PIKA佐剂在体内外诱导小鼠免疫应答的探讨

目的：探讨PIKA佐剂在体内外诱导小鼠的免疫应答。方法：体外将小鼠脾淋巴细胞与不同浓度PIKA佐剂培养后，ELISA检测细胞因子IFN-γ、IL-12p40的产生，FACS检测细胞的增殖及活化。体内将PIKA佐剂经小鼠腹腔注射后，检测不同时间点血清中IFN-γ、IL-12pao、IL-6、TNF-α等细胞因子的产生。结果：PIKA佐剂体外呈剂量依赖性诱导小鼠脾细胞产生IFN-γ和IL-12p40。细胞亚群分析的结果表明，PIKA可显著刺激B细胞和NK细胞活化、增殖。体内注射PIKA佐剂后可诱导细胞因子IFN-γ、IL-12pao、IL-6、TNF-α的产生，但产生的时间点不同。结论：PIKA佐剂体内外直接通过诱导细胞因子的产生，B细胞和NK细胞的活化和增殖，而促进免疫应答反应。     

免疫途径和佐剂对CVB3VP1蛋白免疫效果的影响

目的:观察不同免疫途径和佐剂类型对柯萨奇病毒B组3型(Coxsackievirus group B type 3,CVB3)衣壳蛋白VP1免疫效果的影响.方法:将原核表达质粒pET-his/VP1转入E.coli BL21 (DE3) pLysS中,用异丙基-1-硫代-β呋喃半乳糖苷(IPTG)诱导CVB3VP1蛋白的表达并进行纯化.首先采用不同的免疫途径(皮下,腹腔,肌肉)用VP1蛋白免疫小鼠,每组12只.然后另取小鼠分为PBS组和不同佐剂组(氢氧化铝、弗氏佐剂、Montanide ISA720),每组18只,采用肌肉注射途径免疫.每次每只小鼠注射50μg,共免疫3次,间隔3周.用ELISA和微量中和试验检测血清特异性IgG抗体和中和抗体.用CCK-8法检测淋巴细胞增殖活性和CTL杀伤活性.用致死量的CVB3攻击后,检测血中病毒的滴度并观察小鼠的存活状况.结果:在大肠杆菌中成功表达CVB3VP1蛋白.三种免疫途径比较,肌肉注射组血清中和抗体和特异性IgG抗体的水平明显高于其他组(P＜0.01).采用肌肉注射免疫时,弗氏佐剂组Montanide ISA 720佐剂组的体液免疫和细胞免疫应答的水平明显高于氢氧化铝组(P＜0.05);但血中病毒的滴度低于氢氧化铝组(P＜0.05).弗氏佐剂组小鼠的生存率好于氢氧化铝组(P＜0.05).结论:采用肌肉注射途径,并联合弗氏佐剂或Montanide ISA 720佐剂可以使CVB3VP1免疫获得较好的免疫效果.     

弗氏佐剂与氢氧化铝佐剂对诱导小鼠获得性免疫应答作用的比较

为研究、比较不同佐剂对诱导小鼠产生获得性免疫应答的不同作用,以卵清白蛋白(OVA)为抗原,分别混合完全弗氏佐剂(CFA)或Al(OH)3佐剂,对C57BL/6小鼠进行常规免疫,采用流式细胞技术对细胞内细胞因子IFN-γ和IL-4进行检测;ELISA方法对特异性抗OVA抗体滴度及抗体亚型进行了检测。结果显示在免疫后CFA组产生以IFN-γ为主的细胞因子而Al(OH)3组产生以IL-4为主的细胞因子;两组中均产生特异性抗OVA IgG抗体,但CFA组以IgG2a亚型为主,而Al(OH)3组则以IgG1亚型为主,不产生IgG2a亚型抗体。实验表明,经CFA加抗原免疫后机体产生的免疫应答以Th1型细胞免疫为主,抗体类型为IgG2a;而Al(OH)3佐剂则诱导机体产生Th2型细胞免疫应答,抗体类型为IgG1。     

CpG ODN对rHBsAg免疫小鼠Th1/Th2型免疫应答的影响

目的：初步探讨CpC寡脱氧核苷酸(CpG ODN)与重组乙型肝炎表面抗原(rHBsAg)联合免疫小鼠的Th1／Th2型免疫应答效应。方法：BALB／c小鼠经后腿胫骨前肌免疫2次，ELISA法检测血清乙型肝炎表面抗体(抗-HBs)IgG亚类IgG2a/IgG1的比值；生物活性法检测脾细胞诱生上清中的IFN-γ和IL-2含量；ABC-ELISA法检测小鼠血清中IL-4、IL-10及IL-12含量。结果：加CpG ODN组与单独注射rHBsAg组相比：抗-HBs IgG亚类IgG2a／IgG1比值明显高；Th1型细胞因子IFN-γ和IL-2的表达增强，抑制Th2型细胞因子IL-4和IL-10的产生。结论：CpCODN能够明显增强rHBsAg免疫小鼠Th1型抗体亚类IgG2a的产生，并且诱导Th1型细胞因子的表达，抑制Th2型细胞因子的表达。     

BCG 免疫对2 型糖尿病小鼠血糖和免疫应答的影响

目的:探究BCG 免疫对2 型糖尿病小鼠血糖和免疫应答的影响。方法:BCG 经尾静脉注射小鼠,高糖高脂饮食联合小剂量多次腹腔注射链尿佐菌素建立2 型糖尿病模型。检测小鼠体重、空腹血糖和葡萄糖耐量试验血糖水平,测定小鼠细胞免疫应答水平。结果:BCG 免疫下调2 型糖尿病小鼠空腹血糖和糖负荷后的血糖水平;BCG 免疫促进Th1 型细胞反应,显著抑制2 型糖尿病小鼠Th2 型细胞因子的释放。结论:BCG 免疫可能通过抑制2 型糖尿病小鼠炎症因子的释放下调其血糖水平,有助于延缓糖尿病的发展进程。     

IL-18基因佐剂协同HSV-1 gB DNA疫苗免疫诱导的特异性免疫应答

目的研究IL-18 cDNA协同单纯疱疹病毒Ⅰ型（HSV-1）糖蛋白B（gB）核酸疫苗免疫对机体体液免疫和细胞免疫应答的影响。方法利用pcDNA3载体分别构建HSV-1gB和IL-18的真核表达质粒pgB和pIL-18,分pcDNA3、pgB和pgB＋pIL-18三组,肌肉注射免疫接种BALB/c小鼠3次,每次间隔2周,每次接种质粒100μg,第3次免疫后2周,用ELISA检测特异性抗体滴度;利用^3H-TdR掺入法进行T细胞特异性抗原刺激实验;耳廓肿胀实验检测迟发型超敏（DTH）反应。结果与pgB单独免疫组相比,pIL-18的协同免疫可以显著增强ELISA特异性抗体滴度、T细胞增殖反应和DTH反应。结论IL-18cD-NA的协同免疫可以显著提高pgBDNA疫苗诱导的体液免疫和细胞免疫水平,增强核酸疫苗对机体的免疫保护作用。     

基于枯草芽孢杆菌系统制备的EV71VP1重组抗原诱导小鼠免疫应答的研究

目的观察基于枯草芽孢杆菌系统制备的EV7lVPl重组抗原诱导BALB／c小鼠产生的免疫应答反应。方法通过建立BALB／c小鼠动物模型,分别将前期构建的基于枯草芽孢杆菌系统制备的EV71重组抗原（rVPl）、灭活EV71病毒免疫组（EV71）和空白对照组（PBS）,经鼻腔接种6～8周龄BALB／c小鼠,ELISA方法检测免疫后不同时间产生的特异性IgG、IgA以及IgGl和IgG2a水平。结果EV71重组抗原可以有效诱导小鼠产生高水平的体液免疫和明显的黏膜免疫应答,且能够诱发均衡的Th1、Th2免疫调节。结论肠道病毒71型重组VPl抗原可诱发小鼠产生明显的特异性体液免疫和黏膜免疫应答。     

HIV-1重组核酸疫苗质粒诱导小鼠免疫应答的研究

目的：研究白细胞介素6（IL－6）对人类免疫缺陷病毒（HIV）gp120基因免疫小鼠的调节作用。方法：构建表达中国流行株HIV－1B亚型gp120基因的核酸疫苗质粒pGP及共表达中国流行株HIV－1B亚型gp120基因与IL－6基因的核酸疫苗质粒pGPIL-6，检测其在哺乳动物细胞中的表达并 将上述两种质粒注射Balb/c鼠，检测小鼠产生的CD4^ 和CD8^ T细胞数情况及诱导CTL反应能力。结果：以间接免疫荧光法（IFA）检测到gp120在哺乳动物细胞中得到表达。pGPIL-6免疫鼠，小鼠产生的CD4^＋和CD8^ T细胞数及诱导产生CTL反应的能力明显高于单纯注射gp120的小鼠。结论：协同应用IL－6基因可明显加强HIV－1gp120基因免疫效果，为中国流行株HIV－1核酸疫苗的可行性提供了重要实验依据。     

IgG immune response to B19 parvovirus VP1 and VP2 linear epitopes by immunoblot assay

Human B19 parvovirus recombinant capsid proteins VP1 and VP2 were expressed in E. coli and purified. Recombinant proteins were used to detect a specific IgG immune response against VP1 and VP2 linear epitopes by immunoblot assay. A total of 222 serum samples from 218 apparently immunocompetent subjects with different clinical conditions and laboratory evaluations with regards to B19 infection were analyzed. The sera had previously been tested for B19 DNA and for specific IgM and IgG against VP2 conformational antigens by ELISA assay. The data show that, during the active or very recent phase of infection, IgG anti-VP1 linear epitopes appear in concomitance and with the same frequency as IgG anti-VP2 conformational antigens. IgG against conformational VP2 antigens and against linear VP1 epitopes seem to persist for months or years in the majority of individuals. IgG against VP2 linear epitopes are generally present during the active or very recent phase of infection and during the convalescent phase, while they are present only in about 20% of subjects with signs of a past B19 infection. J. Med. Virol. 57:174–178, 1999. © 1999 Wiley-Liss, Inc.     

人博卡病毒VP2病毒样颗粒诱导特异性细胞免疫反应的研究

目的 探讨酶联免疫斑点试验(ELISPOT)检测人博卡病毒(HBoV) VP2病毒样颗粒(VLPs)诱导特异性细胞免疫反应的最佳条件.方法 HBoV VP2 VLPs免疫小鼠后,用ELISPOT方法检测小鼠的特异性细胞免疫反应,观察不同多肽刺激、不同细胞培养时间、不同细胞浓度以及不同浓度特异性刺激多肽条件下ELISPOT结果.结果 多肽P3(GYIPIENEL)及P5(LYQMPFFLL)刺激HBoV1 VLPs免疫小鼠脾脏淋巴细胞产生斑点数分别为233个/10(6)和157个/10(6)细胞,P8(GYIPVIHEL)刺激HBoV2 VLPs免疫小鼠脾脏淋巴细胞产生斑点数为113个/10(6)细胞;培养时间以24 h为最佳,此时HBoV1与HBoV2实验组特异性分泌IFN-(ｒ)的比率分别为232个/10(6)和119个/10(6)细胞;小鼠脾脏淋巴细胞浓度以5×10(5)为最佳,此时HBoV1与HBoV2实验组特异性分泌IFN-(γ)的比率分别为232个/10(6)和108个/10(6)细胞;刺激物浓度10μg/ml为最佳,HBoV1与HBoV2实验组特异性分泌IFN-(γ)的比率分别为233个/10(6)和96个/10(6)细胞.结论 HBoV1与HBoV2特异性BABL/c小鼠T细胞表位多肽分别为HBoV1:P3;HBoV2:P8.检测人博卡病毒VP2病毒样颗粒诱导特异性细胞免疫反应ELISPOT方法最佳实验条件为:培养时间24 h,细胞浓度5×10(5)细胞/孔,刺激多肽终浓度10 μg/ml,可用于HBoV在小鼠上的细胞免疫研究.     

Effect of doxycycline on immune response in mice.

The effect of doxycycline on immune response has been studied in mice, cell-mediated immunity being evaluated with the split heart allograft technique. Survival duration of heart transplants in animals treated with 2.5 mg of doxycycline per kg per day from the day of transplantation until rejection was slightly but significantly longer than in untreated animals, 18.8 days (P less than 0.05) as compared with 14.5 days. In doxycycline-treated animals, both agglutinating and hemolytic antibody response to sheep erythrocytes was slightly but significantly decreased, though there was no inhibition of splenic production of antibodies to sheep erythrocytes (as measured by the number of plaques of hemolysis detected). The results show the immune response in mice to be only moderately inhibited by doxycycline. The relevance of experiments in mice is also discussed.     

Induction of insert-specific immune response in mice by hamster polyomavirus VP1 derived virus-like particles carrying LCMV GP33 CTL epitope

Hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) carrying model GP33 CTL epitope derived from Lymphocytic choriomeningitis virus (LCMV) were generated in yeast and examined for their capability to induce CTL response in mice. Chimeric VP1-GP33 VLPs were effectively processed in antigen presenting cells in vitro and in vivo and induced antigen-specific CD8+ T cell proliferation. Mice immunized only once with VP1-GP33 VLPs without adjuvant developed an effective GP33-specific memory T cell response: 70% were fully and 30% partially protected from LCMV infection. Moreover, aggressive growth of tumors expressing GP33 was significantly delayed in these mice in vivo. Therefore, HaPyV VP1-derived VLP harboring CTL epitopes are attractive vaccine candidates for the induction of insert-specific CTL immune response.     

ELISAs using human bocavirus VP2 virus-like particles for detection of antibodies against HBoV

Human bocavirus (HBoV) has been identified worldwide in children with lower respiratory tract infections with an incidence of approximately 2-11%. The role of HBoV in pathogenesis, however, is largely unknown, and little is known about the epidemiology of the virus. To study the seroepidemiology of HBoV infection, the capsid protein was expressed in insect cells. Expression of the putative major capsid protein VP2 in insect cells led to the formation of virus-like particles exhibiting the typical icosahedral appearance of parvoviruses with a diameter of approximately 20 nm. The expressed particles were used to establish an enzyme-linked immunosorbent assay (ELISA) method, and serum samples from groups of children of various ages in China were tested for IgG antibodies against HBoV. HBoV antibodies were detected in as high as 36% of healthy children under 9 years. Of children hospitalized with lower respiratory tract infections, 31% were seropositive, and all age groups of these children showed a significantly higher level of HBoV IgG antibody than their healthy counterparts. When divided into age cohorts, results showed that more than 48% of healthy children had seroconverted by age of 4. Thus, HBoV appears to be a common infection in children. The potential pathogenesis of this virus, especially its role in lower respiratory tract infections in children warrants further investigation.     

Effect of Biophytum sensitivum on cell-mediated immune response in mice

Effect of Biophytum sensitivum on cell-mediated immune response was studied in normal as well as Ehrlich ascites tumor bearing BALB/c mice. Administration of Biophytum sensitivum significantly enhanced the proliferation of splenocytes, thymocytes and bone marrow cells by stimulating the mitogenic potential of various mitogens such as Lipopolysaccharide (LPS), Concanavalin A (Con A), Phytohaemagglutinin (PHA) and Poke Weed Mitogen (PWM). Natural killer (NK) cell activity was enhanced significantly by Biophytum sensitivum in both the normal (43.6% cell lysis on day 5) and the tumor bearing group (48.2% cell lysis on day 5), and it was found to be earlier than tumor bearing control animals (maximum of 13.4% cell lysis on day 9). Antibody dependent cellular cytotoxicity (ADCC) was also enhanced significantly in both Biophytum treated normal (35% cell lysis on day 7) as well as tumor bearing animals (40.2% cell lysis on day 7) compared to untreated control tumor bearing animals (maximum of 12.3% cell lysis on day 11). An early antibody dependent complement mediated cytotoxicity (ACC) was also observed in the Biophytum treated normal (22.6% cell lysis, on day 15) and tumor bearing animals (26.4% cell lysis, on day 15). Results of our present study suggest the immunomodulatory property of Biophytum sensitivum.     

Effect of rifampin on the immune response in mice.

In an investigation of the effect of rifampin on the immune response in mice, the cellular immunity was evaluated with the split-heart allograft technique. The survival time of the heart in animals treated with rifampin at a dose of 20 mg/kg per day from the day of the transplantation until the graft was rejected was longer (33.7 days, P less than 0.001) than that of animals not treated with antibiotics (14.5 days). When rifampin was given at a dose of 5 mg/kg per day for the same period, the mean survival time of allografts was 19.5 days. The number of demonstrable plaques of hemolysis and the humoral antibodies to sheep erythrocytes were also reduced by a human therapeutic dose (20 mg/kg per day). However, the suppression of the humoral immune response was probably of more limited biological significane, suggesting a differential sensitivity to rifampin. In contrast to rifampin, benzylpenicillin had no noteworthy inhibiting effect on the cellular or humoral immune response.     

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.