Comparing the immunogenicity of hepatitis B virus S gene variants by DNA immunization.

DNA immunization was used to compare the immunogenicity of hepatitis B virus S gene variants. Four recombinant plasmid DNAs containing the full-length virus genome with different S gene inserts were used to immunize BALB/c and C57/BL/6 mice. These inserts were cloned from 129L (residue 129, glutamine to leucine), 129H (residue 129, glutamine to histidine) 145R (residue 145, glycine to arginine) variants and the wild-type virus. The titer of hepatitis B virus core antibodies (anti-HBc) in immunized mice was used as the control for the efficiency of DNA immunization. Serum hepatitis B surface antibody (anti-HBs) titer and cytokines induced in splenocytes stimulated with hepatitis B surface antigen (HBsAg) were monitored as specific immune responses induced by different plasmid DNAs. 129L DNA induced significantly lower anti-HBs antibodies (IgG, IgG1, and IgG2a) and less interferon-gamma, compared to those in mice immunized with the 129H variant and the wild-type HBV DNA (p < 0.05). Computer modeling showed that a change from glutamine to leucine at 129 residue led to higher hydrophobicity and could result in decreased immunogenicity. Results indicate that DNA immunization can be used to compare the humoral and cellular immunogenicity among different HBV S variants.     

双表达载体研究GM—CSF对HCV C基因免疫应答的影响

目的 HCV C蛋白基因诱导的免疫应答较弱，因而增强HCV C蛋白基因免疫对于开发HCV疫苗具有要其的重要性。方法 将GM－CSF基因与pc154基因插入双表面载体pcDNA3.0 BA构建成双价质粒pcDNA3.0 APC154GM-CSF,肌肉免疫Balb/c小鼠。结果 GM－CSF和pc154在Cos-7细胞中同时获得瞬时表达；pcDNA3.0 BApc154GM-CSF双价质粒较单价pcDNA3.0 BApc154诱导小鼠产生抗体的几何平均滴度显著增高（P＝0.04），而pcDNA3.0 BApc154 pcDNA3.0 BAGMCSF混合质粒较单价质粒pcDNA3.0 BApc154诱导小鼠产生抗体的滴度极显著降低（P＜0.01）。HCV C蛋白抗原特异性的脾淋巴细胞增殖能力，双价质粒较单价及单价混合质粒极显著增高（P＜0.01）。结论 双表达载体同时输送GM－CSF与pc154基因能增强Balb/c小鼠对HCV C蛋白基因的体液免疫应答及免疫鼠脾淋巴细胞对特异性抗原刺激的增殖能力。     

Induction of antibody response by DNA immunization of newborn baboons against influenza virus.

Previous studies showed that DNA immunization of newborn mice with plasmids expressing influenza virus antigens induced protective immunity. We have now extended the study of neonatal responsiveness to DNA vaccines to nonhuman primates. Baboons immunized as neonates with plasmids expressing type A influenza virus hemagglutinin (HA) and nucleoprotein (NP) in doses ranging from 40 microg to 1 mg per plasmid per dose developed virus-specific humoral responses. The titer and kinetics of appearance of virus-specific IgG antibodies were dose dependent. Specific antibodies were detected by enzyme-linked immunosorbent assay (ELISA) as early as 1 month after birth in baboons immunized with the highest and intermediate doses of vaccine. Virus-neutralizing antibodies were detected in the group of baboons immunized with the highest dose. The specificity of virus-neutralizing antibodies was found to be directed against homologous determinants of HA; however, the IgG antibodies also cross-reacted with HA of a drift variant. Thus, DNA vaccination of newborn baboons with a prototype vaccine against influenza virus resulted in induction of specific humoral immunity.     

Co-delivery of GM-CSF gene enhances the immune responses of hepatitis C viral core protein-expressing DNA vaccine: role of dendritic cells.

Hepatitis C virus (HCV) infection has become a critical public health problem worldwide. In Taiwan, it has been estimated that more than 300,000 people, 2% of the general population, have HCV infection. It has been well documented that direct delivery of gene intramuscularly can generate both humoral and cellular immunity, which more closely simulates the conditions of infection. In this study, female Balb/c mice immunized with HCV core plasmid DNA with or without adjuvant GM-CSF cytokine gene could induce both cellular immune response and HCV core-specific antibody titers after injection. Furthermore, the mice immunized with HCV core plus GM-CSF genes showed higher antibody titer and cytotoxic T cell activity compared to those of mice immunized with HCV core gene only (P < 0.05). To explore the effect of GM-CSF gene, the mice were immunized with reporter gene and cytokine gene plasmid. Increased levels of reporter protein and infiltrating cells around muscle tissue were noted. Moreover, the protein could be detected in inguinal node 24 hr after injection, especially in mice immunized with HCV/core plasmid plus GM-CSF gene. It was also observed that reporter protein expressing CD11c(+) dendritic cells could be seen in the inguinal node. These data suggest that the GM-CSF gene did enhance HCV core specific immune response when co-immunized with HCV core DNA plasmid. Although more studies are needed, dendritic cells that appeared around the naked DNA injection area and that local lymph nodes might play a critical role in the immune response induced by naked DNA immunization.     

SARS冠状病毒S蛋白C端片段基因的克隆及其DNA疫苗的免疫学研究

目的：构建抗原基因为SARS冠状病毒（Severe acute respiratory syndrome coronavirus，SARS-CoY）S蛋白C端片段基因的DNA疫苗，采用肌注和滴鼻的方法接种小鼠后观察肌肉注射途径和黏膜途径免疫使机体产生免疫应答的情况。方法：将S蛋白C端片段克隆至真核表达载体pcDNA3．1（-），随之将重组质粒进行小鼠肌肉和黏膜免疫，定期检测外周血中抗SARS-CoV的病毒特异性抗体水平，流式细胞仪观察其淋巴细胞表型变化，免疫组化检测抗原表达分布，脾细胞增殖实验评价CTL效果。结果：疫苗注射后15天就能在血清中检测出病毒特异性抗体。随着时问的延续，抗体水平逐步升高，至30天后达到稳定，以CTL为主的CD8^＋T淋巴细胞的百分比含量增加极显著，引起强大的体液免疫和细胞免疫；疫苗滴鼻后45天可在鼻黏膜检测到抗原表达；而空载体质粒对照组未检测出明显的特异性免疫应答。结论：以S蛋白C端片段基因为抗原基因的DNA疫苗通过肌注和滴鼻能诱导小鼠针对SARS病毒强大的免疫应答。     

我国登革2型病毒43株PrM-E基因的DNA免疫原性研究

目的 观察我国登革２型病毒４３株ＰｒＭ－Ｅ基因的ＤＮＡ免疫原性及非甲基化细菌性ＣｐＧ对ＤＮＡ免疫效果的影响。方法 采用ＲＴ－ＰＣＲ和分子克隆技术构建ＰｒＭ－Ｅ基因片段，并将其插入真核表达载体ｐＢＫ－ＣＭＶ中。在证明其可在哺乳动物细胞中高效表达的基础上，进一步用重组质粒ＤＮＡ免疫小鼠。通过间接免疫荧光法对采集的鼠血清中的病毒特异抗体进行检测。结果 用含ＰｒＭ－Ｅ基因的重组质粒ＤＮＡ免疫小鼠可诱导产生     

Studies on antibody responses following neonatal immunization with influenza hemagglutinin DNA or protein.

Neonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 response following immunization with many vaccine antigens. These studies were undertaken to determine whether 1-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (H1) by either intramuscular (im) or gene gun (gg) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. The newborn mice developed stable, long-lived, protective anti-H1-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult im and gg DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1, IgG2a, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased but did not change these antibody profiles. In contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age.     

HCV/HBV复合DNA免疫的初步研究

目的 探讨HCV/HBV复合抗原PCX/S免疫原性。方法 将已证实具有良好免疫原性的人工合成的HCV复合多表位抗原基因PCX与HBV的S抗原基因融合于真核表达载体pcDNA3.0,构建真核表达载体pcDNA-PCXS。大量提取质粒并免疫小鼠,用ELISA的方法检测抗-HBs和抗-HCVAb;~3H-TdR渗入法检测免疫小鼠T淋巴细胞增殖;用~(51)Cr释放法检测免疫小鼠特异性CTLs杀伤作用。结果 免疫后均能检测到抗-HBsAb和抗-HCVAb,抗体水平随着时间的推移逐渐升高,但后者OD_(450nm)平均值低于抗-HBsAb的OD_(450nm)。免疫小鼠诱发针对PCX或S抗原的CTL反应和淋巴细胞转化。结论 复合型PCX/S抗原基因可诱发特异性免疫应答,为HCV/HBV双价疫苗的研究提供一定实验基础。     

Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases

C57BL/6 mice were immunized intradermally with various doses of purified pCEP4 plasmid DNA containing full-length MUC1 cDNA (22 tandem repeats). Mice immunized with MUC1 DNA three times at weekly intervals had serum antibodies to a synthetic peptide corresponding to the tandem repeats of MUC1. The antibody titer correlated with the plasmid DNA dose. After the third immunization mice were injected intravenously with 5×10⁵ B16-F10 melanoma cells that had been stably transfected with MUC1 cDNA (F10-MUC1-C8 clone cells). The number of lung metastatic nodules three weeks after inoculation of F10-MUC1-C8 cells was significantly lower in mice immunized with MUC1 plasmid DNA than in mice immunized with the vector DNA alone. Thus, the suppression of lung metastasis was antigen-specific. In vivo depletion of lymphocyte subpopulations by specific antibodies revealed that natural killer cells are the major effector cells responsible for the suppression of lung metastasis. CD4⁺ cells and CD8⁺ cells apparently played some roles too. This revised version was published online in July 2006 with corrections to the Cover Date.     

两株登革2型病毒E基因重组质粒免疫BALB/c小鼠产生特异性抗体水平的比较

目的 用含登革2型病毒(Dengue type 2 virus,DEN2)B株和NGC株E基因部分序列pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠,观察免疫小鼠体液免疫应答的差异.方法 用两株含DEN2 E基因部分序列(1～476 bp)的pcDNA3.1重组质粒与含有佐剂的重组质粒共同免疫BALB/c小鼠,初次免疫后第14天、28天分别加强免疫1次,共免疫3次.收集初次免疫后第14、28、42、70和98天外周血标本,间接ELISA法测定小鼠血浆特异性IgM/IgG类抗体水平,细胞病变抑制法检测特异性抗体水平.结果 不同DEN2毒株E基因部分序列的pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠诱导特异性IgM、IgG类抗体的产生存在差异,B株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间.结论 DEN2两毒株E基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异.     

两株登革2型病毒NS1基因真核重组质粒免疫BALB/c小鼠产生特异性抗体水平的比较

目的 利用登革2型病毒(dengue type 2 virus,DENV2)M株和NGC株NS1基因部分扇列PcDNA3.1重组质粒初次和加强免疫BALB/c小鼠,观察免疫小鼠体液免疫应答的差异.方法 分别构建两株DENV2 NS1基因部分序列(1-413 bp)的PcDNA3.1真核重组质粒和pET28a(+)质粒,进行原核蛋白的表达、鉴定、纯化和定量;并用pcDNA3.1重组质粒免疫BALB/c小鼠,初次免疫及第7天、14天分别加强免疫1次,共免疫3次.收集初次免疫后第7、14、28和56天外周血标本,间接ELISA法测定小鼠血清特异性IgM/IgG类抗体水平,细胞病变抑制法检测特异保护性抗体水平.结果 构建了pET28a(+)-NS1m/pET28a(+)-NS1n原核表达重组质粒,SDS-PAGE分析表明,NS1基因部分序列获得表达,其相对分子质量均约22.3×103;Western blot表明该目的 蛋白可与抗His标签单克隆抗体结合;经Ni柱亲和层析法得到纯度达92%的表达蛋白,对C6/36细胞有毒性,并可用于ELASA检测.不同DENV2毒株NS1基因部分序列的pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠诱导特异性IgM、IgG类和中和抗体的产生存在差异,M株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间.结论 DENV2两毒株NS1基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异.

Abstract：

Objective To compare the humoral immune response of BALB/c mice immunized by recombinant plasmids PeDNA3.1-M-NS1 and pcDNA3.1-N-NS1.Methods Dengue type 2 virus(DENV2)NS1 gene were constructed two partial sequences(1-413 bp)of the pcDNA3.1 eukaryotic plasmids and pET28a(+)plasmid for prokaryotic expression,identification,purification and quantification.The BALB/c mice were immunized by pcDNA3.1-M-NS1,pcDNA3.1-N-NS1 recombinant plasmids with adjuvant.Each animal received a primary inoculation and two boosts at 1-week intervals.Then the blood samples of BALB/c mice were collected from different experiment groups at day 7,14,28 and 56,respectively after first immunization.The specific IgM/IgG antibodies for NS1 protein in serum were confirmed by indirect ELISA.And then the activities of the specific protective antibody were determined by cytopathic effect inhibition(CPEI).Results Construction of the pET28a(+)-NS1 m/pET28a(+)-NS1n prokaryotic expression plasmid,SDS-PAGE analysis showed that,NS1 gene partial sequence was expressed,both the relative molecular weight of about 22.3×103:Western blot showed that the protein can bind anti-His tag monoclonal antibody;byNi affinity chromatographywith apurity of 92% protein,on the C6/36 cell toxicity,and can be used ELASA detection.The results showed that the levels of specific IgM/IgG antibody and neutralizing antibody activities were increased in pcDNA3.1-M-NS1 booster immunization group than other groups.The result had been observed longer duration of antibody level in peDNA3.1-M-NS1 booster immunization group.Conclusion Humoral immune response were significantly different between pcDNA3.1-M-NS1 and pcDNA3.1-N-NS1 recombinant plasmid immunized mice groups.     

IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠(H-2d)特异性免疫应答的影响

目的：观察IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠（H-2d）特异性体液免疫和细胞免疫应答的影响.方法：用肌肉注射法将HBV核心区DNA疫苗、IL-12质粒和IL-18 质粒接种BALB/c小鼠;ELISA法检测小鼠血清抗-HBc（IgG）及IgG亚类（IgG1、IgG2a）;LDH释放法检测小鼠脾细胞HBcAg特异性CTL活性.结果：免疫6周后,HBcAg DNA疫苗联合IL-12质粒、IL-18质粒和IL-12＋IL-18质粒组小鼠的血清抗HBc终点滴度均明显高于单纯注射HBcAg DNA疫苗组小鼠（P＜0.05）,抗HBc IgG亚类以IgG2a占优.DNA疫苗免疫的各组小鼠,HBcAg特异性细胞毒性T淋巴细胞杀伤率均高于对照组（P组）,其中C＋IL-18组和C＋IL-12＋IL-18组中CTL值明显高于C组,尤以C＋IL-12＋IL-18组中的CTL杀伤率最高.结论：IL-12和IL-18基因与HBcAg DNA疫苗联合免疫,不仅能增强HBcAg特异性体液免疫应答,而且能增强HBcAg特异性CTL的杀伤活性.     

Comparative evaluation of immunization with recombinant protein and plasmid DNA vaccines of fusion antigen ROP2 and SAG1 from <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in mice: cellular and humoral immune responses

The aim of this work was to evaluate immune responses in BALB/c mice vaccinated subcutaneously by recombinant protein, or intramuscularly by plasmid DNA with fusion antigen of rhoptry protein 2 (ROP2) and major surface protein 1 (SAG1) from Toxoplasma gondii (T. gondii). BALB/c mice were immunized with one of three different antigen formulations respectively, which were rROP2-SAG1, pcROP2-SAG1, and pcROP2-SAG1 boosted with rROP2-SAG1. The production of IgG, IgG subclasses, lymphoproliferation, and level of gamma interferon (IFN-γ) were detected after vaccination. The animals vaccinated with rROP2-SAG1 quickly developed specific anti-TLA (T. gondii lysate antigen) antibodies, which continued to rise after immunization. However, production of IgG against TLA in mice vaccinated with pcROP2-SAG1 was relatively slow and maintained a high level after reaching plateau. There are more vigorous specific lymphoproliferative responses observed in mice of group rROP2-SAG1 than in pcROP2-SAG1. Immune responses in mice of group pcROP2-SAG1 boosted with rROP2-SAG1 were similar to the protein immunization group. Three immunization procedures resulted in a similar level of IFN-γ production. Our results indicate that BALB/c mice vaccinated by three immunization procedures induce similar humoral and cellular immunity against infection of T. gondii. Mice immunized with recombinant protein rROP2-SAG1 produce more humoral immune responses than mice immunized with other antigen formulations.     

壳聚糖介导增强幽门螺杆菌Lpp20 DNA疫苗黏膜免疫效果的研究

目的 探讨壳聚糖包裹DNA疫苗黏膜免疫效果.方法 采用复凝聚法制备载幽门螺杆菌脂蛋白 Lpp20基因的壳聚糖(CS)纳米粒(NPs),并对CS/DNA纳米粒的特质进行研究;用载基因壳聚糖纳米粒黏膜免疫(滴鼻和口服)小鼠,检测免疫小鼠的细胞和体液免疫水平.结果 裸质粒pcDNA3.1(+)/Lpp20与CS/DNA NPs通过黏膜免疫均能诱导小鼠产生有效的免疫应答.CS/DNANPs滴鼻和口服免疫组诱导的抗体明显升高,与裸质粒组相比有明显差异(P＜0.05),同时CS/DNANPs滴鼻和口服免疫组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数及培养上清中IFN-γ和IL-4含量明显高于裸质粒组、壳聚糖组和PBS组,且滴鼻免疫组高于口服组.结论 壳聚糖纳米粒能增强pcDNA3.1(+)/Lpp20核酸疫苗的黏膜免疫(滴鼻和口服免疫)效果;载Lpp20基因壳聚糖纳米粒滴鼻免疫比口服免疫能诱导更强的细胞和体液免疫应答.

Abstract：

Objective To investigate the immune response of mucosal immunization of new chitosan(CS) nanoparticles coating DNA vaccine. Methods The chitosan nanoparticles containing plasmid DNA encoding H. pylori lipoprotein Lpp20 gene were prepared using a complex coacervation method and then its speciality were analyzed. We then administered the naked plasmid DNA and chitosan-DNA nanoparticles to 6-week-old female BALB/c mice by intranasal or oral mucosal routes to observe the humoral and cellular immune responses. Results Naked plasmid pcDNA3.1 ( + )/Lpp20 and chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles both induced effective immune response in mice through mucosal vaccination. Specific IgG and sIgA antibodies of chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles groups were higher than that of naked pcDNA3.1 ( + )/Lpp20 group. The concentration of cytokines IFN-γ and IL-4 in cultural supernatant of T lymphocytes from chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles immunized mice increased greatly than that of control groups. After stimulated by corresponding antigen, the stimulation index of intranasal or oral delivery of chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles group was significantly higher than that of pcDNA3.1( + )/Lpp20 group, CS group and PBS control group. Moreover, systemic and mucosal immune responses in mice induced by intranasal immunization were stronger than that of oral immunization. Conclusion Chitosan nanoparticles enhanced the immune response of pcDNA3.1 ( + )/Lpp20 DNA vaccine by intranasal or oral administration in BALB/c mice. Compared to oral administration, intranasal delivery of chitosan-pcDNA3.1 ( + )/Lpp20 DNA nanoparticles could induce stronger cellular and humoral immune responses in BALB/c mice.     

Immune responses induced by gene gun or intramuscular injection of DNA vaccines that express immunogenic regions of the serine repeat antigen from Plasmodium falciparum.

The liver- and blood-stage-expressed serine repeat antigen (SERA) of Plasmodium falciparum is a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein.     

假结核耶尔森菌侵染素蛋白作为DNA疫苗佐剂的免疫效果

目的探讨假结核耶尔森菌侵染素(invasin)羧基端397个氨基酸(Inv397)对抗原免疫原性的影响,以评价其作为DNA疫苗佐剂的效果。方法利用PCR扩增Inv397编码基因(inv397),分别构建编码猪丹毒丝菌表面抗原氨基端蛋白(spaA-N)和Inv397蛋白的重组真核表达质粒pcDNA3.1-spaA-N,pcDNA3.1-spaA-N-inv397。同时构建重组载体pET-30a-inv397,并在大肠杆菌BL21中诱导表达,经过Ni Sepharose 6 Fast Flow亲和层析纯化重组Inv397蛋白。将纯化后重组Inv397蛋白与包含spaA-N的重组真核质粒滴鼻免疫ICR小鼠,ELISA法检测血清特异性抗体水平,细胞增殖实验分析T淋巴细胞增殖反应。结果 Inv397蛋白与spaA-N重组质粒滴鼻免疫组的血清spaA-N特异性IgG水平明显高于spaA-N其它对照组(P<0.01),且T细胞增殖水平也高于其他各组,但无显著差异。结论 Inv397蛋白具有一定程度的免疫增强作用,有可能成为一种新的DNA疫苗佐剂。     

Single-shot plasmid DNA intrasplenic immunization for the production of monoclonal antibodies. Persistent expression of DNA

Monoclonal antibodies (Mc. Abs.) were generated against a 18-kDa protein from Brucella abortus 48 h and 25 days after a single intrasplenic injection of a DNA plasmid containing the expression vector for the protein. Hybridomas were also obtained from spleens injected 3, 5, and 10 days before fusion. Somatic cell fusion of spleen cells from mice, injected with the plasmid DNA, in saline, with the NS-0 myeloma cell line resulted in Mc. Abs of the IgG and IgM Isotypes. IgG antibodies were of the IgG2b and IgG1 subtype. Hybridoma tissue culture supernatants were strongly positive by ELISA at dilutions of up to 1/1200 and produced intense specific bands in immunoblotting. All these antibodies recognized the native recombinant protein (the screening antigen) and some of them also recognized the heat-denatured recombinant 18-kDa protein. When compared to standard procedures of immunization, as well as to intramuscular or gene gun DNA immunizations, this technique results in very early, time saving, strong Mc Abs. It is common knowledge that in order to generate specific hybridomas; spleen cells from immunized animals have to be fused no later than 5 days after the last boost. The fact that through single-shot intrasplenic immunization (SSI) specific hybridomas are generated 25 days after one single injection indicates that the gene coding the p18 protein is being expressed in the spleen for at least 20 days. We propose that plasmid DNA intrasplenic immunization can be a helpful tool for the production of specific hybridomas. This route of immunization could also be helpful in the further understanding of early events of the immune response to genetic immunization by naked DNA injection.     

No evidence of hepatitis B virus activity in patients with anti-HBc antibody positivity with or without anti-hepatitis C virus antibody positivity.

BACKGROUND: The serological pattern of anti-HBc antibody positivity without both, HBsAg and anti-HBs antibody positivity may be present in up to 4% of the population of Europe and the United States. OBJECTIVES: The aim of the present study was to determine the hepatitis B virus (HBV) activity by detection of serum HBV DNA in patients with anti-HBc antibody positivity only and with confirmed anti-hepatitis C virus (anti-HCV) antibody positivity or without anti-HCV antibody positivity. STUDY DESIGN: A total of 141 patients positive for anti-HBc antibodies only, were investigated on serum HBV DNA load. Patients were classified into two groups: patients with confirmed positive anti-HCV antibodies (group 1) and patients without anti-HCV antibodies (group 2). RESULTS: Demographic data of patient groups were similar. In 66 of 70 patients with anti-HBc antibodies and anti-HCV antibodies (group 1), serum HCV RNA was detected; the remaining 4 patients were HCV RNA negative but the presence of anti-HCV antibodies was confirmed by the line probe assay. In none of the patients, with anti-HBc antibodies and without anti-HCV antibodies (group 2), serum HCV RNA was detected. In none of the patients, serum HBV DNA was detected. CONCLUSION: In this study, serum HBV DNA could not be detected in patients with anti-HBc antibodies only. There seems to be no need for determination of serum HBV DNA in patients without clinical evidence of chronic liver disease. Nevertheless, it would be useful to test patients with progressive liver disease and those, which belong to high-risk groups such as hemophiliacs, intravenous drug abusers, patients on hemodialysis, and immunocompromised patients.     

小鼠及家兔对丙型肝炎病毒多表位DNA疫苗免疫应答的研究

目的 探讨HCV复合多表位DNA疫苗的可行性。方法 把HCV多表位抗原基因PCX克隆到真核表达载体pREP9(RSV启动子）及pcDNA3(CMV启动子）中,构建真核表达载体pREP9/PCX及pcDNA3/PCX。将其肌肉注射免疫小鼠及家兔,检测特异性体液免疫和细胞免疫的水平,并观察免疫小鼠的安全性。结果 质粒pREP9/PCX及pcDNA3/PCX于免疫后第6wk和第10wk时,开始可检测到抗GZ-PCXIgG,随后抗体滴度逐渐升高,但水平较低持续时间较短,pcDNA3/PCX肌肉注射免疫家兔后,于第8wk开始出现特特抗体,至3个月后滴度升至1：3200随后也开始下降,免疫小鼠及家免可诱发针对GZ-PCX融合蛋白的迟发型超敏反应,并刺激淋巴细胞转化,免疫后小鼠的体重正常,肝脾脏未见明显肿大,具有良好的安全性。结论 HCV多表位基因可诱发特异性免疫应答且安全性。结论ＨＣＶ我表位基因可诱发特异性免疫应答且安全性好,为ＨＣＶ疫苗的研究提供一定的理论及实验依据。