Immunogenicity and protective efficacy of DNA vaccine against visceral leishmaniasis in BALB/c mice

The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis.Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1(+) encoding T cell epitopes of gp63 and Hsp70 individually and in combination.Animals were challenged intracardially with 10~7 promastigotes of Leishmania donovani 10 days post immunization and sacrificed 1,2 and 3 months post challenge.The immunized animals revealed a significant reduction(P < 0.05)in splenic and hepatic parasite burden as compared to the infected controls.Maximum reduction in parasite load(P < 0.05) was observed in animals treated with a combination of pcDNA/gp63 and pcDNA/Hsp70.These animals also showed heightened DTH response,increased IgG2a,elevated Th1 cytokines(IFN-γ and IL-2) and reduced IgG1 and IL-10 levels.Thus,mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens.     

柯萨奇病毒B3 VP1蛋白、rAd/VP1和pcDNA3/VP1的免疫效果比较

目的 观察柯萨奇病毒B3(Coxsackievirus B3,CVB3)衣壳蛋白VP1、表达VP1蛋白的重组腺病毒rAd/VP1和重组质粒pcDNA3/VP1的免疫效果.方法 用原核细胞表达VP1蛋白并纯化、扩增重组腺病毒rAd/VP1,扩增并提取真核表达质粒pcDNA3/VP1.BALB/c小鼠随机分为4组,每组18只,分别在股四头肌注射VP1蛋白、rAd/VP1、pcDNA3/VP1和PBS.VP1蛋白组和pcDNA3/VP1组免疫3次,间隔3周;rAd/VP1组免疫2次,间隔2周.VP1蛋白、pcDNA3/VP1和rAd/VP1每次每只注射剂量分别为50μg、100μg和1.2×107PFU.用ELISA法和微量中和试验法检测各次免疫后血清CVB3特异性IgG抗体和中和抗体滴度;末次免疫后3周,CCK-8法检测脾脏淋巴细胞的CTL杀伤活性;用致死量CVB3攻击小鼠后,检测血中病毒滴度并观察动物的存活情况.结果 VP1蛋白组血清特异性IgG抗体和中和抗体滴度明显高于其他实验组(P＜0.05),而脾脏淋巴细胞CTL杀伤活性低于rAd/VP1组(P＜0.05);致死量病毒攻击后,VP1蛋白组血中病毒滴度低于pcDNA3/VP1和rAd/VP1组(P＜0.05),生存率明显高于这两组(P＜0.05).结论 VP1蛋白疫苗能诱导较高水平的体液免疫应答,对动物有明显的免疫保护作用,免疫效果优于质粒pcDNA3/VP1和重组腺病毒rAd/VP1.

Abstract：

Objective To compare the immune effects of Coxsackievirus B3 (CVB3) capsid protein VP1 expressed bacterially, recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1which express VP1 protein in mice. Methods After expressed in prokaryotic cells, VP1 protein was purified. Recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1 were amplified and extracted. Six to 8-week-old, male BALB/c mice were divided into four groups randomly. Each group contained 18 mice. The mice of pcDNA3/VP1 group or VP1 protein group were immunized intramuscularly with three injections at three weeks apart, of recombinant plasmid pcDNA3/VP1 at a dose of 100 μg/mouse or recombinant protein VP1 at a dose of 50 μg/mouse. The mice of rAd/VP1 group were immunized intramuscularly twice at two weeks interval with rAd/VP1 at a dose of 1.2 × 107 PFU. The control group was mock-immunized with 100 μl of PBS intramuscularly. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. ELISA and micro-neutralization test were used to detect levels of CVB3-specific IgG antibody and neutralizing antibody titers in the sera of immunized mice. Three weeks after the last immunization, the cytotoxic T lymphocyte(CTL) killing activity of spleen lymphocytes was detected with CCK-8 assay. Subsequently, virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose( CCID50 ) assay on HeLa cell monolayers and percentage of animals surviving were observed after lethal CVB3 attack over a period of 21 days. Results The titers of specific IgG antibody and neutralizing antibody in sera of VP1 protein immunized mice were higher than other groups( P ＜0.05 ). While CTL killing activity of spleen lymphocytes of VP1 protein immunized mice was lower than mice in rAd/VP1 group( P ＜0. 05). Virus titers in sera of VP1 protein immunized mice were lower than the mice in pcDNA3/VP1 or rAd/VP1 groups ( P ＜ 0.05 ), while survival rate was significantly higher than these two groups ( P ＜ 0.05 ).Conclusion VP1 protein induced higher level of humoral immune response and acquired obvious immune protection effects in mice. The immunizing potency of VP1 protein vaccine surpassed plasmid pcDNA3/VP1or recombinant adenovirus rAd/VP1. It appeared to be a promising candidate among the three different vaccines.     

人巨细胞病毒gB和pp150融合基因真核表达载体的构建及其免疫活性研究

Objective To provide experimental evidence for development of human cytomegalovirus (HCMV) nucleic acid vaccine,HCMV surface protein(gB),membrane protein(pplSO),and gB-pp150 fused gene eukaryotie expression vector were constructed.Methods gB and pp150 genes were amplified and fused into gB-pp150,then were cloned into pcDNA 3.1(+) to obtain recombinant expression plasmids pcDNA 3.1(+)-gB,pcDNA 3.1(+)-pp150 and pcDNA 3.1(+)-gB-pp150,which were encapsulated with chitosan.Mouse were vaccinated and the humoral and eell immune response were determined by ELISA,specific proliferative response of plenie lymphocytes.Results The gB,pp150 and gB-pp150 fusion gene eukaryotie expression vector were successfully constructed.The antibodies A value induced by peDNA3.1(+)-gB or peDNA3.1(+)-gB-pp150 were much higher than that of peDNA3.1(+)(P<0.01).The IFN-γ levels induced by pcDNA3.1(+)-pp150 and peDNA3.1(+)-gB-pp150 were significantly higher than that of pcDNA3.1(+).There are significant diferenee between the stimulating indexes of pcDNA3.1(+)-pp150 or peDNA3.1(+)-gB-pp150 immunized and normal mice.Conclusion The DNA vaccine pcDNA3.1(+)-gB can induce significant humeral immunity response.and pcDNA3.1 (+)-pp150 can induce high cellular immune response,whereas pcDNA3.1(+)-gB-pp150 can induce both humeral and cellar immune responses in BALB/c mice.     

人巨细胞病毒gB和pp150融合基因真核表达载体的构建及其免疫活性研究

Objective To provide experimental evidence for development of human cytomegalovirus (HCMV) nucleic acid vaccine,HCMV surface protein(gB),membrane protein(pplSO),and gB-pp150 fused gene eukaryotie expression vector were constructed.Methods gB and pp150 genes were amplified and fused into gB-pp150,then were cloned into pcDNA 3.1(+) to obtain recombinant expression plasmids pcDNA 3.1(+)-gB,pcDNA 3.1(+)-pp150 and pcDNA 3.1(+)-gB-pp150,which were encapsulated with chitosan.Mouse were vaccinated and the humoral and eell immune response were determined by ELISA,specific proliferative response of plenie lymphocytes.Results The gB,pp150 and gB-pp150 fusion gene eukaryotie expression vector were successfully constructed.The antibodies A value induced by peDNA3.1(+)-gB or peDNA3.1(+)-gB-pp150 were much higher than that of peDNA3.1(+)(P<0.01).The IFN-γ levels induced by pcDNA3.1(+)-pp150 and peDNA3.1(+)-gB-pp150 were significantly higher than that of pcDNA3.1(+).There are significant diferenee between the stimulating indexes of pcDNA3.1(+)-pp150 or peDNA3.1(+)-gB-pp150 immunized and normal mice.Conclusion The DNA vaccine pcDNA3.1(+)-gB can induce significant humeral immunity response.and pcDNA3.1 (+)-pp150 can induce high cellular immune response,whereas pcDNA3.1(+)-gB-pp150 can induce both humeral and cellar immune responses in BALB/c mice.     

Comparative immunization in BALB/c mice with recombinant replication-defective adenovirus vector and DNA plasmid expressing a SARS-CoV nucleocapsid protein gene

In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus （SARS-CoV）-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ （IFN-γ） secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd-N was significantly stronger than that of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific （IFN-γ） secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirns boosting could be used as a potential SARS-CoV vaccine.     

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.     

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.     

A rapid and simple approach to preparation of monoclonal antibody based on DNA immunization

Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb).But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt to replace theprotein inoculation with DNA immunization in the preparation of mAb.The eukaryotic expression vectorspcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNAimmunization.Female BALB/c mice developed a well antibody response to the target antigen after muscleinjection with corresponding plasmids.The mice with effective antibodies induced were used for preparation ofmAb.We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted byintrasplenic injection with the same plasmid could be exploited for preparation of mAb.And positivehybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed.Our studies demonstrate that geneimmunization may provide a convenient and efficient way to prepare mAbs.Cellular & Molecular Immunology.2004;1(4):295-299.     

Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod.gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod.gag, rAd5-mod.gag or DNA and were boosted after 3 weeks. To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIV-specific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.     

Studies on the Specific Degranulation of Mast Cell Sensitized by Several Allergens in vitro

Food allergy is a major health issue worldwide. Mast cells play a very important role in the immediate hypersensitivity for which mast cell degranulation needs to be studied extensively. In this study, an approach was taken to study the characteristics of sensitized mast cell degranulation in vitro, which associated with the study of mast cells and animal models. BALB/c mice were immunized respectively by several food allergens, then blood and peritoneal mast cells were collected at different time points. A dynamic determination was carried out between mast cells and serumal IgE. Comparative analysis on sequential time points showed that there was a close coincidence between mast cell degranulation and IgE antibody titers in sensitized BALB/c mice. Furthermore, it is interesting that sensitized mast cells could implement specific degranulation against the challenges in vitro, but the closely tropomyosins induced mast cell degranulation displayed cross reactions. This is very similar to IgE resisting the allergens in vivo. The study disclosed some characteristics on mast cells, coming from sensitized BALB/c mice, degranulation in vitro.     

登革病毒2型NS1蛋白DNA疫苗的构建及其免疫效果观察

目的　以登革病毒 2型 (denguevirus2 ,DV2 )非结构蛋白 (non structrulprotein 1,NS1)为靶基因 ,构建DV2 NS1的候选DNA疫苗 ;并探讨其在小鼠体内诱导特异性体液免疫和细胞免疫的作用。方法　将登革病毒 2型NS1 NS2a基因片段克隆至含AG强启动子的真核表达载体pCXN2上 ,构建成重组体pCXN2 NS1 NS2a。在体外将重组质粒转染Cos 7细胞 ,间接免疫荧光检测其在真核细胞中的表达。大量提取空质粒和重组质粒 ,进行动物免疫实验。结果　重组质粒可在真核细胞中有效地表达NS1蛋白。免疫接种小鼠后可诱发机体产生针对NS1蛋白的特异性体液免疫和细胞免疫。末次免疫前已有抗体产生 ,4周后达高峰。抗体依赖补体介导的溶细胞作用 (antibody dependentcomple ment mediatedcytolysis,ADCC)试验结果显示产生的抗体在体外具有特异的杀细胞作用。淋巴细胞增殖实验结果显示 ,实验组小鼠的淋巴细胞增殖能力与对照组比较差异有显著性。流式细胞计数仪(FACS)检测DNA免疫鼠CD4 + 、CD8+ T淋巴细胞变化情况 ,与注射空载体pCXN2的阴性鼠相比 ,CD4 + 、CD8+ 细胞水平有较大升高 (P <0 .0 1)。动物保护性实验结果显示 ,当用致死剂量登革病毒攻击免疫鼠时 ,有 6 6 .6 %的免疫鼠受到保护。结论　NS1 NS2a基因重组质粒免疫小鼠可以诱     

空肠弯曲菌DNA疫苗构建及其疫苗免疫程序研究

目的:构建空肠弯曲菌peb1A基因真核表达载体,探讨DNA疫苗不同免疫程序的免疫效果。方法:以重组pET28a(+)-peb1A质粒为模板,将空肠弯曲菌peb1A基因克隆入真核表达载体pcDNA3.1(-),经酶切和测序鉴定后,将重组质粒转染COS-7细胞中表达,Western blot鉴定表达蛋白;昆明小鼠随机分组:设空白对照组、空载体对照组、pcDNA3.1(-)-peb1A实验组,用ELISA法检测免疫后的昆明小鼠血清IgG和IgM抗体水平。结果:重组质粒pcDNA3.1(-)-peb1A经双酶切和PCR鉴定,构建正确。peb1A基因测序结果与GenBank中peb1A基因序列一致。重组质粒成功转染COS-7细胞,并能正确表达重组蛋白。裸DNA组IgG水平均高于两对照组,有显著性差异(P<0.05)。同一剂量短时间(0、10、20天)免疫程序优于长时间(0、15、25、35天)免疫程序(P<0.05)。结论:成功构建空肠弯曲菌真核表达重组质粒pcDNA3.1(-)-peb1A,其经肌肉注射免疫小鼠,具有较好的免疫原性,短时间免疫程序免疫效果好。     

DNA priming E and NS1 constructs--homologous proteins boosting immunization strategy to improve immune response against dengue in mice

DNA priming-protein boosting is a strategy used to establish strong immunity to a specific pathogen by the use of two different antigens through sequential delivery systems. In this work, two recombinant plasmids were used, one encoding for the dengue virus E protein, which is know to induce neutralizing antibodies (pcDNA 3.1/E), and the other encoding for the Dengue virus nonstructural protein 1 (pcDNA 3.1/NS1), as a source of B- and T-cell epitopes possibly involved in protective immunity. We showed that immunization of BALB/c mice with three priming doses of both plasmids pcDNA 3.1/E and/or pcDNA 3.1/NS1 were able to induce antibody responses to E protein with a single plasmid; in contrast to the antibody response to NS1 protein we observed an additive effect in terms of antibody response. Moreover, using a prime-boost protocol in which both plasmid constructs were co-administrated followed by a boost of homologous GST-E and GST-NS1 recombinant proteins, we observed an increased antibody response to NS1 and to E protein compared to animals vaccinated with the proteins or with dengue constructs alone. If neutralizing antibodies play an important role in dengue infection, antibodies generated with this regimen was also significantly better than the administration of the mix of proteins alone. These results suggest that NS1 and E proteins together could be considered in a design of subunit recombinant vaccines.     

蛋白表达的亚细胞定位影响基因免疫诱导的体液免疫应答水平的研究

目的研究表达产物的不同亚细胞定位对基因免疫诱导的体液免疫应答水平的影响,为基因疫苗的设计提供参考。方法利用分子克隆技术,分别构建能表达不同细胞定位的EGFP-HPVl6L1融合蛋白和截短型EGFP-HPV16L1△NLS融合蛋白的重组pcDNA-EGFP-HPV16L1和pcDNA-EGFPHPV16L1△NLS真核表达载体;通过转染CHO细胞,并在倒置荧光显微镜下观察表达产物的细胞定位;用重组pcDNA载体免疫BALB/c小鼠;EGFP作为抗原,ELISA法检测血清抗体吸光度。结果①重组pcDNA-EGFP-HPV16L1转染的CHO细胞核内可见到绿色荧光,pcDNA-EGFP-HPV16L1△NLS真核表达载体转染的CHO细胞质内可见到绿色荧光:②两种不同的重组pcDNA载体均可诱导BALB/c小鼠体液免疫反应,但重组pcDNA-EGFP-HPV16L1△NLS真核表达载体免疫组小鼠IgG抗体A_(450)值显著高于重组pcDNAEGFP-HPV16L1真核表达载体免疫组小鼠IgG抗体A_(450)值(P0.001)。结论表达产物的不同细胞定位可影响基因免疫诱发的体液免疫应答水平,定位于细胞质内的蛋白分子较定位于细胞核的蛋白分子能诱导机体更强的体液免疫反应,这为以增强体液免疫反应为目的的基因疫苗的设计提供了参考。     

IL-23、IL-27对RSV重组蛋白疫苗免疫原性的影响

目的:以编码IL-23和IL-27的真核表达质粒pcDNA3.1-IL-23(以下简称IL-23)和pcDNA3.1-IL-27(以下简称IL-27)为佐剂,与呼吸道合胞病毒(RSV)重组蛋白疫苗G1F/M2共免疫小鼠,观察IL-23和IL-27对疫苗的免疫原性的影响。方法:以IL-23、IL-27质粒和Al(OH)3为免疫佐剂,与G1F/M2共免疫BALB/c小鼠,末次免疫后10天杀死小鼠,用ELISA检测特异性IgG、IgG1和IgG2a水平;流式细胞术检测小鼠脾细胞CD4+、CD8+细胞的变化;用乳酸脱氢酶(LDH)释放法检测特异性小鼠脾细胞杀伤活性。结果:G1F/M2+Al(OH)3+IL-23和G1F/M2+Al(OH)3+IL-27可诱导高效价的IgG、IgG1、IgG2a抗体,且显著高于这三种佐剂单独使用诱导的抗体效价;流式细胞检测结果显示G1F/M2+IL-23和G1F/M2+Al(OH)3+IL-23刺激的CD4+T细胞和CD8+T细胞水平显著高于其他组;单独的IL-23或IL-27对G1F/M2诱导的脾细胞杀伤活性没有增强作用,而Al(OH)3+IL-23和Al(OH)3+IL-27佐剂组的杀伤率显著高于单独的IL-23、IL-27或Al(OH)3组。结论:这些结果表明:IL-23或IL-27质粒与传统铝盐佐剂Al(OH)3联合使用能显著增强RSV重组疫苗G1F/M2的免疫原性。     

不同免疫方案制备抗HAb18G/CD147胞外区多克隆抗血清的比较

目的: 制备针对肝癌相关抗原HAb18G/CD147胞外区不同表位的抗血清, 比较不同免疫方案的免疫效果。方法: 以原核表达的GST -HAb18GEF融合蛋白、重组真核表达质粒pcDNA3 /HAb18G及HCC细胞为免疫原, 分别采用蛋白常规免疫、DNA肌肉免疫及pcDNA3 /HAb18G -HCCbooster(DNA -cellbooster)免疫接种BALB/c小鼠。采用间接ELISA和细胞ELISA, 同时测定免疫血清中抗变性和天然HAb18GEFIgG抗体的滴度和Ig亚类。用Westernblot检测各免疫方案制备的抗血清, 与变性HAb18GEF抗原结合的特异性。用免疫荧光法验证DNA- cellbooster免疫接种法制备的抗血清, 与细胞膜上天然HAb18G抗原的结合特异性。结果:以GST- HAb18GEF常规免疫后, 可诱导高滴度的IgG1抗体产生, 但针对的多为HAb18GEF的变性或线性表位; 以pcD -NA3 /HAb18G肌肉免疫后, 可诱导针对其天然表位的IgG2a抗体产生, 但滴度较低; 以DNA -cellbooster免疫后, 可诱导中等滴度、针对其天然表位的IgG2a和IgG1抗体产生。结论: 不同免疫方案可诱导针对HAb18GEF不同表位、不同滴度的多克隆抗血清, 为淘筛针对其不同表位的多样性抗体奠定了基础。     

T-bet佐剂对Ag85B抗结核DNA疫苗的免疫调节

目的:构建以Th1转录因子T-bet为基因佐剂的Ag85B新型DNA疫苗,并研究其免疫调控作用。方法:RT-PCR法扩增出Ag85B基因和T-bet基因,克隆入pcDNA3.1质粒构建T-bet和Ag85B真核表达质粒,脂质体法转染重组质粒至RAW264.7细胞系,Western blot法检测质粒蛋白表达情况。3次肌肉注射免疫BALB/c小鼠,末次免疫2周后,ELISA法检测血清中抗Ag85B抗体滴度。同时将脾脏淋巴细胞悬液于Ag85B刺激下培养,ELISA法检测培养液中细胞因子分泌情况。结果:质粒蛋白成功表达,并且质粒剂量与质粒蛋白表达水平呈正相关。此外,T-bet/Ag85B不仅诱导IgG2a滴度显著增高伴随IgG1显著降低,而且还刺激IL-2/IFN-γ(Th1类)分泌增加伴随IL-4/IL-10(Th2类)减少。结论:T-bet增强抗Ag85B特异性IgG2a抗体反应,并诱导显著的Th1细胞优势免疫。     

Inverted terminal repeat sequences of adeno-associated virus enhance the antibody and CD8(+) responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera

The immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) have been examined for the effect of the addition of the inverted terminal repeat (ITR) sequences of the adeno-associated virus (AAV) to the DNA plasmid construct, and of packaging the LAMP/gag gene as a recombinant AAV vector (rAAV). DNA plasmids encoding Gag and the LAMP/Gag protein chimera were constructed in two vectors, the pcDNA3.1 and a corresponding plasmid containing the ITR sequences (pITR) flanking the expression elements of the plasmid, and the pITR LAMP/gag DNA plasmid was encapsidated in the rAAV vector. Human 293 cells transfected in vitro with LAMP/gag plasmids either in pcDNA3.1 or pITR produced much Gag protein in cell extracts (1.6 and 2.2 ng of Gag/mg of protein, respectively). The immune responses of mice to immunization with these constructs were examined under three protocols: DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. The results demonstrated that under DNA prime/DNA boost protocol, the "naked" DNA vaccines encoding the LAMP/gag chimera, either as pcDNA3.1 or pITR DNA plasmid constructs, elicited strong CD4(+) T cell responses. In contrast, significantly higher levels of CD8(+) and antibody responses were observed with the pITR-DNA constructs. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG(1) isotype resulting from the activation of the Th2 subset of CD4(+) T cells, that was sustained for at least 5 months after immunization.