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。      

口服型HCV融合抗原DNA疫苗在小鼠诱导免疫应答

将编码一个外源信号肽、一个通用型辅助性T淋巴细胞抗原表位和HCV核心 包膜蛋白E2融合抗原基因的真核表达质粒pST CE2t(DNA疫苗 )转化到减毒鼠伤寒沙门菌SL72 0 7.将该重组菌口服接种BALB c小鼠 3次 .小鼠的抗HCV核心和E2抗体阳转率分别达 6 0 %和 70 % .体外以重组HCV核心或E2抗原刺激小鼠脾细胞 ,均使之发生明显的增殖反应 ,且小鼠脾细胞能有效杀伤表达HCV核心抗原的同系骨髓瘤细胞SP2 0 .这为研制高效免疫、成本低廉、接种方便的HCV疫苗提供了一个新的可行途径     

携带HCV核心蛋白原核表达质粒与真核表达质粒的减毒伤寒沙门菌诱导小鼠免疫应答之比较

丙型肝炎病毒(HCV)核心蛋白是丙肝疫苗的重要候选抗原,然而,该蛋白因具有免疫调控作用而影响免疫应答的诱导。构建了HCV核心蛋白的两种表达质粒,一种是体内激活型原核表达质粒pZW-C,另一种是真核表达质粒pCI-C。将该两种质粒转化减毒鼠伤寒沙门菌SL7207,得到重组菌SL7207/pZW-C和SL7207/pCI-C,分别将重组菌口服接种小鼠,检测小鼠的免疫应答,结果发现:①SL7207/pCI-C免疫鼠的CD3 CD4 T细胞持续降低,而SL7207/pZW-C免疫鼠的CD3 CD4 T细胞无明显改变;②SL7207/pCI-C免疫只诱导低水平抗HCV核心蛋白抗体,加强免疫对抗体阳转率及抗体水平无明显影响,而SL7207/pZW-C免疫组所有小鼠均产生较高水平的抗核心蛋白抗体。③SL7207/pCI-C免疫鼠脾细胞的体外增殖活性、细胞毒性T细胞活性以及加强免疫对细胞免疫应答的增强作用均明显不及SL7207/pZW-C免疫鼠。结果提示:携带真核表达质粒pCI-C的沙门菌因在小鼠细胞内表达天然形式(结构以及磷酸化修饰)的HCV核心蛋白,可能通过对T细胞的免疫抑制作用而弱化免疫应答。而以携带原核表达质粒pZW-C的沙门菌免疫可避免这一问题,并具有接种方便,成本低廉等优点,从而可望作为基于HCV核心蛋白为靶抗原的HCV疫苗的候选免疫方式。     

携带HCV核心蛋白原核表达质粒与真核表达质粒的减毒伤寒沙门菌诱导小鼠免疫应答之比较

丙型肝炎病毒（HCV）核心蛋白是丙肝疫苗的重要候选抗原,然而,该蛋白因具有免疫调控作用而影响免疫应答的诱导。构建了HCV核心蛋白的两种表达质粒,一种是体内激活型原核表达质粒pZW-C,另一种是真核表达质粒pCI-C。将该两种质粒转化减毒鼠伤寒沙门菌SL7207,得到重组菌SL7207/pZW-C和SL7207/pCI-C,分别将重组菌口服接种小鼠,检测小鼠的免疫应答,结果发现：① SL7207/pCI-C免疫鼠的CD3⁺CD4⁺ T细胞持续降低,而SL7207/pZW-C免疫鼠的CD3⁺CD4⁺ T细胞无明显改变;② SL7207/pCI-C免疫只诱导低水平抗HCV核心蛋白抗体,加强免疫对抗体阳转率及抗体水平无明显影响,而SL7207/pZW-C免疫组所有小鼠均产生较高水平的抗核心蛋白抗体。③ SL7207/pCI-C免疫鼠脾细胞的体外增殖活性、细胞毒性T细胞活性以及加强免疫对细胞免疫应答的增强作用均明显不及SL7207/pZW-C免疫鼠。结果提示：携带真核表达质粒pCI-C的沙门菌因在小鼠细胞内表达天然形式（结构以及磷酸化修饰）的HCV核心蛋白,可能通过对T细胞的免疫抑制作用而弱化免疫应答。而以携带原核表达质粒pZW-C的沙门菌免疫可避免这一问题,并具有接种方便,成本低廉等优点,从而可望作为基于HCV核心蛋白为靶抗原的HCV疫苗的候选免疫方式。     

Immune responses with DNA vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in BALB/c mice

To analyze the immune responses of DNA vaccine encoded different gene fragments of severe acute respiratory syndrome coronavirus (SARS-Cov), SARS-Cov gene fragments of membrane (M), nucleocapsid (N), spike a (Sa), and spike b (Sb) proteins were cloned into pcDNA3.1 (Invitrogen) vector to form plasmids pcDNAM, pcDNAN, pcDNASa, and pcDNASb, respectively. After mice were immunized intramuscularly with pcDNAM, pcDNAN or pcDNASa-pcDNASb plasmid, blood was collected and serum was separated. Humoral immune response was detected with the enzyme-linked immunosorbent assay, and cellular immune response of SARS-Cov DNA vaccines was detected with lymphoproliferation assay and cytotoxic T lymphocyte assay. Results show that cellular and humoral immune responses can be detected after immunization with pcDNAM, pcDNAN or pcDNASa-pcDNASb plasmids in BALB/c mice. However, pcDNAM stimulated the highest cellular immune response than other plasmids, and pcDNASa-pcDNASb stimulated the highest humoral immune response in week 12. The present results not only suggest that DNA immunization with pcDNAM, pcDNAN or pcDNASa-pcDNASb could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also to illustrate that gene immunization with these SARS DNA vaccines different immune response characters.     

Immure response induced by oral DNA vaccination against FMDV delivered by attenuated Salm onella choleraesuis C500

A recombinant strain of Salmonella choleraesuis C500,containing a eukaryotic expression plasmid pBO1 with the immune-dominant epitope of foot-and-mouth disease virus,was constructed.Specific immune response to this recombinant strain was evaluated by oral administration of the recombinant live bacteria pBO1/S.cho in rabbits.Results showed that T cell response and specific antibody production were elicited.This approach may present a general strategy for eliciting immune responses with DNA vaccine delivered by live bacterial vectors.The stimulated indexes of T lymphoproliferation by specific antigens of FMDV in rabbits,can reach up to 11.0 and an antibody titer of 1/32 as detected in the erum with liquid block ELISA.     

重组恶性疟原虫DNA质粒免疫小鼠及抗原表达的调控

将恶性疟原虫MSP1-31基因序列,引入四环素(Tc)控制的真核表达载体pTRE,获得重组质粒pTRE-31。将MSP1-31的原核表达载体pDS56T1转化大肠杆菌表达MSP1-31,亲和纯化后作检测用抗原。pTRE-31与辅助质粒pTet-off(tTA)肌肉注射4周龄BALB/c小鼠,观察DNA介导免疫情况。结果显示,四环素饲喂的小鼠4周时血清抗体阳转率为7.1%(1/14),而不饲喂四环素组可达100%(14/14),表明用pTRE-31/pTet-off重组质粒组合直接注射小鼠有效地引发了针对疟原虫MSP1-31抗原的体液免疫反应,且可受控于四环素。不饲喂四环素组小鼠在12周后仍能维持抗体阳性,倍比稀释ELISA显示血清抗体滴度在4周、8周和12周内持续上升。饲喂四环素组小鼠4周后停止饲喂Tc,第8周和第12周检测仍有部分(60%)小鼠血清抗体阳转,而继续饲喂Tc的小鼠未有抗体阳转,暗示重组质粒DNA在小鼠体内可持续存在至少4周并仍具备表达功能。     

稳定携带H5亚型禽流感病毒候选DNA疫苗减毒沙门氏菌的构建及其免疫原性

采用PCR技术从重组质粒pVAX1-HA扩增出禽流感病毒JSGO(H5N1)株的血凝素(HA)基因,将其克隆入真核表达质粒pmcDNA3.1 中,获得重组表达质粒pmcDNA3.1-HA。通过电穿孔转化法将重组质粒转入减毒鼠伤寒沙门氏菌SL7207*,构建成功携带DNA疫苗的重组沙门氏菌SL7207*(pmcDNA3.1-HA)。经体内体外试验证实,重组质粒pmcDNA3.1-HA在沙门氏菌中的稳定性显著高于pcDNA3.1-HA。将重组菌SL7207*(pmcDNA3.1-HA)和SL7207*(pcDNA3.1-HA)分别以2×109CFU剂量两次口服免疫BALB/c小鼠,免疫小鼠可产生针对禽流感病毒HA蛋白的黏膜抗体。重组菌以5×109CFU剂量两次口服免疫试验鸡,免疫鸡的小肠样品中可测到针对禽流感病毒HA蛋白的黏膜抗体,且SL7207*(pmcDNA3.1-HA)免疫组的抗体效价高于SL7207*(pcDNA3.1-HA)免疫组。免疫保护试验结果显示,SL7207*(pmcDNA3.1-HA)和SL7207*(pcDNA3.1-HA)免疫组的免疫保护率均与空载体组之间存在显著性差异(P<0.05),且SL7207*(pmcDNA3.1-HA)免疫组的保护率较SL7207*(pcDNA3.1-HA)免疫组提高了22.6%,说明稳定携带H5亚型禽流感病毒DNA疫苗的减毒沙门氏菌具有良好的免疫原性和免疫保护性。     

用体内激活的启动子调控HCV核心抗原表达可增强重组鼠伤寒沙门氏菌的免疫原性

为提高抗原表达质粒在重组伤寒沙门氏菌中的稳定性以增强重组伤寒沙门氏菌诱导的免疫应答 ,克隆鼠伤寒沙门氏菌pagC基因启动子 ,以其为转录调控元件构建HCV核心抗原表达质粒 ,转化到减毒鼠伤寒沙门氏菌中。体外培养时 ,Mg2 能够剂量依赖性抑制该重组菌表达HCV核心抗原。将该重组菌和组成性表达的重组菌分别口服接种BALB/c小鼠 ,观察质粒的稳定性和小鼠的免疫应答。结果表明 ,体内激活的pagC基因启动子能明显提高质粒在重组鼠伤寒沙门氏菌中的稳定性和增强重组菌诱导的体液和细胞免疫应答 ,这为发展高效免疫、成本低廉的口服丙肝疫苗提供了一个新思路     

稳定携带新城疫病毒DNA疫苗减毒沙门氏菌的构建及其免疫原性

采用PCR技术从重组质粒pVAX1-F中扩增出新城疫病毒JS5株的融合蛋白(F)基因,将其克隆入真核表达质粒pmcDNA3.1 中,获得重组表达质粒pmcDNA3.1-F.通过电穿孔转化法将重组质粒转入减毒鼠伤寒沙门氏菌SL7207,构建成功携带DNA疫苗的重组沙门氏菌SL7207(pmcDNA3.1-F).体内、体外试验结果表明,重组质粒pmcDNA3.1-F在沙门氏菌中的稳定性显著高于pcDNA3.1-F.将重组菌SL7207(pmcDNA3.1-F)和SL7207(pcDNA3.1-F)分别以1×109 CFU剂量两次口服免疫BALB/c小鼠,免疫小鼠可产生针对新城疫病毒F蛋白的血清抗体和黏膜抗体.重组菌以5×109 CFU剂量两次口服免疫4日龄SPF鸡,免疫鸡产生的针对新城疫病毒F蛋白的血清抗体和小肠黏膜抗体效价水平与空载体组之间存在显著性差异(P＜0.05).免疫保护试验结果显示,SL7207(pmcDNA3.1-F)和SL7207(pcDNA3.1-F)免疫组的免疫保护率均与空载体组之间存在显著性差异(P＜0.05),且SL7207(pmcDNA3.1-F)免疫组的保护率较SL7207(pcDNA3.1-F)免疫组提高了20.0%,说明稳定携带新城疫病毒DNA疫苗的减毒沙门氏菌具有良好的免疫原性和免疫保护性.     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体(pmSEA),对HBVDNA疫苗诱导Balbc小鼠(H2d)免疫应答的调节作用。肌内注射空载体pcDNA3、HBVDNA疫苗加pmSEA佐剂(pHBVS2S+pmSEA)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs;ELISPOT检测分泌IFNγ的脾淋巴细胞;4h51Cr释放法检测小鼠脾细胞CTL活性。HBVDNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBVDNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFNγ的分泌量是不加佐剂组2～3倍。CTL细胞杀伤活性(E:T=100)佐剂组与不加佐剂组分别为:69.77%±7.5%、42.81%±7.7%,差异显著(P<0.05)。HBVDNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA疫苗的免疫应答,有望成为DNA疫苗的免疫佐剂。     

基因免疫制备西尼罗病毒NS5蛋白特异性单抗

研究了NS5蛋白在西尼罗病毒的特异性检测方面的应用及NS5在黄病毒复制中的作用机理。采用RT．PCR方法扩增了西尼罗病毒株的NS5基因片段,将其克隆至真核表达载体pVAX1,构建真核表达质粒。以重组质粒免疫BALB／c小鼠后取脾脏进行杂交瘤细胞融合,建立能稳定分泌西尼罗NS5单克隆抗体的杂交瘤细胞株。构建了真核表达质粒pVAX1-WNV—NS5,免疫动物后获得了28289等4株稳定分泌特异性抗体的杂交瘤细胞株,均为IgM型。真核表达质粒免疫后成功地诱导了针对NS5蛋白的体液免疫应答,单抗特异性分析显示4株单抗与其他黄病毒存在一定交叉反应。     

Enhancing the immunogenicity of exogenous hepatitis B surface antigen-based vaccines for MHC-I-restricted T cells

Vaccination with either exogenous hepatitis B surface antigen (HBsAg) lipoprotein particles without adjuvants, or plasmid DNA encoding secreted small HBsAg stimulate long-lasting, potent antibody responses in H-2d (BALB/c) and C57Bl/6 (H-2b) mice. Vaccination with exogenous HBsAg primes MHC-I restricted cytotoxic T lymphocyte (CTL) responses to HBsAg in H-2d but not H-2b mice, while DNA vaccination primes HBsAg-specific CTL responses in both mouse strains. We defined vaccination strategies that could elicit CTL responses to exogenous HBsAg in 'low responder' C57Bl/6 mice. We found that the bacterial plasmid DNA itself, synthetic oligodeoxynucleotides containing immunostimulating sequences, or recombinant Th1 cytokines (IL12, IFNgamma) efficiently support priming of CTL responses to exogenous HBsAg in 'low responder' H-2b mice, but have only minor effects on CTL priming in 'high responder' H-2d mice in the high dose range tested. These molecularly well defined adjuvants can thus efficiently support priming of anti-viral T cell responses under 'low responder' conditions.     

Immune responses to plasmid DNA encoding the hepatitis C virus core protein.

Hepatitis C virus (HCV) is a major causative agent of parenterally transmitted non-A, non-B hepatitis. The genomic region encoding the virion-associated core protein is relatively conserved among HCV strains. To generate a DNA vaccine capable of expressing the HCV core protein, the genomic region encoding amino acid residues 1 to 191 of the HCV-1 strain was amplified and cloned into an eukaryotic expression vector. Intramuscular inoculation of recombinant plasmid DNA into BALB/c mice (H-2d) generated core-specific antibody responses, lymphoproliferative responses, and cytotoxic T-lymphocyte activity. Our results suggest that the HCV core polynucleotide warrants further investigation as a potential vaccine against HCV infection.     

In vitro and in vivo stability of plasmids in attenuated Salmonella enterica serovar typhimurium used as a carrier of DNA vaccine is associated with its replication origin.

The ability of live attenuated Salmonella enterica serovar typhimurium (S. typhimurium) as a carrier of DNA vaccine was evaluated using model plasmid encoding beta-galactosidase (beta-Gal) and BALB/c mice. We constructed pBRCMVbeta, beta-Gal expression apparatus having a replication origin from low copy pBR322. Comparison of the plasmid stability showed that pBRCMVbeta remained stable in Salmonella even after oral administration, while pUC-based pCMVbeta tended to be lost quickly. However, titers for beta-Gal specific IgG in sera did not significantly increase in mice orally administered S. typhimurium harboring pBRCMVbeta. These data suggest that the stability of plasmid in S. typhimurium is associated with its replication origin. Further studies are required to scientifically establish this methodology.     

Immure response induced by oral DNA vaccination against FMDV delivered by attenuated Salm onella choleraesuis C500

A recombinant strain of Salmonella choleraesuis C500, containing a eukaryotic expression plasmid pBO1 with the immune-dominant epitope of foot-and-mouth disease virus, was constructed. Specific immune response to this recombinant strain was evaluated by oral administration of the recombinant live bacteria pBO1/S. cho in rabbits. Results showed that T cell response and specific antibody production were elicited. This approach may present a general strategy for eliciting immune responses with DNA vaccine delivered by live bacterial vectors. The stimulated indexes of T lymphoproliferation by specific antigens of FMDV in rabbits, can reach up to 11.0 and an antibody titer of 1/32 as detected in the erum with liquid block ELISA. __________ Translated From Journal of Fudan University(Natural Science), 2005,44(4)[译自: 复旦学报(自然科学版), 2005,44(4)]     

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].     

Live attenuated Salmonella as a vector for oral cytokine gene therapy in melanoma

Systemic administration of cytokines has shown therapeutic benefits in cancer patients; however, serious adverse effects associated with direct protein administration prevent the wide use of this approach. We have assessed the capacity of live attenuated Salmonella to act as a vector for oral cytokine-gene therapy. Salmonella orally administered to melanoma-bearing mice was found to accumulate within the tumor, reaching up to 10(5) bacteria per gram of tumor by day 21 after bacterial inoculation. Numbers of bacteria recovered from tumor did not differ from those recovered from liver or spleen at any time point. Recombinant bacteria carrying eukaryotic expression vectors encoding the murine IL-4 or IL-18 genes were administered to groups of mice with established subcutaneous melanoma tumors. We found that a single oral dose of Salmonella carrying any of the cytokine-encoding plasmids resulted in significantly increased survival time, as compared with mice that received Salmonella carrying the parental plasmid or PBS. Increased levels of IFNgamma were found in sera of animals receiving either of the cytokine-encoding bacteria, but not in mice receiving Salmonella alone or PBS. Co-administration of both recombinant bacteria maximized the production of IFNgamma. Overall these results suggest that cytokine-encoding Salmonella can be an effective and safer alternative to systemic administration of cytokines for immunotherapy of cancer.     

T7噬菌体转运真核表达载体入胞表达平台的构建

[目的]构建携带锚定序列的真核表达载体,研究T7噬菌体识别、包裹和转运真核表达载体进入细胞实现蛋白表达的可行性,为DNA疫苗研发建立新的技术平台.[方法]本研究通过重叠延伸PCR方法获得候选锚定序列并插入真核表达载体;建立荧光定量PCR方法比较T7噬菌体识别、包裹真核表达载体的效率;激光共聚焦显微镜观察T7噬菌体转运真...     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.