Administration of multiple cytokine genes with anti-tumor activity inhibits both tumor incidence and tumor growth.

The finding of reporter gene expression in muscle cells after intramuscular injection of a reporter gene containing DNA has suggested that injection of a certain gene in its naked form could induce an expression of the injected gene. The result proposed the concept, namely DNA or genetic vaccine technology, that injection of an antigen gene could induce a specific immune response against the antigen. Although the concept was initially applied to vaccination technology, the result also means that administration of cytokine genes with anti-tumor activity could exert their functions when they are applied as a naked form of DNA. To test the possibility, plasmid vector containing granulocyte macrophage-colony stimulation factor (GM-CSF) and interleukin-12 (IL-12) genes, which are known as one of the most potent anti-tumor cytokines, were constructed and injected into mice together with syngeneic tumor cells. When the cytokine gene containing plasmid was injected on the same day of tumor cell injection, a tumor mass developed in 4 out of 5 mice tested. Even among the 4 mice, the tumor mass of a mouse disappeared 2 weeks after tumor development. In addition, tumor generation was significantly delayed in cytokine gene injected mice and the average tumor size was about 51.5% that of vector control injected mice. These results suggested that tumor treatment through the injection of multiple cytokine genes with potent anti-tumor activity significantly inhibits tumor development and growth, and that the method could be considered as one of the tools for efficient tumor treatment.     

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。     

表达丙型肝炎病毒NS3和Core融合蛋白基因的DNA疫苗免疫方案优化

目的 本研究旨在构建一种包含丙型肝炎病毒(HCV)保守区基因的新型DNA疫苗,并在小鼠模型中使用电转技术优化其免疫原性.方法 首先,我们构建了包含HCV非结构蛋白NS3和核心蛋白Core部分基因序列的DNA疫苗,并证实了其表达;然后采用不同的体内电转方式于第0、4周分别免疫BALB/c小鼠,比较分析不同免疫方案的体液免疫(特异性IgG与抗体亚类)与细胞免疫应答(IFN-γ ELISPOT)的效果.结果 使用电转技术可显著增强新型DNA疫苗免疫原性,采用皮内注射加卡钳电极电转的方式产生最强NS3特异性T细胞免疫反应.结论 包含HCV保守区基因的新型DNA疫苗可通过优化电转技术增强免疫应答效果.这为我们下一步优化HCV DNA疫苗的免疫方案提供了依据.     

Amplification of T-Cell and Antibody Responses in DNA-Based Immunization with HIV-1 Nef by Co-Injection with a GM-CSF Expression Vector

Intradermal inoculation of mice with naked plasmid DNA encoding the regulatory HIV-1 Nef protein was shown to induce Nef-specific T and B cell responses. Co-inoculation with an expression vector encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine known to facilitate the induction of primary immune responses, resulted in a markedly enhanced response to Nef. This was manifested both as an increase in Nef-specific T cell responses and antibody levels. DNA immunization with the Nef and GM-CSF vectors induced primarily a Th1 response as judged by the raised levels of both IFN-γ and IL-2 from re-stimulated T cells. The immunostimulatory activity of GM-CSF DNA was locally restricted and was observed only if both plasmid vectors were injected at the same site.     

丙肝病毒(HCV)及乙肝病毒(HBV)双表达载体的构建及表达

目的 :为探索HCV/HBV高效联合基因免疫策略 ,构建具有 2套独立表达单元的HCV/HBV真核表达载体。方法 :分别将与HCV核心区基因互补的cDNA和HBV核心区基因克隆于具有 2套独立表达单元的真核表达载体pRSC的巨细胞病毒启动子和RSV启动子下游 ,称为pRSC HBV/HCV ,转染SP2 / 0细胞 ,通过免疫荧光和Western印迹法观测蛋白的表达 ,免疫Balb/c小鼠 ,用酶联免疫小鼠体液免疫应答。结果 :pRSC HBV/HCV转染SP2 / 0细胞可见HBcAg及HCV核蛋白染色阳性荧光细胞 ,SDS Page电泳显示在 14kD及 2 1kD处均可见蛋白条带 ,与HBcAg及HCV核蛋白的的理论预期值一致 ,Western印迹分析显示在 14kD及 2 1kD处可见特异性的蛋白条带。 5只免疫鼠中全部出现抗 HCV及抗 HBV抗体 ,而对照组全阴性。结论 :pRSC HBV/HCV可分别表达HBcAg及HCV核蛋白 ,免疫Balb/c小鼠后可诱导其体液免疫应答 ,为进一步开展HCV/HBV联合基因免疫奠定了实验基础。     

The effect of vaccination with DNA encoding murine T-cell epitopes on the Der p 1 and 2 induced immunoglobulin E synthesis

BACKGROUND: Immunization with naked plasmid DNA leads to strong and persistent cell-mediated and humoral immune response to plasmid encoded antigen. Vaccination of DNA encoded whole allergen has been tried, but little information is currently available on the efficacy of DNA encoding T-cell epitopes in allergic disease. The purpose of this study was to determine whether the vaccination of naked plasmid DNA encoding only T-cell epitopes suppresses the allergic reaction as effectively as naked DNA encoding whole segments of allergen. METHODS: We immunized mice with a mixed naked plasmid DNA encoding the five classes of murine T-cell epitopes on Der p 1 and Der p 2 three times at weekly intervals via an intramuscular injection of BALB/c mice. Control mice were injected with the pcDNA 3.1 blank vector. After 3 weeks, the mice were actively sensitized twice and allowed to inhale the Der p extracts intranasally six times at weekly intervals. RESULTS: The vaccinated mice showed a significant attenuated induction of Der p-specific immunoglobulin E synthesis compared to controls. In terms of the Der p-specific IgG2a antibody response, the vaccinated mice showed more prominent responses than the control mice group. In addition, analysis of the cytokine profile after Der p stimulation of the lymph-node cells revealed that the level of the mRNA expression of the interferon-gamma gene was higher in the vaccinated mice than in the controls. Histologic studies showed a much reduced infiltration of inflammatory cells in lung tissue of the gene-vaccinated mice in comparison with the controls. CONCLUSIONS: These results suggest that vaccination with DNA encoding T-cell epitopes effectively inhibits allergen-induced IgE synthesis and reduces cell infiltration in lung tissue. Thus, gene therapy using T-cell epitope-encoding DNA presents an ideal way of combating allergic disease in the future.     

布氏杆菌pCDNA3.1-L7/L12核酸疫苗的构建及其免疫学评价

目的 获得布氏杆菌保护性抗原L2／L12重组蛋白及pCDNA3．1-L7／L12重组质粒，并比较其诱导特异性免疫应答的能力。方法 PCR扩增布氏杆菌核蛋白L7／L12基因分别构建至原核表达载体PET32a( )和真核表达载体pCDNA3.1( )中；pET32a-L7／L12重组质粒转化BL21(DE3)，所表达蛋白经SDS-PAGE、免疫印迹分析、纯化后免疫小鼠；pCDNA3.1-12／L12重组质粒配以GM-CSF同时肌肉注射免疫小鼠，3次免疫后测定免疫功能进行免疫效果的评价。结果ELISA、Western blot检测到免疫鼠体内有特异性抗体产生，蛋白苗所诱导的抗体效价远远高于DNA疫苗；通过淋巴细胞增殖实验、细胞因子和CD分子测定表明DNA疫苗以诱发TH1型免疫为主。结论 所构建的布氏杆菌DNA疫苗和蛋白苗均具有诱导特异性细胞和体液免疫应答的能力，DNA疫苗诱导产生的细胞免疫反应强于蛋白苗，可作为潜在的布氏菌新型疫苗，有进一步研究的意义。     

含有丙型肝炎病毒核心基因表达质粒的构建及其基因免疫

目的：研究丙型肝炎病毒（ＨＣＶ）核心（Ｃ）基因免疫诱生特异性免疫应答的可行性。方法：将ＨＣＶ Ｃ基因片段插入真核表达载体ｐｃＤＮＡ３质粒ＣＭＶ启动子的下游,构建真核表达载体ｐｃＤＮＡＨＣＶ－Ｃ,分别转染小鼠骨髓瘤细胞ＳＰ２／０和人肝癌细胞７７２１进行瞬时表达,用免疫荧光法和Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测表达产物,将重组质粒注射,ＢＡＬＢ／ｃ（Ｈ－２＾ｄ）小鼠股四头肌,ＥＬＩＳＡ法检测血清中抗体产生水     

Comparison of antibody- and cell-mediated immune responses after intramuscular hepatitis C immunizations of BALB/c mice

Current treatments for hepatitis C infection have limited efficacy, and there is no vaccine available. The goal of this study was to compare the immune response to several immunization combinations against hepatitis C virus (HCV). Six groups of mice were immunized at weeks 0, 4, and 8 with different combinations of a candidate HCV vaccine consisting of 100 microg recombinant HCV core/E1/E2 (rHCV) DNA plasmid and/or 25 microg rHCV polyprotein and 50 microL Montanide ISA- 51. Four weeks after the last injection, all groups of mice were sacrificed and blood samples and spleens were collected for measuring the levels of specific HCV antibodies (total IgG, IgG1, and IgG2a). Cell proliferation and intracellular interferon-gamma were also measured. Among the groups of immunized mice, only the mice immunized with rHCV DNA plasmid, rHCV polyprotein, and montanide (group D) and mice immunized with rHCV polyprotein and montanide (group F) demonstrated a significant increase in the total IgG titer after immunization. IgG1 was the predominant antibody detected in both groups D and F. No IgG2a was detected in any of the groups. Proliferation assays demonstrated that splenocytes from group D and group C (rHCV DNA primed/rHCV polyprotein boost) developed significant anti-HCV proliferative responses. The combination of an rHCV DNA plasmid, rHCV polyprotein, and montanide induced a high antibody titer with a predominance of IgG1 antibodies and recognized the major neutralization epitopes in HVR1. In contrast, group C did not show an increase in anti-HCV antibodies, but did show a proliferative response.     

DNA immunization in relB-deficient mice discloses a role for dendritic cells in IgM → IgG1 switch in vivo

A single intraspleen inoculation of plasmid DNA coding for an immunoglobulin heavy chain gene initiates immunity and establishes immunologic memory against the antigenic determinants of transgenic immunoglobulins, somatic transgene immunization. During priming mice produce IgM but not IgG1 antibodies. Since IgM → IgG1 class switch occurs spontaneously during the primary immune response to protein antigens we investigated possible mechanisms for failure of spontaneous isotype switch in vivo in this model of immunity. We found that inoculation of plasmid DNA in the form of a chimeric gene coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) was able to drive IgG1 class switch readily after priming. Since GM-CSF activates cells of the dendritic lineage we tested the possibility that dendritic cells (DC) may be involved in regulating IgM → IgG1 switch. To this end we used bone marrow chimeras constructed from mice carrying the null mutation for the relB member of the NF-κB/Rel family as these mice lack bone marrow-derived mature DC. RelB (-/-) mice and (-/-) bone marrow chimeras inoculated with DNA/GM-CSF did not produce IgG1 antibodies during the primary immune response. Since relB (-/-) bone marrow chimeras lack DC of donor origin but possess resident follicular dendritic cells we conclude that Ig class switch in vivo is regulated by the function of interdigitating dendritic cells (IDC). Thus, IDC may contribute to the qualitative aspects of the emerging immune response.     

鼠疫耶尔森菌重组质粒pVAX1/Fl-V的构建及其免疫效果的研究

目的获得含有鼠疫F1和V抗原编码基因的重组真核表达质粒pVAX1／F1-V，并测定其诱导特异性免疫应答的能力。方法PCR扩增鼠疫菌Fl和V编码基因，分别与pGEM-T连接测序，构建pVAX1／F1-V融合重组质粒，转染Cos-7细胞，用Western blot方法鉴定目的蛋白的表达，重组质粒pVAXI／F1．V加GM．CSF佐剂免疫BALB／c小鼠，观察免疫效果，400个半数致死量（uk）强毒鼠疫菌皮下攻毒观察保护率。结果pVAX1／F1-V在Cos-7细胞中表达，免疫鼠体内产生特异性抗体，通过抗体亚型分析、细胞因子等指标的测定表明所构建DNA疫苗以诱发TH1型免疫为主，攻毒保护率达60％。结论成功构建F1-V融合蛋白真核表达载体，具有诱导特异性细胞免疫和体液免疫应答的能力，对强毒鼠疫菌皮下攻毒有一定的保护效力，为鼠疫菌新型疫苗研制奠定了基础。     

Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM-CSF gene adjuvant

Granulocyte-macrophage clony-stimulating factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells to the site of antigen synthesis as well as stimulate the maturation of dendritic cells.This study evaluated the utility of GM-CSF as a plasmid DNA replicon vaccine adjuvants for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In balb/c mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) carrying the Hc gene of BoNT/A (AHc), both antibody and lymphoproliferative response specific to AHc were induced, the immunogenicity was enhanced by co-delivery or coexpress of the GM-CSF gene. In particular, when AHc and GM-CSF were coexpressed within the SFV based DNA vaccine, the anti-AHc antibody titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased, and further enhanced by coimmunization with aluminum phosphate adjuvant.     

DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus

The immune response to a naked plasmid DNA encoding the nucleoprotein (N protein) of porcine transmissible gastroenteritis virus (TGEV) was investigated in this study. A complementary DNA of the entire N gene was amplified by RT-PCR, and inserted into a mammalian expression vector (pcDNA3.1) to construct a recombinant plasmid (pcDNA/N). To evaluate the immunogenicity of the construct, BALB/c mice were intramuscularly immunized with different doses (50, 100 and 200 μg/mouse) of pcDNA/N twice at a 5-week interval. An optimal antibody response was achieved with 100 μg of pcDNA/N. The response lasted at least 11 weeks after primary immunization. By western blotting analysis, the antibodies specifically recognized a 47 kDa protein corresponding to the viral N protein, but they did not reveal neutralizing activity against infectious TGEV in vitro. Immunoglobulin G2a was predominant among these antibodies, which was indicative of Th1 type cell activation in pcDNA/N immunized mice. Moreover, spleen cells from these mice showed stronger immune responses than those from live vaccine or parental vector immunized mice. These results suggest that the construct can elicit both humoral and cell-mediated immune (CMI) responses against TGEV N protein in mice.     

HCV核心-包膜E2抗原融合基因DNA疫苗的构建及对小鼠的免疫应答试验

目的：观察一种结构新颖的HCV融合抗原DNA疫苗在BALB/c小鼠的免疫效果，探讨其用于防治丙型肝炎的可行性。方法：用重叠延伸PCR拼接编码小鼠IgG kappa链信号肽和通用型辅助性T细胞表位PADRE的DNA片段，PCR分别扩增HCV核心抗原基因和包膜E2抗原基因，将3段基因插入真核表达载体pcDNA3.1，构成重组表达质粒pST-CE2t，转染COS7细胞，免疫组化检测HCV抗原的表达。将pST-CE2t和HCV核心抗DNA疫苗pcDNA3.1core分别肌肉注射接种BALB/c小鼠，检测小鼠的血清抗体、T细胞增殖和CTL反应。结果：pST-CE2t可在COS7细胞内表达HCV核心抗原和E2抗原，接种于BALB/c小鼠能有效诱导体液和细胞免疫应答，其中抗HCV核心抗原免疫应答的强度明显超过pcDNA3.1core，且更趋向于TH1型免疫应答。结论：pST-CE2t对于丙型肝炎的防治有潜在的应用价值。     

Evaluation of Brucella abortus DNA vaccine by expression of Cu-Zn superoxide dismutase antigen fused to IL-2

The Cu-Zn superoxide dismutase (SOD) antigen of Brucella abortus was previously identified to be a T cell antigen which induces both proliferation of and gamma interferon (IFN-gamma) secretion by T cells from infected mice. In an earlier study, we demonstrated that intramuscular injection of mice with a plasmid DNA carrying the gene for SOD leads to the development of significant protection against B. abortus challenge. It has been reported that the antigen-specific immune responses generated by a DNA vaccine can be enhanced by co-delivery of certain cytokine genes. In this study, we evaluated the effect of delivering IL-2 on the efficacy of SOD DNA vaccine by generating a plasmid (pSecTag-SOD-IL2) that codes for a secretory fusion protein of SOD and IL-2. Another plasmid (pSecTag-SOD) that codes for only SOD as a secretory protein was used for comparison. BALB/c mice injected intramuscularly with pSecTag-SOD or pSecTag-SOD-IL2, but not the control plasmid pSecTag, developed SOD-specific antibody and T cell immune responses. Upon in vitro stimulation with recombinant SOD (rSOD) antigen, T cells from mice immunized with pSecTag-SOD-IL2, in comparison with those from mice immunized with pSecTag-SOD, exhibited a lower proliferation response but produced significantly higher concentrations of IFN-gamma. Both DNA vaccines, however, induced similar levels of SOD-specific antibodies and cytotoxic T cell response. Although mice immunized with pSecTag-SOD-IL2 showed increased resistance to challenge with B. abortus virulent strain 2308, this increase was not statistically significant from that of pSecTag-SOD vaccinated mice. These results suggest that a SOD DNA vaccine fused to IL2 did not improve protection efficacy.     

两种HCV不同结构基因重组质粒DNA诱导免疫应答的比较研究

目的：探讨丙型肝炎病毒（HCV）包膜基因E1E2,对核心基因CDNA疫苗诱生的免疫应答有无增强作用。方法：构建包含HCVC或CE1E2基因片段的真有达载体pHCV-C和pHCV-CE1E2,分别接种于Balb/c小鼠股四头肌（以空载体pcDNA3作为对照）,每间隔2wk l次,用ELISA法检测免疫小鼠血清中抗HCVC特异性抗体的产生。以pHCV-C转染并表达HCcAg r S p2/0细胞为靶细胞,采用^51Cr翻译放试验检测特异性CTL的杀伤作用。结果,两个实验免疫的20只小鼠均产生抗HCV C特异性抗体,当前／靶细胞比例为100：1时,CTL的杀伤率均明显高于对照组（P＜0．01）;pHCV-CE1E2与pHCV-C组之间,无论是抗HCV C抗体的滴度还是CTL的杀伤率均无显著性差异（P＞0．05）。结论E1E2基因的加入,并没有增加HCV C基因DNA疫苗诱导的抗HCcAg特异性抗体的滴度和CTL的杀伤作用。     

Efficient induction of mouse immune responses to hepatitis C virus by viral core protein-carrying attenuated Salmonella typhimurium

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for inclusion in a protective vaccine. However, this protein may attenuate the development of systemic immune responses because of its immunomodulatory properties. In this study, a eukaryotic expression plasmid, pCI-C, and an in vivo-inducible prokaryotic expression plasmid, pZW-C, for HCV core protein were constructed and transformed into attenuated Salmonella typhimurium aroA strain SL7207. The recombinant expression plasmids SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and immune responses specific to core protein were assessed. Immunization with bacteria SL7207/pCI-C led to a persistent decrease in the percentage of CD3(+)CD4(+) T cells and triggered weak anti-core IgG production. Splenocytes from SL7207/pCIC-immunized mice developed relatively weak proliferation response, low interferon-gamma secretion, and inferior cytotoxic activity compared with those from mice immunized with SL7207/pZW-C. Boost immunization with SL7207/pCI-C yielded limited improvement in the immune response, whereas boost with bacteria SL7207/pZW-C enhanced immune responses significantly. These results suggest that de novo host synthesis of native HCV core protein may cut down the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.     

tPA信号肽和鼠疫杆菌保护性抗原V融合基因的构建及免疫效果鉴定

获得含有鼠疫杆菌V抗原编码基因以及tPA信号肽编码序列的重组质粒,并测定其诱导特异性免疫应答的能力。采用PCR扩增鼠疫菌杆菌V基因构建到pVAX1质粒中产生pVAX1/V重组质粒,PCR扩增tPA信号肽编码序列片段并将其插入到pVAX1/V中V基因的上游,构建tPA-pVAX1/V重组质粒;转染COS-7细胞,免疫细胞化学方法鉴定V蛋白的表达;二重组质粒分别加mGM-CSF质粒免疫BALB/c小鼠,观察免疫应答反应;以400个LD50强毒鼠疫杆菌皮下攻击免疫小鼠观察保护效率。结果显示,tPA-pVAX1/V在COS-7细胞中表达了V蛋白;免疫小鼠血清产生了特异性抗体和细胞免疫应答;攻毒保护率达80%。成功构建了分泌型V蛋白的真核表达质粒载体,具有诱导特异性细胞免疫和体液免疫应答的能力,对强毒鼠疫杆菌攻毒有一定的保护效力,为鼠疫杆菌新型疫苗研制奠定了基础。     

鼠疫菌重组质粒pcDNATE/F1-V的构建及其免疫效果的研究

目的 获得含有鼠疫F1和V抗原编码基因的重组质粒pcDNATE／E1-V,并测定其诱导特异性免疫应答的能力。方法PCR扩增鼠疫菌F1和V编码基因,分别与pGEM-T连接测序,构建pcDNATE／F1-V融合重组质粒,转染COS-7细胞,用Western blot方法鉴定目的蛋白的表达,重组质粒pcDNATE／E1-V加集落刺激因子(GM-CSF)免疫Balb／c小鼠,观察免疫效果。结果pcDNATE／F11-V在COS-7细胞中表达,免疫鼠体内产生特异性抗体,抗体亚型分析、细胞因子等指标的测定结果表明所构建DNA疫苗以诱发Th1型免疫为主。结论成功构建重组真核表达质粒pcDNATE／F1-V,其具有诱导特异性细胞免疫和体液免疫应答的能力,为鼠疫菌新型疫苗研制奠定了基础。