISPOT after the last vaccination. RESULTS: The average titer of serum anti-HBsAg antibody in the mice immunized with HBsAg together with gp96 or its N-terminal fragment were much higher than those immunized with HBsAg alone detected by ELISA. The cellular immune response of the mice immunized with HBsAg together with gp96 or its N-terminal fragment was not different with those immunized with HBsAg alone measured by ELISPOT assay. CONCLUSION: gp96 or its N-terminal fragment greatly improved humoral immune response induced by HBsAg, but failed to enhance the CTL response, which demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment humoral immune response against HBV infection.     

Bacterial motif DNA as an adjuvant for the breakdown of immune self-tolerance to pyruvate dehydrogenase complex

Bacterial DNA containing unmethylated CpG dinucleotide motifs is immunostimulatory to mammals, skewing CD4(+) T-cell responses toward the Th1 phenotype. Autoreactive T-cell responses seen in primary biliary cirrhosis (PBC) are typically of the Th1 phenotype, raising the possibility that bacterial DNA might play a role in the generation of pathologic autoimmunity. We therefore studied the effects of CpG motif-containing oligodeoxynucleotides (ODN) on responses to pyruvate dehydrogenase complex (PDC, the autoantigen in PBC) in a murine model. Sensitization of SJL/J mice with non-self-PDC has been shown to result in induction of autoreactive T-cell responses to PDC sharing characteristics with those seen in patients with PBC. Administration of CpG ODN to SJL/J mice at the time of sensitization with PDC resulted in a significant skewing of splenic T-cell response to self-PDC, with significant augmentation of the Th1 cytokine response (interleukin [IL] 2 and interferon [IFN] gamma) and reduction of the Th2 response (IL-4 and IL-10). In fact, CpG ODN seemed to be more effective at biasing the response phenotype and as effective at inducing liver histologic change as complete Freund's adjuvant (CFA), the standard adjuvant used for induction of Th1 responses in murine autoimmune and infectious immunity models. In conclusion, our findings raise the possibility that bacteria play a role in the development of autoimmunity (in PBC at least) through the potential of their DNA to shift the T-cell responses toward the phenotype associated with autoimmune damage. Moreover, this study suggests caution in the therapeutic use of CpG ODN as vaccine adjuvants.