CpG ODN增强乙型肝炎表面抗原免疫小鼠的抗体产生

目的：探讨合成含CpG基序的寡核苷酸（CpG ODN)对重组乙型肝炎表面抗原（rHBsAg)及乙型肝炎疫苗增强小鼠特异性抗体产生的效应。方法：采用非纯系（Km)及纯系（Balb/c)小鼠作为免疫对象，经后腱胫骨前肌免疫2次，ELISA法检测血清乙型肝炎表面抗体（抗－HBs)效价。结果：加CpG ODN组，其抗-HBs效价均较单独注射rHBsAg和疫苗组明显增高，持续时间长，且纯系鼠的抗体效价明显高于非纯系鼠。结论：CpG ODN对小鼠抗－HBs产生具有增强作用，具与疫苗中的铝佐剂有协同效应。     

CpG ODN对rHBsAg免疫小鼠淋巴细胞增殖反应的影响

目的：进一步探讨以合成含CpG基序的寡核苷酸（CpG ODN）作为免疫增强剂与重组乙型肝炎表面抗原（rHBsAg）联合免疫小鼠,观察其淋巴细胞增殖反应效应.方法：经后腿胫骨前肌初次免疫BALB/c小鼠两周后加强免疫1次;MTT和3H-TdR掺入法分别检测免疫小鼠胸腺及脾脏淋巴细胞增殖反应.结果：加CpG ODN组与单纯注射抗原及疫苗组相比,淋巴细胞增殖反应显著增强.而且,除胸腺特异性增殖中的CpG加疫苗组和疫苗组外,其他各实验组与对照组相比,淋巴细胞增殖反应也都显著增强.结论：CpG ODN能够明显刺激小鼠胸腺及脾脏淋巴细胞增殖反应,显示出强而有效的免疫增强效应,有望成为一种新型的免疫佐剂.     

CpG ODN佐剂促进HBsAg疫苗诱导小鼠细胞免疫应答的探讨

目的 探讨cpG ODN对重组乙型肝炎表面抗原（HBsAg）诱导小鼠细胞免疫应答的影响。方法HBsAg和HBsAg＋CpG ODN分别肌肉注射免疫Balb／c小鼠后,从淋巴组织（脾和淋巴结）和非淋巴组织（肺）分离淋巴细胞,体外经HBsAg抗原刺激后,利用ELISA检测细胞培养液中IFN-γ的水平,再利用流式细胞仪在单个细胞水平上检测IFN-γ^＋细胞的频率,分析IFN-7^＋细胞亚群。结果对照组和HBsAg免疫组IFN-γ产生的水平很低,当HBsAg加强免疫1～2次后,IFN-γ表达仍然没有明显升高;而CpG ODN＋HBsAg免疫1次或加强免疫后,CpG显著促进HBsAg诱导的IFN-γ产生。进一研究结果表明CpG促进HBsAg诱导淋巴器官和非淋巴器官内CD4^＋和CD8^＋T细胞IFN-γ的表达。结论CpG免疫佐剂不仅能诱导细胞免疫应答同时也诱导体液免疫应答,提示CpG ODN可以用于HBsAg预防和治疗性佐剂。     

CpG ODN联合乙型肝炎疫苗与乙型肝炎疫苗免疫效果对比研究

目的探讨CpG ODN乙型肝炎疫苗与市售乙型肝炎疫苗对Balb/c小鼠的免疫作用效果。方法将乙肝疫苗、乙肝疫苗+100μgCpG ODN分别肌肉注射到4～6周龄,16～18g Balb/c小鼠体内,于第1次免疫后28d以同样剂量加强免疫1次。分别于第1次免疫后28、42、63 d收集小鼠血清,用ELISA方法检测抗HBs IgG抗体。结果实验组产生的抗HBs IgG与对照组HBsIgG相比P<0.05,具有显著性差异。28、42、63 d抗HBs IgG分别是对照疫苗的114倍、4.73倍、5.41倍以上。结论 CpG ODN能够显著增强小鼠对乙肝疫苗的免疫应答,在较短时间内显著的提高乙肝抗体的产生水平,免疫效果是乙肝疫苗的5倍以上。     

CpG ODN作为乙型肝炎疫苗佐剂的研究进展

随着对含有CpG寡聚脱氧核苷酸(CpG ODN)的结构、功能以及作用机理的深入研究,CpGODN已经应用于疾病的治疗和预防等许多方面。本文就CpG ODN作为乙型肝炎疫苗佐剂的研究进展做一综述     

CpG ODN生物学活性和免疫作用的研究进展

CpG ODN为人工合成的含胞嘧啶和鸟嘌呤二核苷酸的寡聚脱氧核苷酸,能与Toll样受体9(TLR9)结合而发挥作用.CpG ODN能直接或间接激活树突状细胞、单核细胞和巨噬细胞以及B淋巴细胞、NK细胞、T淋巴细胞等,使之分泌许多生物活性因子和细胞因子,从而在感染性疾病和肿瘤的免疫治疗中发挥重要作用.     

CpG ODN增强免疫抑制小鼠对乙肝疫苗的免疫应答效果

环磷酰胺是肿瘤治疗中最常见的烷化剂 ,严重影响骨髓、胸腺和脾脏的功能导致患者免疫力低下 ,容易患各种感染性疾病 ,疫苗接种成功率也很低 ,因此 ,寻找有效疫苗佐剂来提高这群人的疫苗接种成功率具有重要的实际意义。近年来 ,大量的研究显示细菌DNA中存在非甲基化的CpG二核苷酸序列 (即CpG基序 )能激活免疫系统 ,诱导天然免疫应答分泌免疫球蛋白和各种细胞因子。人工合成含CpG基序的脱氧寡核苷酸 (CpGODN )与蛋白疫苗同时接种正常小鼠 ,能提高抗原特异性免疫应答 ,诱导出高水平保护性抗体。本实验应用CpGODN与乙肝疫苗同时肌肉注射…     

CpG ODN的免疫剌激机制

非甲基化CpGODN的免疫增强作用已经为许多研究所证实,CpGODN作为一种新型的免疫刺激剂,主要剌激多种免疫活性细胞分泌细胞因子,使机体产生快速的细胞免疫及体液免疫。CpGODN的特殊结构诱导激活细胞信号转导网络系统,介导了其免疫剌激活性及调节作用模式。     

破伤风类毒素非磷脂脂质体疫苗的实验研究

以二氧乙烯十六烷醚、胆固醇和油酸为原料制备非磷脂脂质体 ,试验证明其包裹率高、稳定性好。与破伤风类毒素(TT )铝佐剂疫苗相比较 ,TT非磷脂脂质体疫苗二针免疫小鼠产生的抗体应答水平高一倍左右 ,抗体亚类以IgG1为主 ,加入IFN γ可增加IgG2a亚类应答。但免疫脾细胞IL 2产量不高。此外 ,该疫苗鼻粘膜接种可诱导显著的血清抗体应答。因而这种新型脂质体值得疫苗工作者重视。     

CpG ODN的体外免疫活性及其作为乙肝疫苗佐剂的研究新进展

为了增强乙肝疫苗的免疫效力,开发研制新型疫苗佐剂是当前国内外学者研究的热点之一。近年来的研究表明：含CpG基序的寡脱氧核苷酸（CpG ODN）在体外实验中,对人和鼠的多种免疫细胞有活化作用;在动物实验中,CpG ODN不仅可增强乙肝疫苗的体液免疫应答,还可增强其细胞免疫应答,这正是铝佐剂所达不到的,且CpG ODN与铝佐剂具有协同效应,今后可考虑双佐剂联合乙肝疫苗接种,能提高接种人群的应答率,刺激机体产生更有效的保护作用;动物实验还显示,CpG ODN能增强HBV转基因小鼠对乙肝疫苗的免疫应答和抗病毒效应,说明乙肝疫苗辅以CpG ODN可望作为临床上免疫治疗慢性HBV感染的可行性途径;Ⅰ期临床试验表明CpG ODN对人体较安全。     

HBV和HIV的复合型DNA免疫可诱发BALB/c小鼠的细胞免疫应答

本研究是通过细胞免疫检测来研究含有两种不同病毒基因的复合型DNA免疫的效果 ,同时和单一病毒基因的DNA免疫作比较。在本实验中 ,将HBV的S基因和 (或 )HIV 1的gp12 0基因插入到真核表达载体pcDNA3中 ,得到能表达HBsAg和gp12 0融合蛋白的重组体pcDNA S 12 0 ,和能表达HBsAg的重组体pcDNA S。在实验中 ,将重组质粒DNA直接注射到BALB/c小鼠股四头肌内 ,按每只小鼠 10 0 μg/ 10 0 μl的剂量经 3次免疫后 ,在T细胞增殖实验中 ,用HBsAg蛋白刺激体外培养的被免疫小鼠T细胞后 ,出现了明显的T细胞增殖。采用51Cr释放法检测特异性CTL杀伤作用时 ,发现致敏的CTL对HIVgp12 0多肽孵育后的靶细胞P815有明显的杀伤作用。     

The role of interleukine-12 in protection induced by CpG ODN against Listeria monocytogenes in BALB/c and C57BL/6

Synthetic oligodeoxynucleotides containing CpG motifs have several immune effects such as cytokine production in normal mice. In this study, we demonstrated the protective effect of CpG ODN against Listeria monocytogenes in BALB/c and C57BL/6. With a single dose of 40 μg/mouse of CpG ODN 48 h before bacterial challenge, protection was achieved in both strains of mice based on survival rates compared with controls. Serum IL-12 from each mouse was measured by using enzyme-linked immunosorbent assay (ELISA), at day 0 (48 h after CpG treatment) and at days 5, 11, and 15 after bacterial challenge. It was shown that serum IL-12 was only elevated at day 0 in BALB/c mice. However, for C57BL/6 mice, IL-12 was elevated at days 0, 5, and 11. These data support the hypothesis that CpG DNA motifs activate protective innate immune defenses.     

抗HBs-HBsAg复合物诱生体液免疫应答的研究

目的研究抗ＨＢｓ－ＨＢｓＡｇ复合物在ＢＡＬＢ／ｃ小鼠诱生体液免疫应答的特点。方法分别用鼠抗ＨＢｓ－ＨＢｓＡｇ免疫原性复合物（ＩＣ）或单独ＨＢｓＡｇ免疫ＢＡＬＢ／ｃ小鼠（加或不加氢氧化铝），分析其诱生的抗ＨＢｓＩｇＧ效价及ＩｇＧ亚类。结果用ＩＣ加氢氧化铝为佐剂免疫小鼠诱生的抗ＨＢｓ中以ＩｇＧ１亚类为主，而用ＨＢｓＡｇ免疫鼠则以ＩｇＧ２ｂ亚类较高。ＩＣ不加氢氧化铝佐剂诱生的抗ＨＢｓ中ＩｇＧ１及ＩｇＧ２ａ均高于单独ＨＢｓＡｇ免疫鼠。研究还显示，抗ＨＢｓＩｇＧＦｃ段是巨噬细胞（ＭΦ）等抗原提呈细胞摄取ＩＣ的主要途径。结论抗ＨＢｓ－ＨＢｓＡｇ复合物免疫诱生的体液免疫应答较单独ＨＢｓＡｇ免疫强。抗ＨＢｓ主要通过Ｆｃ段的介导作用，改变了机体抗原提呈细胞对ＨＢｓＡｇ的摄取、加工及抗原提呈，并可增强ＨＢｓＡｇ的免疫原性。     

The Wild-Type Hepatitis C Virus Core Inhibits Initiation of Antigen-Specific T- and B-Cell Immune Responses in BALB/c Mice

In this study, the effects of wild-type and deletion mutant hepatitis C virus (HCV) core proteins on the induction of immune responses in BALB/c mice were assessed. p2HA-C145-S23, encoding a core protein with the C-terminal 46 amino acids truncated, significantly produced stronger antibody and cellular responses than p2HA-C191-S23. The induction of immune responses by p2HA-C145-S23 was dose dependent. However, increasing the doses or repeated administration did not enhance immune responses by the wild-type core protein. In addition, p2HA-C191-S23 was apparently able to interfere with the priming of specific immune responses by p2HA-C145-S23 when the two were coadministered. These results demonstrated that the wild-type HCV core protein itself could inhibit the priming of immune responses in the course of a DNA vaccination, whereas the truncated HCV core protein could provide potential applications for the development of DNA- and peptide-based HCV vaccines.Hepatitis C virus (HCV) has been identified as the major cause of posttransfusion and sporadic non-A, non-B hepatitis (4). One of the remarkable features of HCV infection is the high rate of persistent infections that eventually progress to liver cirrhosis and hepatocellular carcinoma (1, 36). The frequent progression of HCV infection to the chronic disease course has been largely attributed to the inability of the host immune system to clear the initial HCV infection (38).Current data indicate that HCV-specific T-cell responses play a critical role in the control of HCV infection (5, 24). Robust HCV-specific CD4⁺ and CD8⁺ T-cell activation is associated with viral clearance in acute infection. However, HCV-specific T-cell clones in chronic HCV-infected persons are directed to many viral determinants, occur with low frequency, and are apparently functionally ineffective. Additional immune response abnormalities in chronic HCV infections include inadequate activation of the innate immune system, which includes excessive proinflammatory cascades in monocytes and altered dendritic cell (DC) functions (47). Therefore, effective new therapies and improved vaccines aimed at preventing HCV infection should induce intense, multispecific, and long-lasting T-cell immune responses that can suppress the replication of HCV in the early stages of infection.Genetic immunization is a potent vaccine strategy for inducing effective antigen-specific CD4⁺ and CD8⁺ T-cell responses. Induction of HCV-specific T-cell responses by plasmid DNA vaccines has been demonstrated in a variety of experimental systems (13, 25). However, compared with DNA vaccines like those coding for hepatitis B virus proteins, HCV DNA vaccines appeared to be less efficient and induced only transient and weak responses (18, 20, 37). The failure of HCV DNA vaccines may be explained by the fact that HCV proteins are able to interfere with host cellular functions and thereby prevent the efficient induction of immune responses (6, 10, 23).The HCV core gene is highly conserved among the various HCV genotypes and contains several well-characterized B-cell and cytotoxic T-lymphocyte (CTL) epitopes. Antibodies against the core proteins often appear first during natural HCV infections (35). In chronically infected individuals, cellular immune responses against HCV core proteins are always attenuated (11, 39). This implies that HCV core protein-specific immune responses may be important for the control of HCV infections. Therefore, it is worthwhile to test HCV core genes as candidate HCV vaccines for the prevention and therapy of HCV infections. However, the immune response induced by the HCV core in DNA vaccination is always weak and transient. Interaction of the HCV core protein with a wide variety of cellular proteins has been reported to influence host cell functions (26, 34). The HCV core protein is also able to suppress host immunity through several mechanisms, such as impairment of the function of dendritic cells in vitro, promotion of apoptosis of immune cells, or suppression of type I interferon (IFN) signaling, among other mechanisms (7, 14, 22, 28, 31, 32, 52). In the case of DNA vaccination, a high level of expression of the HCV core protein is desired for the priming of specific immune responses. However, the overexpressed viral protein may interfere even more strongly with host functions (2).In this study, we began by investigating whether HCV core-based DNA vaccines could be improved by truncating the HCV core protein. The removal of the carboxy-terminal region significantly enhanced specific T- and B-cell responses. Through coadministration with other DNA vaccines, it was shown that the full-length HCV core protein was able to inhibit the initiation of antigen-specific T- and B-cell responses. Our findings imply that the HCV core protein could interfere with host immune responses and therefore contribute to the development of chronic infection.     

Local Immune Responses to Chlamydia pneumoniae in the Lungs of BALB/c Mice during Primary Infection and Reinfection

Cell-mediated immune (CMI) responses play a major role in protection as well as pathogenesis of many intracellular bacterial infections. In this study, we evaluated the infection kinetics and assessed histologically the lymphoid reactions and local, in vitro-restimulated CMI responses in lungs of BALB/c mice, during both primary infection and reinfection with Chlamydia pneumoniae. The primary challenge resulted in a self-restricted infection with elimination of culturable bacteria by day 27 after challenge. A mild lymphoid reaction characterized the pathology in the lungs. In vitro CMI responses consisted of a weak proliferative response and no secretion of gamma interferon (IFN-γ). The number of lung-derived mononuclear cells increased substantially during the primary infection; the largest relative increase was observed in B cells (B220⁺). After reinfection, the number of lung-derived mononuclear cells increased further, and the response consisted mainly of T cells. The reinfection was characterized in vivo by significant protection from infection (fewer cultivable bacteria in the lungs for a shorter period of time) but increased local lymphoid reaction at the infection site. In vitro, as opposed to the response in naive mice, acquired immunity was characterized by a strongly Th1-biased (IFN-γ) CMI response. These results suggest that repeated infections with C. pneumoniae may induce Th1-type responses with similar associated tissue reactions, as shown in C. trachomatis infection models.     

Systemic Administration of Olygodeoxynucleotides with CpG Motifs at Priming Phase Reduces Local Th2 Response and Late Allergic Rhinitis in BALB/c Mice

Oligodeoxynucleotides (ODN) with CpG motifs (CpG ODN) induce T helper (Th)1-type reaction. We aimed to evaluate the therapeutic effect of CpG ODN in the development of late allergic rhinitis induced by ovalbumin (OVA), which is one of Th2 diseaes, in BALB/c mice. Effects of a single dose of synthetic CpG-ODN (50 μg) intraperitoneally (i.p.) at the priming phase (on day 0) by OVA on the development of late eosinophilic rhinitis at respiratory areas were compared to the control mice treated with its vehicle (ODN without CpG motifs; 50 μg). Animals were again sensitized by OVA (on day 10) i.p., and 4 days after second sensitization animals were challenged by OVA intranasally (on day 14). Four days after challenge, eosinophilic reactions, nasal lesions and local cytokine values were examined. Compared to the control group, the CpG ODN-administration increased production of OVA-specific Th1 cytokine (interferon-γ) and decreased productions of ovalubmin-specific Th2 cytokines [interleukin (IL)-5 and IL-13] in nasal cavity fluids, supernatants of splenocytes and/or sera. Also, eosinophilia and increased total IgE values were decreased in mice treated with the CpG ODN compared to the control group. Moreover, nasal lesions with infiltration of eosinophils were prominently reduced by the CpG ODN-treatment compared to the control mice. The present study suggests that the systemic administration of CpG ODN at the priming phase may reduce local OVA-specific Th2 responses, resulting in decreased nasal pathology in the late allergic eosinophilic rhinitis. The authors wish it to be known, in their opinion, Toshiharu Hayashi and Keiko Hasegawa contributed equally to this work.     

Molecular Analyses of Anti-DNA Antibodies Induced by Polyomavirus BK in BALB/c Mice

In the present experiments, two groups of BALB/c mice (five individuals in each group) were hyperimmunized through four consecutive immunizations with either BK virus (Group 1) or BK dsDNA complexed with methylated BSA (Group 2). All immune sera taken after the fourth immunization from both groups reacted strongly with polyomavirus BK dsDNA as well as with calf thymus dsDNA, and all sera contained antibodies that bound in the Crithidia luciliae assay. This indicates that polyomavirus BK was able to induce antibodies with binding characteristics similar to SLE anti-DNA antibodies. To further characterize these induced anti-DNA responses, 10 monoclonal anti-DNA antibodies (four from Group 1, and six from Group 2) were generated and selected for reactivity with Sl-nuclease digested CT dsDNA. Their specificity for BK and CT dsDNA molecules, as well as their light and heavy chain variable region cDNA nucleotide sequences were analysed to compare them with known SLE derived anti-DNA antibodies. All of the 10 antibodies bound strongly to BK dsDNA, while seven also bound to CT dsDNA in competitive ELISA experiments. V-region analysis revealed that the induced antibodies resembled anti-DNA antibodies characteristic for murine SLE, and all but one contained arginine in the V_H CDR3 region. The arginines present in the monoclonal antibodies originated either from an RF shift from RF1RF3 of the D-genes or from N-sequence additions. Taken together, the data demonstrate that anti-DNA antibodies in response to hyperimmun-ization with polyomavirus BK have the same characteristics as of those occuring spontaneously in SLE. As virus infection/replication in vivo implies expression of immunogenic (non-self) DNA-binding proteins that may render DNA immunogenic, the present results may therefore suggest one physiological mechanism for production of SLE-related anti-DNA antibodies.     

CpG ODN对ZP~(121-140)表位合成肽诱导的免疫应答和免疫避孕效应影响

目的:探讨CpG ODN对透明带(四)抗原表位ZP~(121-140)合成肽诱导的免疫应答和免疫避孕效应影响.方法:应用人工合成ZP2~(121-140)表位肽,与20μg CpG ODN或等量完全弗氏佐剂(CFA)混悬后左胫前肌免疫雌性BALB/c小鼠,初次免疫后2、4、6周各加强免疫一次,共免疫四次.在每次加强免疫前和末次免疫后两周断尾取血,分离血清,分析特异性lgG抗体及非特异性细胞因子IFN-γ、TNF-Ⅱ,IL-10水平;收集小鼠阴道冲洗液,离心取上清,分析特异性IgA抗体水平.免疫小鼠生育实验结束后,取卵巢组织行病理学分析.结果:CpG ODN组诱导的特异性IgG和IgA水平较CFA组有增高趋势,但无显著性差异(P＞O.05).CpG ODN组诱导的非特异性IFN-γ和TNF-水平明显高于CFA组(P＜0.05);而CpG ODN组诱导的IL-10水平显著低于CFA 组(P＜0.05).两种佐剂对小鼠的受孕率影响没有显著性差异,但CpG ODN组孕鼠每胎产仔数明显低于CFA组(P＜O.05).病理学分析显示实验小鼠卵巢组织无病理性变化.结论:CpG ODN对Zp~(121-140)合成肽诱导的免疫应答和免疫避孕效应略优于CFA,更适合用于避孕疫苗佐剂研究.     

HBsAg重组腺病毒转染的树突状细胞诱导BALB/c小鼠抗HBV免疫的研究

目的 研究HBsAg重组腺病毒转染的小鼠树突状细胞（DC）体内外免疫刺激活性和诱导小鼠抗HBV免疫的特点。方法 BALB／c小鼠的骨髓细胞体外扩增为DC，转染HBsAg重组腺病毒Ad-S或被HBsAg蛋白冲击后，流式细胞术分析DC的表型，混合淋巴细胞反应（MLR）检测DC的刺激活性；或与pcDNA3．1（＋）-S质粒分别免疫小鼠后，LDH法测定脾细胞CTL活性，放免法检测血清抗．HBs；流式细胞术分析体外自体MLR中及免疫后小鼠脾脏T细胞内细胞因子。结果 DC／Ad-S、DC／HBsAg和各对照组DC之间的表型和MLR差异无统计学意义，并均刺激TH和Tc分泌IFN-γ。DC／Ad-S免疫后2周能诱导比DNA疫苗更强产生IFN-γ的TH（P〈0．05），以及比DC／HBsAg和DNA疫苗更强分泌IFN-γ的Tc（均P〈0．01）和特异性CTL（P〈0．05，P〈0．01）。但DC／Ad-S和DC／HBsAg诱导的CTL反应及Tc分泌IFN-γ在免疫后4周均明显减弱，且诱导抗．HBs作用弱于DNA疫苗。结论 HBsAg重组腺病毒转染的DC比HBsAg冲击的DC及DNA疫苗能诱导更强的TH1/Tcl（Ⅰ型）细胞免疫和特异性CTL反应，是诱导抗HBV细胞免疫的有效刺激细胞。