C3d-P28增强乙型肝炎病毒特异性基因免疫效果的研究

目的　观察补体C3d P2 8对基因免疫的调节作用及其不同拷贝数对调节作用的影响 ,为增强基因免疫效果寻求新方法。方法　PCR法获得补体C3d P2 8编码基因并以头尾串连方式将1～ 4拷贝C3d P2 8编码基因克隆至pVAON33,构建pVAON33 P2 8.[1～ 4 ]重组质粒 ,然后将HBV preS2 S编码基因分别插入pVAON33和pVAON33 P2 8.[1～ 4 ]质粒获得pVAON33 S2 S和pVAON33 S2 S P2 8.[1～ 4 ]重组质粒。肌肉注射各重组质粒DNA(每只 10 0 μg 10 0 μl)初次免疫小鼠 ,并以pVAON33为对照 ;12周后皮下注射HBsAg蛋白加强免疫各组小鼠 ,ELISA法检测免疫小鼠血清特异性抗 HBs IgG。结果　pVAON33 S2 S重组质粒免疫小鼠可诱导产生特异性抗 HBs IgG ,含不同拷贝C3d P2 8编码基因的重组质粒可诱导更高的特异性抗体 ,其中pVAON33 S2 S P2 8.4重组质粒诱导的抗体水平最高 (P <0 .0 1)。蛋白加强免疫后 ,含C3d P2 8编码基因重组质粒免疫组抗 HBs IgG迅速上升 ,并明显高于pVAON33 S2 S重组质粒免疫组 (P <0 .0 5 ) ,pVAON33 S2 S P2 8.4重组质粒诱导的抗体仍维持最高水平。结论　不同拷贝的C3d P2 8能不同程度地增强HBV preS2 S基因免疫诱导的特异性体液免疫及其蛋白加强后的回忆反应 ,其中 4拷贝C3d P2 8的增强作用较为显著。     

HBV-preS2/S-C3d联合基因疫苗诱导的特异性免疫应答及其意义

目的：研究HBV—preS2／S—C3d联合基因疫苗诱导的特异性体液和细胞免疫应答及其意义。方法：CR2结合实验检测C3d融合蛋白的结合特性；分别用TR421、TR421-preS2／S和TR421—preS2／S—C3d3重组质粒免疫小鼠．ELISA法检测特异性抗HBs—IgG的亲合力，^3H-TdR掺入法检测其特异性淋巴细胞增殖活性（SI），半定量RT—PCR法检测IL-4、IFN-γ、T—bet和GATA-3基因表达的水平。结果：C3d融合蛋白可有效地结合CR2；TR421—preS2／S—C3d3质粒基因免疫诱导的抗HBs—IgG水平、亲合力以及SI均明显高于TR421-preS2／S组，并且前者诱导的IL-4、IFN-γ、T—bet和GATA-3基因表达水平也均高于后者。结论：C3d通过结合CR2、上调IL-4和转录因子的表达、促进抗体亲合力成熟来增强基因免疫诱导的HBV特异性免疫应答。     

C3d-P28增强乙肝病毒基因免疫诱导的特异性细胞免疫应答

目的 :研究补体C3d中P2 8分子对HBV基因免疫诱导的细胞免疫应答的调节作用 ,为增强基因疫苗细胞免疫的效果寻求新方法。方法 :分别分离获取C3d P2 8和HBV preS2 /S编码基因 ,并克隆入真核表达载体 pVAON33中 ,构建相应重组质粒pVAON33 S2 /S (仅含HBV preS2 /S编码基因 )和pVAON33 S2 /S P2 8.4 (含HBV preS2 /S和 4拷贝C3d P2 8的编码基因 ) ,并以PCR、酶切和DNA序列测定进行鉴定。以肌肉注射法对BALB/c小鼠实施 3次基因免疫 ( 10 0 μg/10 0 μL·只 ) ,间隔 3wk ,并以空质粒免疫小鼠作为对照。免疫小鼠脾细胞体外经HBsAg刺激后 ,用3 H TdR掺入法和同位素释放法 ,分别检测特异性淋巴细胞增殖和CTL杀伤活性。结果 :pVAON33 S2 /S和 pVAON33 S2 /S P2 8.4免疫小鼠的脾细胞 ,均显示较强的特异性增殖活性和CTL杀伤活性 ,但后者显著强于前者 (P <0 .0 5 )。结论 :C3d P2 8可增强HBV preS2 /S基因免疫诱导的特异性细胞免疫应答     

C3d-P28对基因免疫诱导的HBV特异性细胞免疫应答的调节作用

目的：观察补体C3d-P28对基因免疫诱导的HBV特异性细胞免疫应答的调节作用，为增强基因免疫效果寻求新方法。方法：将质粒pVAON33-S2／S质粒(仅含HBV-preS2／S编码基因)和pVAON33-S2／S-P28．4质粒(含HBV-prS2/S和4拷贝C3d-P28的编码基因)以肌肉注射法对BALB／C小鼠实施基因免疫(100μg/100μl只)，并以空载质粒为对照。定期采集免疫小鼠血清、ELISA法检测其中特异性抗HBs-IgG及其亚型的水平；免疫小鼠脾细胞经特异性抗原(HBsAg)刺激后，采用，H-TdR掺入法、半定量RT-PCR法、同位素释放法分别检测其特异性淋巴细胞增殖活性、IL-4和IFN-γ基因表达的水平、CTL杀伤活性。结果：pVAON33-S2／S-P28．4质粒基因免疫诱导的特异性淋巴细胞增殖活性、抗HBs-IgG水平以及特异性CTL杀伤活性均明显高于pVAON33-S2／S质粒；C3d-P28未改变基因免疫诱导的抗HBs-IgG各亚型水平的格局，同时增强IgG1、IgG2a、IgG2b的水平；pVAON33-S2／S-P28．4质粒诱导的IL-4和IFN-γ的基因表达水平均明显高于pVAON33-S2／S质粒。结论：C3d-P28可在提高体液免疫应答的同时增强基因免疫诱导的HBV特异性细胞免疫应答。     

HBV与HCV融合DNA疫苗的构建及其体液免疫应答

目的 构建含乙型肝炎病毒（HBV）表面抗原基因（S区基因）与丙型肝炎病毒（HCV）核心抗原基因（C区基因）的嵌合真核表达载体,观察preS1和preS2基因对HBV表面抗原及HCV核心抗原体液免疫的影响。方法 用PCR方法,分别扩增HBV S区基因和HCV C区基因。将S区基因克隆入真核表达载体pcDNA3.1,酶切鉴定后,大量提取质粒并免疫Balb/c小鼠,用ELISA法检测抗HBs和抗HCV抗体。结果 成功地扩增出目的基因片段,克隆后酶切鉴定结果正确,序列分析与文献报告相一致。免疫后检测到抗HBs和抗HCV抗体。preS1与preS2基因对构建的融合DNA疫苗的体液免疫应答有一定的抑制作用。抗HBs抗体的产生低于只含S基因的真核表达载体;preS1基因对抗HCV抗体的产生具有抑制作用,而preS2无影响。结论 不同长度的HBV S区基因可影响抗HBs和抗HCV抗体的产生。     

全反式维甲酸对基因免疫应答的调节作用

目的　研究全反式维甲酸 (ATRA)对TR4 2 1 hCGβ质粒基因免疫诱生的特异性细胞免疫与体液免疫应答的调节作用。方法　肌肉注射重组质粒TR4 2 1 hCGβDNA(每只鼠 5 0 μg 10 0 μl)初次免疫小鼠 ,以灌胃的方式给予ATRA ,并以灌溶剂和TR4 2 1质粒免疫为对照 ;3周与 6周后经同样的方式加强免疫各组小鼠 ,采用ELISA方法对基因免疫小鼠血清中IgG抗体水平进行动态观察 ,分析小鼠血清中IgG抗体亚类 ;3H TdR掺入法测定特异性细胞增殖和细胞杀伤功能。结果　ELISA结果表明 ,TR4 2 1 hCGβ质粒基因免疫诱生较高的抗hCGβ抗体水平 ,ATRA增强TR4 2 1 hCGβ质粒基因免疫诱生的抗hCGβ特异性IgG抗体水平并且伴随IgG2a IgG1显著性降低 ;TR4 2 1 hCGβ质粒基因免疫诱生较强的淋巴细胞增殖活性和CTL活性 ,ATRA抑制TR4 2 1 hCGβ质粒基因免疫诱生的特异性细胞增殖和细胞杀伤功能。结论　ATRA促进基因免疫诱生的TH2免疫应答 ,抑制TH1型免疫应答 ,为改变基因免疫诱生的特异性免疫应答类型提供了一条新的途径。     

C3d对原癌基因HER-2/neu基因免疫的调节作用

目的：研究C3d对HER-2／neu基因疫苗诱导的免疫应答的调节作用。方法：分别构建含人HER-2／neu胞外区（hECD）的重组质粒TR421．hECD和hECD与C3d的融合质粒TR421-hECD-C3d3、pcDNA3-hECD-C3d3。以质粒基因免疫小鼠，定期收集血清，以ELISA法检测抗HER-2／neu抗体，3H-TdR掺入法检测细胞免疫应答，观察C3d对hECD免疫应答的调节作用，并以C3d对HBV免疫应答的调节作为对照。结果：hECD-C3d3融合质粒诱导的抗HER-2／neu抗体水平显著低于hECD质粒诱导的抗体水平，其诱导的淋巴细胞增殖反应也显著低于hECD质粒免疫组，但C3d能增强HBV基因免疫诱导的免疫应答。结论：C3d负调控HER-2／neu基因免疫诱导的免疫应答。     

重组质粒pcDNA3.1IL-2/Fc对HBV preS2S DNA疫苗免疫作用的影响

目的 探讨IL-2/Fc融合表达后对HBVpreS2S基因疫苗诱导免疫反应的佐剂效应.方法 采用HBV preS2S DNA疫苗作为基础免疫,重组质粒pcDNA3.1IL-2/Fc作为佐剂加强免疫BALB/c小鼠,采用0、2、4周的方案接种,检测各次接种后抗体水平.初次免疫后7周,测定免疫脾细胞的杀伤活性、增殖活性和细胞因子的分泌水平.结果 pcDNA3.1IL-2/Fc作为佐剂在HBV preS2S注射3 d后免疫组小鼠抗体滴度、免疫脾细胞的杀伤活性和增殖活性、TH1型细胞因子的分泌水平,均比各对照组明显增强.结论 IL-2/Fc是有效的HBV preS2S DNA疫苗佐剂之一.     

体内电转染增强基因免疫诱导的HBV特异性体液免疫应答

为观察体内电转染对HBV基因免疫诱导的特异性体液免疫应答的调节作用,将HBV-preS2/S编码基因插入pVAON33载体构建重组质粒pVAON33-preS2/S,运用肌肉注射法对BALB/c小鼠进行基因免疫(100ug质粒DNA.100ul.只)。以pVAON33-preS2/S、pVAON33-preS2/S(体内电转染)为实验组,并以pVAON33空质粒为对照。按期采集免疫小鼠血清。采用ELISA法检测免疫小鼠血清特异性抗HBs-IgG抗体。结果显示,pVAON33-preS2/S免疫小鼠后可诱导产生特异性抗HBs-IgG抗体,到第7周时其OD值为0.73±0.18(P/N为2.13),而pVAON33-preS2/S(体内电转染)组诱导了更高水平的特异性抗HBs-IgG抗体,第7周时OD值为1.30±0.45(P/N为3.79),两组比较有显著性差异(P<0.05)。本研究表明体内电转染可明显增强HBV基因免疫诱导的体液免疫应答。     

重组乙型肝炎病毒变异s基因疫苗诱导小鼠产生特异性体液免疫应答

目的：构建乙型肝炎病毒(HBV)变异s基因真核表达载体，检测其诱导小鼠产生特异性体液免疫应答。方法：利用限制性内切酶定位克隆构建s基因nt587 G→A的真核表达载体pcMV-S2．S 145R(PR).用其转染人肝癌细胞系Hep G2后，用EIA、EILISA及免疫细胞化学法，观察其抗原性。以重组变异型s基因真核表达载体(PR)和载体pcDNA3．0分别免疫C57BL／6小鼠各5只。每只小鼠各肌肉注射纯化质粒100μg.用ELISA法检测血清抗-HBs及抗-HBs2抗体的效价。结果：体外实验证实，变异型HBs矩可与抗-HBs结合；PR免疫小鼠可诱导其产生抗-HBs抗体及抗．HBs2抗体，但抗-HBs2抗体的出现早于抗-HBs抗体约1～2wk。结论：HBV变异s基因(nt587G→A)的真核表达载体的表达产物具有良好的抗原性，能够诱导C57BL／6小鼠产生体液免疫应答。     

Primary humoral antibody response to Coxiella burnetii, the causative agent of Q fever.

Of 147 patients with acute Q fever diagnosed during a major outbreak in Birmingham, England, in early summer 1989, 41 provided sets of sera which allowed us to make a detailed analysis of the primary humoral immune response. Antibody titers specific for Coxiella burnetii were measured by the complement fixation test and by an immunoglobulin M (IgM)- and IgG-specific indirect immunofluorescence test. The relative avidity of specific IgGs was determined by the indirect immunofluorescence test with and without treatment of antigen-antibody complexes with 8 M urea. The IgG subclass responses after primary infection and their avidities were also determined for a limited number of paired serum specimens. Specific IgM titers persisted for more than 6 months in the majority of cases and were therefore not a sufficient criterion for the diagnosis of recent infection. However, for serial samples the antibody titer ratios (IgG/IgM) and the ratios (IgG titer with treatment/IgG titer without treatment) that indicated relative avidity changed significantly, depending on the time postinfection. Within the IgG class, the C. burnetii-specific antibody response over time was almost exclusively represented by subclass 1 molecules, which thus showed affinity maturation.     

Serum immunoglobulin G antibody to Porphyromonas gingivalis in rapidly progressive periodontitis: titer, avidity, and subclass distribution.

Porphyromonas gingivalis is a suspected pathogen in rapidly progressive periodontitis (RPP). We have determined the anti-P. gingivalis serum immunoglobulin G (IgG) isotype response and avidity and the subclass titer distributions for 30 RPP patients and 30 age-, sex-, and race-matched healthy subjects by using enzyme-linked immunosorbent assay technology. Patients and control subjects were classified as seropositive if their total IgG response to P. gingivalis was twofold or more than the median response in healthy subjects. The predominant antibody responses for both patients and healthy subjects were IgG2 and IgG3, with a subclass order of IgG2 greater than IgG3 greater than IgG1 greater than IgG4. The avidity of the IgG response was highest for the seropositive healthy subjects and was no different between seronegative and seropositive RPP patients. The subclass antibody responses did not depend on gender, and there were no correlations between titer, avidity, or subclass with disease severity in the RPP patients as measured by pocket depth or bone loss on dental X rays. The seronegative RPP patients exhibited antibody responses that were greater than the responses of seronegative healthy subjects for all four subclasses, while the seropositive RPP patients had higher IgG1 and IgG4 levels than seropositive healthy subjects. These findings are consistent with the hypothesis that both carbohydrate and protein antigens are important in the IgG response to P. gingivalis. The relative predominance of IgG2, a subclass which lacks strong complement fixation and opsonic properties, and the low avidity of patient anti-P. gingivalis IgG antibodies suggest that humoral responsiveness to infection with P. gingivalis may be ineffective in clearing this organism.     

C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge

The ability of the C3d component of complement to enhance antibody responses and protective immunity to influenza virus challenges was evaluated using a DNA vaccine encoding a C3d fusion of the hemagglutinin (HA) from influenza virus. Plasmids were generated that encoded a transmembrane HA (tmHA), a secreted form of HA (sHA), or a sHA fused to three tandem copies of the murine homologue of the C3d (sHA-3C3d). Analysis of the titers, avidity maturation, and hemagglutinin-inhibition activity of raised antibody revealed that immunizations with sHA-3C3d DNA accelerated both the avidity maturation of antibody to HA and the appearance of hemagglutinin-inhibition activity. These accelerated antibody responses correlated to a more rapid appearance of protective immunity. They also correlated to complete protection from live virus challenge by a single vaccination at a dose ten times lower than the protective dose for non-C3d forms of HA.     

不同HBsAg疫苗联合免疫后诱导小鼠细胞和体液免疫应答的研究

目的：了解HBsAg的蛋白疫苗（P）、痘苗病毒疫苗（V）、DNA疫苗（D）联合免疫小鼠诱导的特异性体液和细胞免疫应答。方法：以P、V或D疫苗中的一种疫苗初次免疫BALB／c小鼠后，于第2、5、8、11周再用另一种疫苗加强，共产生9种免疫组合：即PP、PV、PD、VP、VV、VD、DP、DV及DD。于初免后第2、5、8、11周采血检测血清中抗HBsAgIgG的总滴度及其IgG1和IgG2a亚类，并于每次加强免疫后第7天，检Nd,鼠脾脏的CTL对P815S细胞的特异性杀伤率。结果：在P、V、D3种疫苗中，V疫苗诱导产生抗HBsAg抗体的速度最快，P疫苗诱导的体液免疫回忆反应最强，D疫苗诱导产生的抗体最弱。除PP疫苗组合诱导的抗体明显倾向于IgG1外，其他均无明显的倾向性。各种免疫组合中，VD和DV疫苗组诱导的CTL应答最强，对P815S的特异性杀伤率分别为71％和64％。结论：在各种联合免疫组合中，PV、PD、VP和VD疫苗组的抗体应答较好；而DV和VD疫苗组诱导的CTL杀伤效应最强。     

IgG2 subclass restriction of antibody to pneumococcal polysaccharides

Studies in experimental animals suggest that antibody responses to certain polysaccharide antigens may be restricted in IgG subclass distribution. To determine if human antibodies to pneumococcal polysaccharides are similarly restricted we measured the IgG subclass specific response to immunization with purified polyvalent pneumococcal polysaccharide vaccine. For the type 3 pneumococcal antigen, the geometric mean titre of IgG2 antibody was significantly greater than that of IgG1, IgG3 or IgG4, in both pre-immunization and post-immunization sera. A significant rise in mean titre, comparing pre- to post-immunization sera was observed only for IgG2 antibody. Similar predominance of IgG2 antibody was found for pneumococcal polysaccharides types 6, 18, 19 and 23. In contrast, antibody to the protein antigen tetanus toxoid was exclusively of the IgG1 subclass. Patients with IgA/IgG2 deficiency demonstrated a normal IgG response to tetanus, a normal IgM response to pneumococcal polysaccharides, but no IgG antibody to pneumococcal antigens. IgG2 subclass restriction of antibody to pneumococcal polysaccharides suggest that these antigens may elicit an immune response analogous to the murine type 2 T-cell independent immunogens which show IgG subclass restriction and the requirement of a mature B cell subset defined by the Lyb5+ alloantiserum. Our findings support the possibility of subclass-specific inducing or regulating mechanisms for human responses to carbohydrate or polysaccharide antigens.     

Avidities of hapten-specific antibodies when the responses are modulated by anti-carrier antibodies.

Either alone or in combination with antibodies having specificity for the carrier erythrocyte, TNP-ORBC were injected i.p. into CBA/J mice. Five days later, their spleens were removed and evaluated for TNP-specific PFC. The spleens from animals receiving 19S antibody (IgM) with carrier specificity displayed 3-4-fold more direct and indirect hapten-specific PFC than control animals receiving the TNP-erythrocyte conjugate only. Animals receiving 7S antibodies (IgG) with carrier specificity displayed very little change in their direct PFC response to the hapten. However, the indirect response to the hapten was suppressed as much as 16-fold by carrier specific IgG. Evaluation by haptenic inhibition of the relative avidity of the antibodies being secreted by these cells revealed the following: the relative avidity of antibodies secreted by indirect PFC in the spleens of animals receiving TNP-ORBC only was approximately 20-fold higher than antibodies secreted by the direct PFC. The 3-4-fold potentiation of the hapten-specific PFC response by carrier-specific IgM antibody did not result in a change in relative avidity of direct or indirect PFC. IgG with carrier specificity did not change the relative avidity of the antibodies secreted by direct PFC having specificity for the hapten. However, evaluation of the remaining PFC in spleens from animals whose indirect hapten-specific PFC response had been suppressed by carrier-specific IgG revealed that the remaining PFC had a lower avidity than indirect PFC from animals receiving TNP-ORBC only. In other words, carrier-specific IgG selectively induced suppression of high avidity hapten-specific IgG antibody secreting cells.