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1.
目的 探讨含HIV-1 gp120基因的重组腺相关病毒(rAAV)和重组腺病毒(rAdV)疫苗在BALB/c小鼠中联合免疫的效果。方法 将密码子优化的HIV-1 gp120基因分别插入腺相关病毒(AAV)和腺病毒(AdV)载体质粒,构建含该基因的rAVV和rAdV载体疫苗。将两种疫苗以不同的联合方式免疫BALB/c小鼠,ELISA检测小鼠血清中的gp120特异性抗体,细胞内细胞因子染色法检测小鼠的特异性细胞毒性T淋巴细胞(CTL)应答。结果 两种重组病毒均可表达目的基因gp120;在小鼠体内两种重组病毒联合免疫可诱导特异性的CTL应答和血清1gG抗体反应,但用rAAV初免2次,再用rAdV加强3次所诱发的CTL和血清1gG反应最强。结论 rAAV和rAdV疫苗联合免疫可在小鼠体内诱导特异性的CTL应答和血清1gG抗体反应。  相似文献   

2.
目的:确定新型chitosan-DNA疫苗的有效免疫途径。方法:将chitosan-pcDN3-VPI疫分别苗以肌注、口服、滴鼻3种免疫方式免疫Balb/c小鼠;以ELISA检测免疫小鼠血清中IgG、IgM、、IgA,评估其特异性体液免疫应答;以特异性淋巴细胞增殖反应和CTL活性反映其诱导细胞免疫;以5LD50致死剂量CVB3攻击免疫小鼠,评价不同免疫途径的免疫保护效果。结果:①在诱导CVB3特异性体液免疫方面:chitosan-pcDNA3-VPI疫苗肌注组诱生了高水平IgM和IgG,但未能诱生黏膜IgA;口服免疫组仅诱生低水平的黏膜IgA,未能诱生特异性IgM和IgG;滴鼻组可诱生低水平的I埘及高水平的IgG和黏膜IgA。②在诱导CVB3特异性细胞免疫方面:仅滴鼻组诱导了较高水平的淋巴细胞特异性增殖反应和CTL活性;口服组的淋巴细胞增殖活性和CTL活性稍弱;肌注组几乎不能诱导特异性细胞免疫应答。③免疫保护作用:滴鼻组可保护33.3%小鼠长期存活;口服组仅达到16.7%的保护率;肌注组无保护作用。结论:滴鼻免疫途径可能是chitosan-pcDNA3-VPI基因疫苗最合适的诱导全面免疫应答的免疫途径。  相似文献   

3.
汉滩病毒核酸疫苗滴鼻及肌注免疫小鼠效果的比较研究   总被引:1,自引:0,他引:1  
观察汉滩病毒DNA疫苗滴鼻及肌注免疫诱导机体产生的免疫应答,探索汉滩病毒DNA疫苗新的免疫途径。以pcDNA3.1B-S1.3进行肌肉注射及滴鼻免疫小鼠,采用ELISA、淋巴细胞转化试验及特异性CTL杀伤活性测定等方法检测其诱导的系统和黏膜免疫反应的差异。实验结果表明,滴鼻组粪IgA滴度明显高于肌注组(P<0.05);而肌注组血IgG滴度均值虽高于滴鼻组,但两者统计上无显著意义(P>0.05);滴鼻组及肌注组小鼠脾细胞分别经ConA刺激后,其刺激指数作统计分析,结果无显著性差别(P>0.05);肌注组与滴鼻组效应细胞对靶细胞的杀伤力用方差分析进行比较,无显著性差异(P>0.05)。这些均表明,滴鼻免疫对特异的黏膜免疫激发作用明显优于肌肉注射,疫苗的滴鼻免疫途径较肌注途径有着明显的优势。  相似文献   

4.
目的 :检测表达HIV 1gag gp12 0嵌合基因的DNA疫苗在小鼠体内的免疫应答效果。方法 :将真核表达质粒pVAXGE肌肉注射BALB C小鼠 ,观察免疫小鼠脾T淋巴细胞亚群的数量、特异性CTL杀伤率及小鼠免疫后不同时间点血清中IgG抗体滴度。结果 :重组质粒pVAXGE免疫组小鼠脾淋巴细胞进行了增殖 ,脾特异性CTL杀伤率显著高于对照组 (P <0 0 1) ;小鼠免疫后于第 8周血清抗体达到最高。结论 :表达HIV 1gag gp12 0嵌合基因的DNA疫苗质粒可诱导BALB C小鼠发生免疫应答  相似文献   

5.
鼠疫F1-V重组蛋白疫苗滴鼻免疫应答效果的研究   总被引:2,自引:0,他引:2  
目的 以重组霍乱毒素B亚单位(rCT-B)为鼠疫F1-V重组蛋白的佐剂制备黏膜疫苗,观察小鼠诱导的黏膜免疫和系统免疫应答效果。方法以制备的鼠疫黏膜疫苗滴鼻免疫小鼠4次免疫后,采用间接ELISA检测血清特异性抗F1-V的IgG和IgA抗体及抗体亚型分类,检测鼻咽喉、肺、小肠及阴道灌洗液中特异性抗F1-V的黏膜分泌型IgA;采用流式细胞术检测鼻相关淋巴组织淋巴细胞、脾淋巴细胞、肠系膜淋巴结及小肠PP结T淋巴细胞表型的变化。结果以rCT-B为佐剂的鼠疫F1-V重组蛋白黏膜疫苗滴鼻免疫后,能够诱导血清中IgG、IgA抗体比正常对照组显著升高(P〈0.01),同时诱导鼻咽、肺、小肠和阴道内特异性黏膜抗体升高,尤其是肺和生殖道冲冼液内抗体升高极为显著(P〈0.01)。与单纯的F1-V组相比,不同剂量比例疫苗组都能诱导较高、较快的血清IgG、IgA和黏膜sIgA,其中1:2疫苗组能诱导更强的系统免疫和黏膜免疫,但是相比之下,5:1疫苗组是最合适的免疫剂量。结论rCT-B佐剂不仅能提高鼠疫F1-V黏膜疫苗的系统全身免疫应答,还能促进诱导呼吸道、消化道和生殖道等局部黏膜sIgA抗体,增强局部免疫应答,提示rCT-B佐剂能显著提高鼠疫感染的免疫应答作用,这为下一步疫苗的免疫保护评价奠定了基础。  相似文献   

6.
目的 探讨HIV-2核心蛋白基因gag重组DNA疫苗与重组鸡痘病毒进行联合免疫引起小鼠的免疫应答,为研究HIV-2基因重组疫苗的免疫策略提供实验基础。方法 大量制备并纯化HIV-2 gag重组DNA疫苗和重组鸡痘病毒,以肌肉注射的方式免疫BALB/c小鼠,ELISA法检测小鼠血清HIV-2抗体,流式细胞仪测定CD4^+、CD8^+T淋巴细胞亚类数量,乳酸脱氢酶(LDH)释放法检测脾CTL对HIV-2靶细胞的杀伤活性。结果 重组DNA疫苗和重组鸡痘病毒单独免疫及二者联合免疫均刺激小鼠产生HIV-2特异性抗体,脾T细胞亚类数量增加,并产生针对HIV-2靶细胞的特异性CTL杀伤活性,但联合免疫组在各项指标上均高于单独免疫组。结论 以HIV-2gag重组DNA疫苗进行基础免疫、以HIV-2gag重组鸡痘病毒进行加强免疫能诱导小鼠产生更强的特异性细胞和体液免疫应答。  相似文献   

7.
目的评价毕赤酵母表达的HIV-1中国株CN54 Gag蛋白在Balb/c小鼠诱导体液和细胞免疫的能力。方法利用重组Gag蛋白单独及与重组DNA或痘苗联合免疫Balb/c小鼠,检测小鼠产生的特异性结合抗体和IgG1/IgG2a,同时检测T淋巴细胞增殖反应和CTL反应。结果3个蛋白单独免疫组均能诱导小鼠产生抗体和T淋巴细胞增殖反应,其中不加佐剂的免疫组诱导免疫反应低于其它两组。但3组的CTL杀伤活性均不明显。重组蛋白和重组DNA及痘苗联合免疫均诱导产生了较好的体液和细胞免疫反应,而且重组痘苗初始免疫一蛋白加强免疫组还诱导产生了较强的CrL反应。结论酵母表达的HIV-1 Gag蛋白具有良好的免疫原性,在与重组DNA和痘苗疫苗联合使用时具有协同作用,能刺激动物产生更强的HIV-1特异性体液和细胞免疫反应。本研究为构建HIV-1蛋白疫苗和探讨各类疫苗的联合免疫方式提供了有价值的参考数据。  相似文献   

8.
小鼠对HIV-2 gp105核酸疫苗免疫应答的研究   总被引:2,自引:2,他引:2  
目的: 探讨HIV- 2gp105基因核酸疫苗在小鼠体内的免疫应答, 为开发HIV- 2核酸疫苗提供实验依据。方法:将HIV- 2外膜蛋白 (gp105 )基因插入真核表达质粒载体pVAX1中, 构建pVAX1 gp105重组表达质粒。将其肌注免疫BALB/c小鼠, 用ELISA法检测小鼠血清抗HIV -2抗体, 用流式细胞仪测定CD4 、CD8 T细胞亚群数, 以乳酸脱氢霉释放法检测脾特异性CTL的杀伤活性。结果: 重组质粒pVAX1 -gp105免疫组小鼠的血清抗体滴度、脾T细胞亚群的数量及特异性CTL的杀伤活性, 均明显高于对照组, 分别为P<0. 01, P<0. 05和P<0. 01。结论: HIV -2gp105核酸疫苗能诱导小鼠产生特异性细胞和体液免疫。  相似文献   

9.
恶性疟原虫AMA1不同类型疫苗组合免疫接种研究   总被引:2,自引:0,他引:2  
目的 探索应用恶性疟原虫DNA疫苗、重组痘苗病毒疫苗和重组蛋白疫苗进行组合免疫接种,诱导针对恶性疟原虫红内期抗原AMA1的保护性抗体。方法PCR扩增云南株恶性疟原虫AMA1编码基因,构建和制备DNA免疫质粒VR1020/E(疫苗D)、重组改良安卡拉痘苗病毒rMVME(疫苗V)和重组原核表达AMAl胞外域蛋白E(疫苗P)。用疫苗D单独或与小鼠GM-CSF表达质粒共同对小鼠进行初始免疫,用疫苗V和疫苗P进行单次或先后各一次追加强化。采用疫苗D-V组合方案免疫新西兰白兔。ELISA测定小鼠血清IgG及其亚类IgG1和IgG2a的水平,用免疫动物血清进行疟原虫裂殖子体外入侵抑制实验。结果在疫苗D初始免疫的基础上,采用疫苗V或疫苗P进行强化免疫可显著提高小鼠针对AMA1的抗体应答,小鼠血清特异性IgG平均增加15至137倍,GM-CSF表达质粒对疫苗D-V组合免疫小鼠的抗体应答有显著促进作用。采用疫苗D-V组合免疫在家兔可诱导明显的抗体应答。小鼠和家兔免疫血清在体外可显著抑制疟原虫裂殖子对RBC的入侵。结论将DNA疫苗、重组改良安卡拉痘苗病毒疫苗和重组蛋白疫苗进行组合免疫接种是诱导疟疾红内期保护性抗体的有效方法。  相似文献   

10.
目的 构建能表达野生型和密码子优化型人免疫缺陷病毒Ⅰ型(HIV-1)B亚型中国流行株gp120基因的非复制型腺病毒。方法 按哺乳动物细胞偏好的密码子对HIV-1B亚型中国流行株Ch gp42的gp120基因进行优化,合成优化基因。将野生型和密码子优化的gp120基因插入穿梭质粒,再与腺病毒骨架质粒pAdEasy-1共转化E.coli BJ5183,获得重组子,转染293细胞后获得重组病毒。分别以两种重组腺病毒疫苗免疫小鼠,ELISA检测小鼠血清中的特异性抗体,乳酸脱氢酶法检测小鼠细胞毒性T淋巴细胞(CTL)反应。结果 获得两株重组腺病毒rAd-wt.gp120和rAd.mod.gp120,能正确表达Gp120。rAd-mod.gp120比rAd-wt.gp120蛋白表达水平明显提高。重组腺病毒免疫小鼠后能产生HIV-1特异性的抗体及CTL反应,rAd-mod.gp120组明显优于rAd-wt.gp120组。结论 成功构建了表达野生型和密码子优化的HIV-1 gp120基因的重组腺病毒,能诱导HIV-1特异性体液和细胞免疫反应。  相似文献   

11.
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。  相似文献   

12.
目的 通过高致病性人禽流感H5N1全病毒-MF59佐剂疫苗滴鼻免疫Balb/c小鼠,评价该疫苗所诱导的系统免疫与黏膜免疫应答效果.方法 以不同剂量抗原按比例与MF59佐剂配伍制成粘膜疫苗,滴鼻免疫Balb/c小鼠,二免2周采血检测血清IgG、IgM效价及血清中HAI(HA inhibitor)的中和抗体效价,同时收集鼻、肺灌洗液,检测其lgG和slgA抗体效价.结果 H5NI+MF59组血清抗体效价较H5NI组有显著升高(P<0.01);在各剂量组中,随着剂量的增加抗体效价呈上升趋势.12μg腭后抗体效价呈下降趋势,以HSNI+MF59(12μg)组效价最高;肺鼻灌洗液中,均可检测到特异性分泌型IrA、IsG,其中特异性分泌型IgA效价略高于IgG;抗体亚型的分布以IgG1、IgG2b为主.结论 灭活高致病性禽流感全病毒H5N1在佐剂MF59作用下可诱导机体产生体液免疫应答,同时还可以在黏膜局部产生特异性分泌型IgA、IsG,为高致病人禽流感病毒I-15N1黏膜疫苗的研制奠定了基础.  相似文献   

13.
目的 探讨表达中国株HIV 1gp12 0基因的核酸疫苗在小鼠体内的免疫反应。方法 将表达HIV 1gp12 0的核酸疫苗质粒pVAXP经肌肉注射免疫Balb c小鼠 ,检测免疫小鼠脾CD4 +、CD8+T细胞亚群的数量 ,脾特异性CTL杀伤活性和血清抗体滴度。结果 重组质粒pVAXP免疫组小鼠脾CD4 +、CD8+T细胞亚群的数值均比对照组高 (P <0 .0 5 ) ;免疫组脾特异性CTL杀伤活性与对照组相比差异极显著 (P <0 .0 1) ;血清抗体滴度显著高于对照组 (P <0 .0 5 )。结论 表达HIV 1gp12 0基因的核酸疫苗质粒pVAXP能诱导小鼠产生特异性细胞和体液免疫。  相似文献   

14.
Streptococcus pneumoniae is a respiratory pathogen, and mucosal immune response plays a significant role in the defense against pneumococcal infections. Thus, intranasal vaccination may be an alternative approach to current immunization strategies, and effective delivery systems to mucosal organism are necessary. In this study, BALB/c mice were immunized intranasally with chitosan-DNA nanoparticles expressing pneumococcal surface antigen A (PsaA). Compared to levels in mice immunized with naked DNA or chitosan-pVAX1, anti-PsaA IgG antibody in serum and anti-IgA antibody in mucosal lavages were elevated significantly in mice immunized with chitosan-psaA. The balanced IgG1/IgG2a antibody ratio in serum, enhanced gamma interferon (IFN-γ) and IL-17A levels in spleen lymphocytes, and mucosal washes of mice immunized with chitosan-psaA suggested that cellular immune responses were induced. Furthermore, significantly fewer pneumococci were recovered from the nasopharynx of mice immunized with chitosan-psaA than for the control group following intranasal challenge with ATCC 6303 (serotype 3). These results demonstrated that mucosal immunization with chitosan-psaA may successfully generate mucosal and systemic immune responses and prevent pneumococcal nasopharyngeal colonization. Hence, a chitosan-DNA nanoparticle vaccine expressing pneumococcal major immunodominant antigens after intranasal administration could be developed to prevent pneumococcal infections.  相似文献   

15.
Antigen‐specific mucosal immunity is generally induced by the stimulation of inductive mucosal sites. In this study, we found that the replication‐deficient vaccinia virus vector, DIs, generates antigen‐specific mucosal immunity and systemic responses. Following intradermal injection of recombinant DIs expressing simian immunodeficiency virus gag (rDIsSIVgag), we observed increased levels of SIV p27‐specific IgA and IgG antibodies in faecal extracts and plasma samples, and antibody‐forming cells in the intestinal mucosa and spleen of C57BL/6 mice. Antibodies against p27 were not detected in nasal washes, saliva, and vaginal washes. The enhanced mucosal and systemic immunity persisted for 1 year of observation. Induction of Gag‐specific IFN‐γ spot‐forming CD8+ T cells in the spleen, small intestinal intraepithelial lymphocytes, and submandibular lymph nodes was observed in the intradermally injected mice. Heat‐inactivated rDIsSIVgag rarely induced antigen‐specific humoral and T‐helper immunity. Moreover, rDIsSIVgag was detected in MHC class II IA antigen‐positive (IA+) cells at the injection site. Consequently, intradermal delivery of rDIs effectively induces antigen‐specific humoral and cellular immunity in gut‐mucosal tissues of mice. Our data suggest that intradermal injection of an rDIs vaccine may be useful against mucosally transmitted pathogens.  相似文献   

16.
Synthetic, biotinylated somatostatin-14 (Somatotropin Release-Inhibiting Factor; SRIF) was conjugated to avidin, and the resulting complex incorporated into immune-stimulating complexes (ISCOMS). The ISCOMS were used to study the systemic and mucosal immune responses induced by parenteral and gastrointestinal vaccination. Mice were immunized by intraperitoneal (IP) and intragastric (IG) routes and subsequently by either IP or IG secondary immunizations (groups-IP/IP; IP/IG; IG/IG). Antigen specific IgG and IgA antibody secreting cells (ASC) from the spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP's) were studied by an enzyme-linked immunospot assay (ELISPOT). Specific proliferative responses of spleen cells to avidin and to SRIF were measured.

Immunization IP/IP evoked the highest serum IgG levels to avidin and to SRIF as well as the highest numbers of splenic IgG isotype ASC. The greatest IgA response in MLN and PP's was induced by IP/IG immunization. Only marginal mucosal immunity and no splenic cell specific proliferative responses were found by IG/IG immunization.

These results indicate that ISCOMS are an effective delivery system for protein-peptide antigens. The ISCOMS system described elicited systemic and mucosal antibody immune responses, and primed specific proliferative response when administered IP/IG. This offers another approach for the design and delivery of mucosally administered peptide vaccines.  相似文献   

17.
In this report, we described induction of HIV envelope (env)-specific systemic and mucosal immune responses by oral vaccination of BALB/c mice with env-encoded plasmid DNA encapsulated in poly(dl-lactide-co-glycolide) (PLG) microparticles. We demonstrated that intragastric administration of the encapsulated plasmid DNA resulted in transduced expression of the env glycoprotein in the intestinal epithelium. Mice immunized orally exhibited env-specific type 1 and cytotoxic T lymphocyte (CTL) responses in spleen and the inductive (Peyer's patches) and effector (lamina propria) mucosal tissues of gut. Oral administration of PLG-encapsulated plasmid DNA encoding gp160 also induced env-specific serum antibodies, and an increased level of IgA directed to gp160 was detected in fecal washes of the immunized mice. In contrast, intramuscular (i.m.) administration of naked or PLG-encapsulated DNA vaccine induced only systemic cellular and humoral responses to the env glycoprotein. Using an HIV env-expressing recombinant vaccinia viral intrarectal murine challenge system, we observed higher resistance to mucosal viral transmission in mice immunized orally than in animals injected i.m. with PLG-encapsulated plasmid DNA encoding gp160. Results of these studies demonstrate the feasibility of using orally delivered PLG microparticles containing plasmid DNA-encoded HIV gp160 for induction of env-specific systemic and mucosal immune responses and protection against recombinant HIV env vaccinia virus challenge.  相似文献   

18.
Previously, 39% of rhesus macaques primed orally, intranasally, and intratracheally with adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinants and boosted with gp120 in monophosphoryl lipid A-stable emulsion (MPL-SE) remained aviremic or cleared or controlled viremia at the threshold of detection following SIV(mac251) intrarectal challenge (Study B). In contrast, no macaques primed orally and intranasally with Ad-SIV recombinants and boosted with gp120 in Quillaja Saponaria-21 exhibited undetectable viremia post-challenge (Study A). We conducted a detailed comparison of the studies to elucidate the effect of different vaccine regimens on induced immunity associated with the different challenge outcomes. Quantitative viral load comparisons were statistically analyzed. All immune responses were assessed at identical timepoints post-immunization, and cellular immunity was re-evaluated on cryopreserved cells from Study B macaques to match Study A data acquired with frozen cells. Study B exhibited greater protective efficacy, increased levels of p11C and p54m tetramer positive cells and a trend toward enhanced interferon-gamma secreting cells in response to Env and Gag peptides, modestly enhanced serum neutralizing antibodies, and greater positivity in anti-gp120 rectal IgA and IgG antibodies. Study A macaques exhibited greater positivity in salivary IgA anti-gp120 antibodies. Thus, the vaccine regimen using oral-intranasal-intratracheal priming and protein boosting in MPL-SE was superior, eliciting greater protective efficacy against pathogenic SIV(mac251) and enhanced SIV-specific immunity, systemically and at rectal sites. The mechanism(s) by which binding antibodies, lacking neutralizing activity against the primary challenge virus, may contribute to protection requires further study.  相似文献   

19.
The acquired immunodeficiency syndrome (AIDS) has now become one of the most serious infectious disease in the world, the safe and effective AIDS vaccines would be the best tools to control the disease. Fowlpox virus has been studied recently as a potential vector for the delivery of heterologous vaccine antigen. In this study, a recombinant fowlpox virus coexpressing gp120 of Chinese prevalent HIV-1 strain and IL-2 was constructed and the cellular immune responses against HIV-1 gp120 in BALB/c mice were evaluated. Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gp120 and IL-2 via homologous recombination, and the recombinant virus was identified by PCR and Western blotting. The specific DNA fragment was amplified by PCR from the genomes of rFPV. Western blotting analysis showed that HIV-1 gp120 and IL-2 was expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, specific CTL activities in the immunized mice were detected in the spleen. The results demonstrated that rFPV had good immunogenicity and could induce BALB/c mice to produce specific cellular immunity. IL-2 played a role of immunoadjuvant and enhanced the cellular immune response. The study provides the basis for the preparation of a Chinese vaccine candidate against HIV-1.  相似文献   

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