禽流感H5N1灭活疫苗与不同纳米化佐剂联合免疫研究

目的 观察禽流感H5N1灭活疫苗加不同佐剂以及纳米化佐剂免疫小鼠后产生的免疫应答的差异,同时观察免疫对异亚型病毒攻击后的保护情况.   方法 用H5N1灭活疫苗分别联合佐剂氢氧化铝、纳米化氢氧化铝、MF59和纳米化MF59通过腹腔注射方式免疫雌性BALB/c小鼠,同时分别以H5N1灭活疫苗免疫小鼠以及PBS腹腔注射处理小鼠作对照.采用ELISA方法分别对各组小鼠免疫后血清特异性IgG及其亚类IgG1、IgG2a水平进行检测,以PR8病毒鼻腔攻击后观察小鼠体重变化情况和生存率.采用t检验作组间比较.    结果 与PBS处理组相比,无论以何种方式免疫H5N1疫苗,免疫后特异性IgG及其亚类水平均明显升高(t=7.4004,P<0.01),以联合MF59后诱导的特异性抗体水平最高.其中疫苗单独免疫组和联合M59免疫组IgG2a水平升高明显,联合纳米化MF59免疫小鼠后IgG2a水平有所下降,IgG1水平有所升高;联合铝佐剂组以IgG1升高为主.攻毒后各组小鼠体重均出现下降,但疫苗单独免疫小鼠以及疫苗与佐剂联合免疫小鼠于攻毒后期体重恢复接近正常或者正常.PBS处理组小鼠攻毒后全部死亡,佐剂联合免疫组小鼠存活率100%,而疫苗单独免疫小鼠存活率为70%.   结论 H5N1疫苗无论是单独免疫或是联合不同佐剂免疫均可诱导较高水平的特异性抗体产生,有非常好的免疫原性.H5N1灭活疫苗诱导的抗体亚类以IgG2a为主,MF59以及纳米化MF59也以诱导IgG2a亚类为主,但纳米化MF59可诱导更均衡的免疫应答.联合铝佐剂或纳米化铝佐剂免疫小鼠后均可诱导以IgG1亚类为主的抗体应答.联合两种佐剂均可对小鼠产生很好的异亚型保护.     

金黄色葡萄球菌锰转运蛋白C的原核表达及小鼠免疫评价

目的在原核系统中表达金黄色葡萄球菌(金葡菌)锰转运蛋白C(manganese transporter C, MntC), 对其免疫原性进行分析, 为筛选金葡菌疫苗候选抗原提供参考。方法合成MntC基因, 克隆至pET-22b(+)载体, 构建重组表达质粒pET-22b-mntc, 转化至E.coli BL21(DE3), 异丙基硫代-β-D-半乳糖苷诱导表达, 镍柱纯化。将纯化产物进行免疫印迹法鉴定, 再采用分子排阻高效液相色谱法进行纯度分析。将铝佐剂吸附重组MntC免疫BALB/c小鼠作为实验组, 将铝佐剂免疫BALB/c小鼠作为对照组。经ELISA检测小鼠血清中特异性IgG抗体水平、中性粒细胞呼吸爆发试验检测小鼠血清特异性抗体功能, 再用细胞因子试剂盒检测小鼠脾淋巴细胞培养上清液中抗原特异性细胞因子水平。免疫后小鼠用致死剂量金葡菌49521菌株攻毒, 观察小鼠存活情况。结果经双酶切及测序鉴定, 重组表达质粒pET-22b-mntc构建正确。重组MntC相对分子质量约为34 000, 以可溶性形式表达, 可与抗His单克隆抗体特异性结合, 纯度大于95%。将铝佐剂吸附MntC免疫小鼠...     

茯苓多糖PCP-Ⅰ和PCP-Ⅱ作为疫苗佐剂的免疫原性

目的研究茯苓多糖(PCP)中PCP-Ⅰ和PCP-Ⅱ作为疫苗佐剂的免疫原性。方法 1采用钥孔戚血蓝蛋白(KLH)和牛血清白蛋白(BSA)为载体蛋白分别与PCP-Ⅰ或PCP-Ⅱ连接制备免疫抗原KLHPCP-Ⅰ和KLH-PCP-Ⅱ及筛选抗原BSA-PCP-Ⅰ和BSA-PCP-Ⅱ。KLH-PCP-Ⅰ和KLH-PCP-Ⅱ分别与弗氏佐剂联用id免疫家兔2次,ELISA检测家兔血清中抗多糖抗体。2PCP-Ⅰ或PCP-Ⅱ单独im免疫小鼠2次,ELISA检测小鼠血清中抗多糖抗体。3PCP-Ⅰ或PCP-Ⅱ为佐剂分别配伍重组乙肝病毒表面抗原(HBs Ag)和猪繁殖与呼吸综合征病毒灭活疫苗(PRRSV)im或sc免疫小鼠2次,ELISA检测小鼠血清中抗多糖抗体。结果1KLH-PCP-Ⅰ或KLH-PCP-Ⅱ与弗氏佐剂联用免疫2次,可产生抗KLH和抗多糖抗体。2PCP-Ⅰ或PCP-Ⅱ单独im免疫小鼠2次,检测出低水平Ig M抗体,未检测出IgG抗体。3HBs Ag或PRRSV抗原联用PCP-Ⅰ或PCP-Ⅱ免疫小鼠2次,未检出抗多糖IgG抗体。结论 PCP-Ⅰ和PCP-Ⅱ本身免疫原性较弱,作为疫苗佐剂可能具有良好的安全性。     

日本血吸虫脂肪酸结合蛋白DNA联合白细胞介素-12的免疫保护作用

目的探讨白细胞介素-12(IL-12)对日本血吸虫脂肪酸结合蛋白(Sj14FABP)DNA疫苗的辅佐作用。方法大量制备pVIVO2、pVIVO2 IL-12、pVIVO2-Sj14FABP质粒DNA。48只雄性Balb/c小鼠随机分为A、B、C、D组，每组各12只，每只小鼠于免疫前1 d在右后腿股四头肌肌内注射075%盐酸布比卡因30 μL。A组给予0.9%氯化钠注射液100 μL，im；B组给予pVIVO2质粒DNA 100 μg，im； C组给予pVIVO2-Sj14FABP质粒DNA 100 μg，im；D组给予pVIVO2-Sj14FABP质粒DNA 50 μg 和pVIVO2-IL-12质粒DNA 50 μg，im。免疫30 d后，每只小鼠以(40±2)条尾蚴感染，感染45 d后，计数成虫及肝内虫卵；用ELISA法检测小鼠血清中IgG抗体水平。结果C、D组的减虫率分别为24.11%和38.83%,减卵率为27.20%和40.27%，差异有显著性（P＜0.05）；免疫30 d后小鼠血清IgG水平无明显升高，各组间差异无显著性 (P＞005)。结论pVIVO2-Sj14FABP能够诱导小鼠产生部分抗血吸虫感染的保护力，IL-12是一种有效的血吸虫病DNA疫苗佐剂。     

杆状病毒系统表达的重组H7N9血凝素亚单位疫苗的免疫保护效果

目的 评价流感病毒H7N9血凝素（hemagglutinin，HA）亚单位疫苗在动物中的免疫效果。方法 利用杆状病毒表达系统表达分泌型的HA片段，单独或联合Al(OH)₃佐剂腹腔注射免疫BALB/c小鼠后，对小鼠进行H7N9毒株的攻击。分析重组H7N9 HA（rH7HA）的免疫保护效果。结果 rH7HA 加Al(OH)₃佐剂与rH7HA单独免疫相比，在血清中诱导了更高的IgG滴度。最高剂量（1.500 μg）rH7HA加佐剂组诱导IgG滴度达2¹⁵，单独诱导IgG滴度达2¹³，但两者之间差异无统计学意义，说明免疫小鼠的rH7HA量达到1.5 μg即能够对同源病毒的感染提供保护。结论 杆状病毒系统表达的分泌型HA通过腹腔注射能够保护小鼠抵抗致死性剂量的同源病毒的感染。     

鼠疫组分疫苗免疫后小鼠血清抗体滴度检测与豚鼠效力观察

目的 根据对小鼠血清抗体滴度检测和对豚鼠的保护力观察,评估鼠疫组分疫苗的免疫效果.方法 通过氢氧化铝佐剂吸附15μg F1、15μg rV、15μg F1+15μg rV抗原,分别制成鼠疫单组分疫苗和双组分疫苗.采用2剂(0、2周)皮下接种途径,分别免疫NIH小鼠和豚鼠.以皮上划痕人用鼠疫活疫苗1剂免疫作为对照.免疫6周后,采用间接ELISA法检测小鼠血清IgG滴度,采用t检验作组间比较.与此同时,使用强毒鼠疫杆菌141株对豚鼠进行皮下攻击,以观察疫苗效力.结果 小鼠血清抗体检测结果表明,鼠疫组分疫苗组血清F1抗体或/和rV抗体滴度均高于鼠疫活疫苗组,差异有统计学意义(t=3.041、3.472、15.958、16.264,P值均＜0.01).豚鼠攻击试验结果显示,F1+rV组的存活率可达到90%(9/10),而F1组和rV组分别为50%(5/10)和20%(2/10).结论 鼠疫F1+rV双组分疫苗是有效的抗鼠疫疫苗,而单组分F1或rV鼠疫疫苗不能有效防御鼠疫杆菌攻击.     

DNA疫苗初免能增强卡介苗抗结核病的保护力

作者用表达分枝杆菌抗原 Ag- 85B的DNA( DNA- 85B)和卡介苗 ( BCG)序贯免疫小鼠获得的预防结核杆菌感染的保护力强于BCG免疫。 C57BL / 6雌鼠初免 DNA- 85B后6周 ,分别加强免疫 DNA- 85B、表达 Ag85B的重组痘苗病毒 ( VV- 85B)、乳化于弗氏不完全佐剂的重组 Ag85B蛋白 ( P- 85B)或BCG,同时另设未免疫 /未免疫、对照 DNA/对照 DNA、BCG/未免疫和 BCG/ BCG对照组。各组小鼠在末次免疫后 6周气雾感染结核杆菌 H37RV,每个鼠肺大约接受 1 0 0个活菌。感染后 4周收集肺、脾和血液进行活菌计数 ( CFU)。结果表明 ,DNA- 85…     

重组质粒Eg.EF-1/pGEX-6P-1的构建及原核诱导表达

将细粒棘球蚴（Echinococcus granulosus,Eg.）延伸因子（Elongation factor 1,EF-1）与pGEX-6P-1构建成重组质粒并进行表达、纯化及对其特性进行鉴定.将构建好的EF-1/pGEM-T经双酶切后获取目的基因片段,定向重组于表达载体pGEX-6P-1并转化大肠杆菌（Escherichia coli）E.coli BL21,IPTG诱导表达,用蛋白亲和层析柱GSTrap FF对表达产物进行纯化,特异性蛋白解离酶PreScission^TM Protease酶切融合蛋白从中获取重组Eg.EF-1;SDS-PAGE和Western blot方法鉴定表达产物.获得的Eg.EF-1/pGEX-6P-1/E.coli BL21菌株,诱导表达融合蛋白和纯化分离得到的31 ku Eg.EF-1均能被细粒棘球蚴天然抗原免疫的兔多克隆抗血清识别.证明成功构建出具有有效表达的Eg.EF-1/pGEX-6P-1菌株,初步证实重组蛋白具有较好的抗原性.     

单剂HBsAg-PLGA控释疫苗微球小鼠体内免疫学研究

目的研究小鼠皮下注射重组乙型肝炎病毒表面抗原(HBsAg)-乳酸/乙醇酸共聚物(PLGA)微球后的体内抗体应答水平和免疫学机制。方法采用复乳法制备疫苗微球后，单剂注射到BALB/c小鼠皮下，在一定时间内检测全抗体、IgG抗体亚型及细胞因子的应答水平。结果HBsAg-PLGA微球在小鼠体内主要引发体液免疫应答；其中单剂注射HBsAg-PLGA50/50-COOH微球在免疫早期产生较高免疫表达，6周后降低，全抗体水平显著低于常规铝佐剂疫苗(P<0.01)；分别单剂注射HBsAg-PLGA50/50微球及HBsAg-PLGA75/25微球后产生的免疫应答在18周内与铝佐剂疫苗相当(P>0.05)。结论PLGA微球作为乙肝疫苗的长效缓释可生物降解载体，具有一定潜在优势。     

姬松茸多糖佐剂诱导OVA抗原特异性Th1免疫应答的机制研究

目的： 探讨姬松茸多糖（ABP-AW1）作为佐剂对卵白蛋白（OVA）免疫鼠细胞免疫反应的调节作用。方法： 以OVA为抗原,ABP-AW1为佐剂经皮下两点免疫小鼠,于0,15 d各免疫一次,第28 d,碳粒廓清实验检测Mφ吞噬能力;ELISA法检测血清中免疫球蛋白及其抗体亚类水平,RT-PCR法分析Th1细胞因子IL-2、IFN-γ mRNA表达水平。结果： ABP-AW1可以增强OVA免疫小鼠抗原递呈细胞的吞噬功能;血清中抗体亚类IgG2b明显高于对照组（P<0.05）;脾细胞分泌Th1细胞因子IL-2、IFN-γ mRNA水平也均高于其他各组（P<0.05）。结论： ABP-AW1可作为OVA抗原的细胞免疫佐剂成分,调节OVA抗原Th1型反应的免疫应答。     

Cannabinoid 2 (CB2) Receptor Involvement in the Down-regulation but not Up-regulation of Serum IgE Levels in Immunized Mice

Marijuana cannabinoids such as Δ(9)-tetrahydrocannabinol (THC) have been shown in experimental systems to bias T helper immunity towards Th2 and away from Th1. This effect if broadly applicable to humans could have important implications in Th2-mediated diseases such as allergy. In the current study, we examined the effect of cannabinoids on serum immunoglobulin IgE levels in immunized mice and also examined the role of cannabinoid receptors in the response. The method involved pre-injecting mice with cannabinoid receptor agonists and antagonists followed 18-24?h later with an immunizing injection with two different antigen/adjuvant combinations. This treatment was followed 2-3?weeks later with a booster injection of antigen and the subsequent bleeding of mice 1-2?weeks later for serum immunoglobulin analysis by ELISA. Our results showed that THC injection enhanced total IgE serum levels in response to antigen immunization even under conditions of deficient cannabinoid receptor 2 (CB2) and cannabinoid receptor 1 (CB1) activity and furthermore the increase in IgE was accompanied by a decrease in serum IgG2a. In addition, we observed that l-α-lysophosphatidyliniositol (LPI) increased serum IgE levels and that IgE levels were higher in CB2 deficient mice and suppressed by the CB2 agonist, Gp1a. These results suggest that in this IgE induction model in mice, non-selective cannabinoids such as THC increase IgE through receptors other than CB1 and CB2 but that CB2 receptors do play a suppressive role in the control of serum IgE levels.     

Suppressive effects of Hochu-ekki-to, a traditional Chinese medicine, on IgE production and histamine release in mice immunized with ovalbumin

We examined the effects of Bu-Zhong-Yi-Qi-Tang (Japanese name: Hochu-ekki-to, HET), a traditional Chinese medicine, on IgE production and histamine release in mice immunized intraperitoneally with a mixture of ovalbumin (OA) and aluminum hydroxide (alum adjuvant). Three groups of mice were orally administered 0, 1.7 or 17 mg of HET on day 13 after the first immunization with a mixture of 1 microg OA and 1 mg alum adjuvant. They were again immunized with the same dose of OA plus alum adjuvant on day 14. The immunological changes in mice treated with OA alone or OA plus HET were examined, and the following findings were obtained. In the HET-treated mice, the elevation of anti-OA IgE in serum, and histamine release from basophils in blood, were significantly suppressed. A significant suppression of interleukin-4 (IL-4) secretion and proliferation of splenic lymphocytes in primary culture was also observed. A tendency to suppress the elevation of anti-OA IgG1 in serum and interleukin-2 (IL-2) secretion from splenic lymphocytes was observed in the HET-treated mice. These findings suggest that oral administration of HET suppresses IgE antibody production and histamine release in type I allergic reaction in mice immunized with OA plus alum adjuvant; this shows the efficacy of HET in treating type I allergic diseases, such as asthma.     

Airway inflammation and adjuvant effect after repeated airborne exposures to di-(2-ethylhexyl)phthalate and ovalbumin in BALB/c mice

BACKGROUND: Epidemiological studies have suggested an association between exposure to phthalate plasticizers, including di-(2-ethylhexyl)phthalate (DEHP), and increased prevalence of asthma, rhinitis or wheezing. Furthermore, studies in mice have demonstrated an adjuvant effect from DEHP after parenteral administration with the model allergen ovalbumin (OVA). OBJECTIVE: Exposures to DEHP were investigated for adjuvant effects and airway inflammation in a mouse inhalation model. METHODS: BALB/cJ mice were exposed to aerosols of 0.022-13 mg/m(3) DEHP and 0.14 mg/m(3) OVA 5 days/week for 2 weeks and thereafter weekly for 12 weeks. Mice exposed to OVA alone or OVA+Al(OH)(3) served as control groups. Finally, all groups were exposed to a nebulized 1% OVA solution on three consecutive days. Serum, bronchoalveolar lavage (BAL) fluid, and draining lymph nodes were collected 24h later. RESULTS: In the OVA+Al(OH)(3) group, significantly increased levels of OVA-specific IgE and IgG1 in serum as well as of eosinophils in BAL fluid were observed. DEHP affected OVA-specific IgG1 production in a concentration-dependent manner, whereas little effect was seen on IgE and IgG2a. Dose-dependent increases in inflammatory cells were observed in BAL fluids, leading to significantly higher lymphocyte, neutrophil and eosinophil numbers in the OVA+13 mg/m(3) DEHP group. Ex vivo cytokine secretion by cultures of draining lymph nodes suggested that DEHP has a mixed Th1/Th2 cytokine profile. CONCLUSION: Airborne DEHP is able to increase serum IgG1 and lung inflammatory cell levels, but only at very high concentrations. Realistic DEHP levels do not have an adjuvant effect or induce allergic lung inflammation in the present mouse model.     

Enhancement of the immune responses of mice to <Emphasis Type="Italic">Bacillus anthracis</Emphasis> protective antigen by CIA07 combined with alum

Anthrax is an acute zoonotic disease caused by infection with Bacillus anthracis. B. anthracis spores are highly resistant to environmental degradation and are used as a biological weapon. In this study, we investigated the adjuvant activity of CIA07 to anthrax protective antigen (PA). A/J mice were immunized intraperitoneally once, or twice with a 4-week interval, with recombinant PA alone or combined with alum, CpG1826, or CIA07 as adjuvant, and serum anti-PA IgG antibody responses were measured 4 weeks after each immunization. All three adjuvants significantly enhanced anti-PA IgG antibody titer 4 weeks after the priming and boosting immunizations, and alum gave the highest titer. In order to evaluate the adjuvant activity of CIA07 in the presence of alum, Balb/c mice were immunized 3 times at 1-week intervals with PA in combination with alum, CIA07 or alum plus CIA07, and the immune responses were assessed 2 weeks after the third immunization. The serum anti-PA IgG antibody titer of the CIA07-treated group was 14-fold higher than the group given PA alone, and the coadministration of CIA07 with alum further increased the titer 3.5-fold (P < 0.05). The toxin neutralizing activity of the sera from the mice given the combination of CIA07 and alum was 109-times higher than the animals given PA alone. The mice given CIA07 plus alum also showed a marked increase in the number of IFN-γ-, IL-2-, and IL-4-producing CD4⁺ T cells among their splenocytes. These data suggest the potential of CIA07 in combination with alum as an adjuvant for the development of a potent anthrax vaccine.     

Immunogenicity of the hookworm Na-ASP-2 vaccine candidate: characterization of humoral and cellular responses after vaccination in the Sprague Dawley rat

The recombinant larval protein Na-ASP-2 from the human helminth parasite Necator americanus is a lead candidate for a human hookworm vaccine. We have characterized the humoral and cellular immune responses elicited by the Na-ASP-2 formulated with the aluminum-based adjuvant Alhydrogel in the Sprague Dawley rat. We demonstrated that Na-ASP-2 vaccine induced a strong antibody response at all doses tested, as evidenced by high specific IgG1, IgG2a and IgM titers. High IgG antibody titers were maintained up to three months post vaccination and were boosted by an additional vaccine dose. Specific cell proliferation and a Th2 cytokine profile were also observed in peripheral blood of all rats immunized with the formulated vaccine. Host IL-6 levels were also significantly elevated. These data provide evidence that the immune response of the Na-ASP-2 vaccine is robust and durable and therefore suitable for the further clinical development of the Na-ASP-2 vaccine.     

Design and evaluation of a hypoallergenic peptide-based vaccine for Salsola kali allergy

BackgroundThe Salsola kali (S. kali) pollen is one of the most important causes of allergic rhinitis in the deserts and semi-desert areas. Immunotherapy with allergen extracts remains the only available treatment addressing the underlying mechanism of allergy. However, given the low efficacy of this method, it is necessary to find more effective and alternative therapeutic interventions using molecular biology and bioinformatics tools. In this study, a hypoallergenic vaccine was designed on the basis of B-cell epitope approach for S. kali immunotherapy.MethodsUsing the Immune Epitope Database (IEDB), a 35-mer peptide was selected and chemically conjugated to a keyhole limpet hemocyanin (KLH) molecule. Specific IgG and IgE from immunized BALB/c mice sera against the vaccine (Sal k 1-KLH), S. kali extract and the recombinant protein, rSal k 1, were measured using ELISA. Also, inhibition of IgE by mouse IgG was evaluated using an inhibitory ELISA. Finally, the IgE reactivity and T-cell reactivity of the designed vaccine were evaluated by dot blot assay and MTT assay.ResultsVaccination with the vaccine produced high levels of protective IgG in mice, which inhibited the binding of patients IgE to recombinant proteins. The result showed that the designed vaccine, unlike the recombinant protein and extract, did not induce T-cell lymphocytes response and also exhibited decreased IgE reactivity.ConclusionThe designed vaccine can be considered as a promising candidate for therapeutic allergen-specific immunotherapy.     

The feeding of beta-carotene down-regulates serum IgE levels and inhibits the type I allergic response in mice

Feed containing beta-carotene was administered orally to BALB/c mice immunized intraperitoneally with ovalbumin (OVA) for approximately 1 month. The titers of OVA-specific IgE, OVA-specific IgG1 and OVA-specific IgG2a in the mouse sera were determined. The OVA-specific IgE titer and OVA-specific IgG1 titer by mice fed beta-carotene were significantly inhibited. On the other hand, the OVA-specific IgG2a titer in mice fed beta-carotene was significantly greater than those of control mice. The OVA-specific IgE suppression of beta-carotene feeding was dose-dependent. We also examined the effect of fed beta-carotene on active systemic anaphylaxis. Feeding beta-carotene to mice immunized with OVA inhibited the immediate reduction of the body temperature induced by antigen stimulation. Furthermore, the increase in serum histamine in the mice fed beta-carotene under active systemic anaphylaxis was lower than in controls. We then examined the pattern of cytokine production by spleen cells from mice followed by restimulation with OVA in vitro. The spleen cells from the mice fed beta-carotene produced more IFN-gamma, IL-12 and IL-2 than those from the control group. In contrast, the spleen cells from the mice fed beta-carotene produced less IL-4, IL-5, IL-6, IL-10 than those from the control group. Furthermore, analysis of IFN-gamma mRNA levels of the splenocytes using the real-time quantitative RT-PCR technique revealed higher levels in the splenocytes from the mice fed beta-carotene. These findings suggest that feeding beta-carotene improves the helper T cell (T(H))1-T(H)2 balance, inhibiting specific IgE and IgG1 production and antigen-induced anaphylactic response.