免疫佐剂增强犬传染性肝炎核酸疫苗免疫效果研究
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摘要
目的:核酸疫苗是90年代从基因治疗研究领域发展起来的一种新的免疫技术。它可诱导机体产生包括体液免疫应答和细胞免疫应答在内的全面的免疫应答,与传统疫苗相比有许多全新的潜在优势,从而被誉为第三次疫苗革命。目前对于核酸疫苗的研究,主要涉及包括病毒、细菌和原虫在内的多种传染性病原的预防。但由于其抗原分子小、纯化程度高,疫苗的免疫原性不够理想。因此需要安全有效的佐剂来增强疫苗的效力。有效的免疫佐剂对DNA疫苗的免疫保护作用及发挥长效免疫效果有增强作用。
CpG寡聚脱氧核苷酸(CpG-ODN)中的CpG基序能够刺激B细胞,T细胞,自然杀伤细胞和抗原提呈细胞发生免疫级联放大反应。CpG-ODN可以诱发机体产生Th1型免疫反应并能诱导致炎因子的产生。在体内,骨髓来源的免疫细胞包括单核细胞,巨噬细胞和骨髓来源的树突细胞均表达TLR9受体,TLR9受体对CpG基序作出反应。溴化二甲基双十八铵(DDA)是一种阳离子脂质,作为一种佐剂它可以吸附抗原到它的表面,能增强IFN-γ的分泌和诱发免疫保护反应,并能增强抗原在体内诱导的体液和细胞免疫反应。DDA的作用类似于微胶囊,缓慢释放包裹的质粒载体,从而达到长时间表达抗原的作用,因此在机体抵抗多种疾病中发挥重要作用。Lipofectamine TM 2000是一种阳离子聚合物,带净正电荷的聚合物与带负电荷的DNA之间相互作用,自发形成了脂质-DNA复合物。脂质体-DNA复合物能与细胞表膜和细胞内膜直接融合或通过内吞作用进入细胞。脂质扩散入靶细胞膜的过程,破坏了膜与DNA间的相互作用,从而使DNA进入细胞浆。作为佐剂Lipofectamine TM 2000可以诱导机体产生Th1反应,已被广泛应用于增强DNA疫苗免疫效果的研究。本室构建的犬传染性肝炎病毒核酸疫苗pVAX1-CpG-Loop免疫小鼠诱导了体液免疫和细胞免疫的产生。
为进一步提高pVAX1-CpG-Loop免疫效果,寻求最佳适配佐剂,本次实验是在本室对核酸疫苗pVAX1-CpG-Loop的研究基础上,用佐剂CpG-ODN,DDA和Lipofectamine TM 2000与质粒pVAX1-CpG-Loop共同免疫小鼠,并检测其诱导小鼠产生的细胞及体液免疫效果。
方法:
1质粒的大量提取
采用SDS-碱裂解法大量提取重组质粒pVAX1–CpG -Loop,采用聚乙二醇沉淀法纯化质粒。并对提取的重组质粒pVAX1-CpG-Loop经分光光度计进行纯度和浓度测定。将原核表达质粒pET28a(+)Loop转化大肠杆菌BL21(DE3),摇菌并诱导重组Loop蛋白的表达, Western blot鉴定。用尿素溶解沉淀,采用镍离子亲和层析柱纯化重组蛋白,透析并复性后浓缩目的蛋白。
2免疫佐剂的准备
CpG-ODN 1826序列为5′TCCATGACGTTC CTGACG TT3′非甲基化,全硫代修饰,PAGE纯化。- 20℃保存备用。PBS溶解并与质粒pVAX1-CpG-Loop混匀后备用。
DDA溶于pH7.4的10mM Tris-Cl,终浓度为1.25 mg/ml,加热80℃,20分钟,并反复颠倒混匀,冷却到室温与质粒pVAX1-CpG-Loop混合后备用。
Lipofectamine TM 2000 40μl/只与质粒pVAX1-CpG-Loop混合后备用。
3小鼠的免疫方案:
方案1:6-8周龄雌性BALB/c小鼠随机分为6组各5只:
①对照组:pVAX1-CpG-Loop 25μg/只。
②CpG-ODN高剂量组: CpG-ODN 50μg/只+质粒pVAX1-CpG-Loop 25μg/只。
③CpG-ODN低剂量组: CpG-ODN 20μg/只+质粒pVAX1-Cp G-Loop 25μg/只。
④LipofectamineTM 2000组: Lipofectamine TM 2000 40ul/只+质粒pVAX1-CpG-Loop 25μg/只。
⑤DDA高剂量组:DDA 250μg/只+质粒pVAX1-CpG-Loop 25μg/只。
⑥DDA低剂量组:DDA 25μg/只+质粒pVAX1-CpG-Loop 25μg/只。
小鼠左右股四头肌注射重组质粒pVAX1-CpG-Loop并混有相应佐剂,两周免疫一次,共免疫三次,末次免疫两周后,无菌取脾,进行T淋巴细胞特异性增殖分析,流式细胞术检测IFN-γ,蛋白特异性CTL活性分析。
方案2:分组同方案1。小鼠左右股四头肌注射重组质粒pVAX1-CpG-Loop并混有相应佐剂,两周免疫一次,免疫三次后,每组小鼠分别于第十三,十五,十八周用pVAX1-CpG-Loop (25μg/只)加强免疫三次,末次免疫后两周,无菌取脾,进行T淋巴细胞特异性增殖分析,流式细胞术IFN-γ检测,蛋白特异性CTL活性分析检测。免疫后每周尾尖采血,分离血清,ELISA测定血清抗体效价。
方案3:6-8周龄雌性BALB/c小鼠随机分为5组每组各5只:
①对照组:pVAX1-CpG-Loop 100μg/只。
②CpG-ODN高剂量组: CpG-ODN 50μg/只+质粒pVAX1-CpG-Loop 100μg/只。
③CpG-ODN低剂量组:CpG-ODN 20μg/只+质粒pVAX1 -CpG-Loop 100μg/只。
④Lipofectamine TM 2000组:Lipofectamine TM 2000 40ul/只+质粒pVAX1-CpG-Loop 100μg/只。
⑤DDA组:DDA 250μg/只+质粒pVAX1-CpG-Loop 100μg/只。
小鼠左右股四头肌注射重组质粒pVAX1-CpG-Loop (100μg/只)并混有相应佐剂,两周免疫一次,共免疫三次,末次免疫两周后,无菌取脾,进行T淋巴细胞特异性增殖分析,流式细胞术检测IFN-γ,蛋白特异性CTL活性分析。免疫后每周尾尖采血,分离血清,ELISA测定血清抗体效价。
4免疫效果评价
4.1 ELISA测定血清抗体效价免疫后每周尾尖采血,分离血清,以犬传染性肝炎病毒抗原每孔100μg/ml包被酶标板,4℃过夜。洗涤封闭后加倍比稀释的小鼠血清100μl,孵育60min后洗涤加HRP标记的二抗孵育60min,OPD显色。酶标仪492nm波长处测吸光度值
4.2 T淋巴细胞特异性增殖分析采用MTT方法检测免疫小鼠的T淋巴细胞增殖活性。
4.3流式细胞术检测IFN-γ的分泌。加入Loop蛋白30μg/ml和10ng/ml interleukin-2孵育72h。收集细胞,加入荧光标记的抗体CD8-PE,IFN-γ-FITC,固定过夜,用流式细胞仪检测。
4.4蛋白特异性CTL活性分析采用LDH释放法。
4.4.1效应细胞制备:调整脾淋巴细胞密度至1×107 /ml ,脾淋巴细胞悬液2ml/孔加入6孔板,加入Loop蛋白30μg/ml和10 ng/ml interleukin-2孵育7天,于第7d收集细胞作为效应细胞,进行特异性杀伤活性测定。
4.4.2靶细胞的制备:胰酶消化CT26细胞,传代于12孔细胞培养板中,次日进行转染。无血清DMEM稀释的质粒(pVAX1-CpG和pVAX1-CpG-loop)分别与无血清DMEM稀释的Lipofectamine TM 2000混匀并在室温下静置20分钟。每个孔加混合液200μl, CO2培养箱,37℃5﹪CO2培养48小时。
4.4.3效应细胞对靶细胞的杀伤活性的检测:上述转染细胞混合作为靶细胞,效应细胞:靶细胞混合比例为100:1, 50:1, 25:1。同时设靶细胞自发释放孔和靶细胞最大释放孔。测492nm波长的吸光值,按下面的公式计算CTL对靶细胞的杀伤活性:实验组释放量-自发释放量最大释放量-自发释放量
结果:
1碱裂解法大量提取的重组质粒pVAX1-CpG-Loop经双酶切和1.2﹪琼脂糖凝胶电泳鉴定,显示860bp左右的×100%电泳条带。所得重组质粒OD260/OD280在1.8–2.0之间。Western blot结果显示诱导表达蛋白与抗六聚组氨酸抗体特异性结合。诱导表达的Loop蛋白经镍离子亲和层析纯化,纯度可达95%以上。
2将重组质粒pVAX1-CpG-Loop与不同免疫佐剂混合后,免疫小鼠,间接ELISA检测显示:各实验组小鼠所产生的抗体效价明显高于pVAX1-CpG-Loop对照组(P<0.05),各组之间无统计学差异(P>0.05)。
3 T淋巴细胞特异性增殖分析,方案2和方案3的小鼠检测到T淋巴细胞的增殖活性明显高于同组的pVAX1-CpG-Loop对照组(P<0.05)。方案1的小鼠无明显增殖活性(P>0.05)。pVAX1-CpG-Loop与CpG-ODN(20μg/只)共免疫组的小鼠脾淋巴细胞的增殖活性明显高于其他实验组。
4免疫小鼠脾淋巴细胞经流式细胞分析,方案2和方案3的小鼠CD8+T细胞被标记IFN-γ的抗体数量高于同组的pVAX1-CpG-Loop对照组(P<0.05)。方案2免疫的小鼠CD8+T细胞被标记IFN-γ的抗体数量高于方案3免疫的小鼠。方案3的小鼠CD8+T细胞被标记IFN-γ的抗体数量高于方案1免疫小鼠。
5蛋白特异性CTL活性分析结果显示,各实验组小鼠LDH释放率高于pVAX1-CpG-Loop对照组(P<0.05)。方案2免疫小鼠LDH释放率高于方案3免疫小鼠。方案3免疫小鼠LDH释放率高于方案1免疫小鼠。方案2免疫小鼠(25μg/只) DDA与pVAX1-CpG-Loop共免疫组小鼠LDH释放率高于(250μg/只)DDA与pVAX1-CpG-Loop共免疫组小鼠。
结论:
1 ELISA检测:各免疫组小鼠均产生了针对抗原病毒的特异性IgG抗体,且pVAX1-CpG-Loop 100μg/只+ CpG -ODN 20μg/只,pVAX1-CpG-Loop 100μg/只+DDA 250μg/只免疫组的抗体水平明显高于其它实验组。以上结果提示重组质粒pVAX1-CpG-Loop诱导小鼠产生了体液免疫应答,免疫佐剂可有效地增强DNA疫苗诱导的体液免疫应答。
2流式细胞分析显示:各实验组小鼠的CD8+T细胞被标记IFN-γ的抗体数量高于pVAX1-CpG-Loop对照组;采用MTT方法检测小鼠脾脏的淋巴细胞增殖活性,实验组小鼠淋巴细胞增殖活性高于pVAX1-CpG-Loop对照组;实验组小鼠LDH释放率高于pVAX1-CpG-Loop对照组(P<0.05)。以上结果提示免疫佐剂能增强重组质粒pVAX1-CpG-Loop诱导小鼠产生特异性的细胞免疫应答。
3 DDA,CPG-ODN,Lipofectamine TM 2000三种免疫佐剂均能增强DNA疫苗pVAX1-CpG-Loop对小鼠的体液免疫和细胞免疫。方案2免疫的小鼠能够产生对Loop蛋白特异性免疫记忆。证明免疫佐剂与低剂量DNA疫苗共免疫结合低剂量DNA疫苗加强免疫的策略是一种非常有效和有潜力的免疫策略。
Objective Nucleic acid vaccine is a new vaccine technique following inactivated vaccines and attenuated vaccines in 1990s. Nucleic acid vaccine is capable of eliciting both cellular and humoral immune responses more complete than conventional vaccination and have the advantage of mass-production easily. It has been actively developed in the past few years against a variety of infectious agents including virus, bacteria and parasites. Beause of it’s small antigen molecule,higher level of purification, short of immunogenicity,the effective adjuvant was needed to strengthen the immune response that induced by DNA vaccine. Efficient adjuvant is beneficial for DNA vaccine to enhance sufficient level of protection and long-lasting immunity.
Synthetic oligodeoxynucleotides (ODNs) that contain immunostimulatory CpG motifs trigger an immunomodulatory cascade that involves B and T cells, natural killer cells and professional antigen-presenting cells. The response to CpG-ODNs skews the host’s immune milieu in favour of T helper 1 (Th1)-cell responses. In mice, immune cells of the myeloid lineage including monocytes, macrophages and myeloid DCs express TLR9 and respond to CpG stimulation.
The cationic surfactant dimethyl dioctadecyl ammonium bromide (DDA) is a novel adjuvant, DDA can adsorb most antigens on their surface and promote a strong cell mediated immune response and a humoral immune response in vivo, which is essential for the induction of protective immunity against most diseases.
Lipofectamine TM 2000 is a cationic lipid, liposome has been widely used to enhance DNA vaccines and preferentially activate Th1 responses. in vitro, the lipide and DNA compounds can fuse with the celluar membrance and help the DNA come into the cytolymph .
In previous studies, we have constructed the DNA vaccine (pVAX1-CpG-Loop) that encoding two major Loop DNA fragments from CAV-1 and it has induced significantly cell mediated and humoral immune responses. In order to get the better immune efficacy in mice, especially induce a more durable T-cell response, we compared the effect of the three adjuvants on pVAX1-CpG-Loop DNA vaccine. This study evaluated the adjuvant effects of CpG-ODN, DDA, and LipofectamineTM 2000 on a DNA vaccine pVAX1-CpG-Loop, which was against canine adenovirus type-1. Selecting the appropriate adjuvant to specific antigenic components will not only enhance the level of immune response but also determine the type of immune response induced.
Methods
1 Plasmid isolation and purification
The plasmid pVAX1-CpG-Loop was isolated by the method of alkaline lysis, and purified by polyethylene glycol precipitation. The fusion protein was purified by Ni-NTA affinity chromatography column.
2 preparation of adjuvants
The CpG-ODN (1826) sequence is 5'-TCCATGACGTTCCTGACGTT-3', which was produced with DNase-protected phosphorothioate bonds backbone. CpG-ODN were dissolved in a sterile phosphate buffered saline solution and stored at -20℃.
DDA adjuvant was prepared as described previously. Briefly, DDA adjuvant was mixed into 10 mM Tris-buffer at pH 7.4 to a concentration of 1.25 mg/ml, heated to 80℃for 20 min with intermittent shaking, then cooled to room temperature.
3 Mice
BALB/c mice were bred and cared in the Animal Facilities of Hebei Medical University, Shijiazhuang. Only female mice ages 6-8 weeks old were used for the experiments.
4 Immunization protocols
Experiment 1: female mice were randomly divided into 6 experimental groups (5 mice /group). The detailed vaccination scheme were as followed. Biweekly mice were injected intramuscularly with a volume of 100μl/mouse for three times. After two weeks, the splenocytes (5 mice / group) were harvested. The Intracellular staining of IFN-γ,In vitro CTL activity assay and The lymphocyte proliferation assay was performed.
Group1. pVAX1-CpG-Loop 25μg/mouse
Group2.pVAX1-CpG-Loop25μg/mouse+CpG-ODN50μg/mous-e
Group3.pVAX1-CpG-Loop25μg/mouse+CpG-ODN20μg/mous-e
Group4.pVAX1-CpG-Loop25μg/mouse+LipofectamineTM 2000 (Invitrogen USA)40μl/mouse
Group5.pVAX1-CpG-Loop 25μg/ mouse +DDA 250μg/mouse
Group6.pVAX1-CpG-Loop 25μg/ mouse +DDA 25μg/mouse
Experiment 2: The grouping project same as Experiment 1. Biweekly mice were injected intramuscularly with a volume of 100μl/mouse for three times. All the groups were re-injected with pVAX1-CpG-Loop (25μg/mouse) at thirteenth, fifteenth and eighteenth week respectively. two weeks later after the final immunization, the splenocytes were harvested. The Intracellular staining of IFN-γ, In vitro CTL activity assay and The lymphocyte proliferation assay was performed.
Experiment 3: The mice were injected intramuscularly (100μg/100μl/mouse, 5 mice /group) with the DNA vaccine and the adjuvants at a 2-week interval for three times. After two weeks, the splenocytes were harvested. The Intracellular staining of IFN-γ, In vitro CTL activity assay and The lymphocyte proliferation assay was performed.
Group1.pVAX1-CpG-Loop100μg/mouse+CpG-ODN50μg/mo- use
Group2.pVAX1-CpG-Loop100μg/mouse+CpG-ODN20μg/mo-use
Group3.pVAX1-CpG-Loop 100μg/ mouse + LipofectamineTM 2000 40μl/ mouse
Group4.pVAX1-CpG-Loop100μg/ mouse +DDA250μg/ mouse
Group5. pVAX1-CpG-Loop 100μg/ mouse
5 The evaluation of the immuned mice
5.1 Enzyme-linked immunosorbent assay
Sera of immunized mice were collected and specific IgG antibody was detected by the method of indirect enzyme linked immunosorbent assay (ELISA) as described previously . Briefly, the dilution of serum sample was 1:40, purifed CAV-1 was used as antigen and specific anti-virus IgG antibody was detected. Absorbance was determined at 492 nm.
5.2 The Loop protein specific lymphocyte proliferation assay
OD 570 was measured by a standard 3-(4,5dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) method. The stimulation index was calculated from the formula: stimulation index (SI) = (Arestimulated -Ablank)/(Aunrestimulated-Ablank)
5.3 Intracellular staining of IFN-γ
1×107 spleen cells from immunized mice were cultured in 0.5 ml of complete DMEM in the presence of the recombinant Loop protein 30μg/ml and 10 ng/ml of interleukin-2 for 72h. The stimulated lymphocytes were harvested and labeled for surface CD8 and intracellular IFN-γusing PE (Jingmei Biotech) or FITC (BioLegend) labeled specific monoclonal antibodies following the detailed instructions provided by the vendor. Spleen cells were analyzed by flow cytometry.
5.4 The Loop protein specific CTL activity assay
5.4.1 preparation of the effectors :The cell line CT26 was transfected with plasmid pVAX1-CpG-Loop or pVAX1-CpG vector with Lipofectamine 2000 according to the manufacturer’s direction as the method described previously. After 48 h incubation at 37℃, 5% CO2, CT26 cells were collected and the transfection products were analysed by RT-PCR and Western blotting respectively.
5.4.2 Preparation of the target cells: splenocytes from primed mice (1×107 /well) were cultured with the recombinant Loop protein (30μg/ml) and recombinant mouse IL-2 (10 ng/ml) in DMEM medium for 7days in 6 -well tissue culture plates.
5.4.3 CTL activity assay: The restimulated splenocytes were effectors. Transfectant CT26 cells (transfected with plasmid pVAX1-CpG-Loop) were used as target cells. The mean percentage of specific lysis in triplicate wells was calculated as follows: % specific lysis = [(experimental release-spontaneous release)/(maximum release-spontaneous release)]×100.
6 Statistical analysis
Unless noted, Comparisons between two groups were performed by two-tailed Student’s t-test. A p-value < 0.05 was considered as significant.
Results
1 The recombinant plasmid pVAX1-CpG-Loop extracted abundantly by alkaline lysis was identified by double enzyme digestion. The electrophoretic band about 860 bp could be observe under ultraviolet light about 1.2﹪agarose gel electrophoresis. The purity and concentration of the extracted recombinant plasmid detected by grating spectrophotometer demenstrated that the ratio of OD260/OD280 was between 1.8 and 2.0. Western blot analysis indicated that the recombinant protein could be recognized by His-specific McAb; The purity of Loop protein was about 95% after purification with Ni-NTA affinity chromatography column;
2 Higher levels of Ag-specific IgG were induced by coimmunization with adjuvants and DNA vaccine. In the experiment2 and 3, in contrast to the pVAX1-CpG-Loop (25 or 100μg/ mouse) group, higher levels of IgG were elicited in DNA vaccine plus adjuvants groups (all p<0.05). However, no significant differences of the levels of IgG in DNA vaccine plus different adjuvants were observed (p>0.05).
3 Splenocytes from immunized mice with pVAX1-CpG-Loop plus adjuvants showed markedly higher proliferative response than those from immunized with pVAX1-CpG-Loop DNA vaccine solely in experiment 2 and in experiment 3 (all p<0.05) . However, the mice showed no porliferative response in experiment 1(all p>0.05) (Fig.2C).
4 11-29% of the splenic CD8+ T cells from mice that in the experiment 2 produced IFN-γ. There were significant differences between control and coimmunization groups, all p < 0.05. Whereas only 1.86-2.87% of the splenic CD8+ T cells from mice vaccinated with the DNA vaccine plus adjuvant produced IFN-γin the experiment 3. Compared with control, all p < 0.05. But only 0.26-0.58℅ of the spleen CD8+ T cells from mice in the experiment 1 produced IFN-γ, all p < 0.05.
5 Significantly higher CTL responses to the Loop protein were generated in mice in the experiment 2. In experiment 3, the mice vaccinated with a higher dose (100μg) of DNA vaccine plus adjuvants induced weaker CTL responses than that in mice in the experiment 2. Significantly lower CTL responses were generated in mice in the experiment 1 than that in the experiment 3. These results further revealed that the lower dose (25μg/ mouse) of DDA plus pVAX1-CpG-Loop DNA vaccine generated significantly higher CTL response than the higher dose (250μg/ mouse) of DDA coinjecting DNA vaccine in mice in the experiment 2.
Conclusions:
1 The higher level of IgG was elicited by the lower dose of DNA vaccine and the adjuvants in experiment 2. when the vaccination frequency was added, our data indicated that the adjuvants had higher efficency in humoral immune responses even on immunization with the lower dose of pVAX1-CpG-Loop.
2 The DNA vaccine plus adjuvant eliciting significantly higher level of T-cell proliferation and CTL response than the control group in experiment2 and 3. The data indicated that higher frequency of CD8+ T cells and higher level IFN-γproduction appeared in experiment2 and 3.
3 These studies showed that DDA, CpG-ODN and LipofectamineTM 2000 had corresponding adjuvant effect and the augmented effect on DNA vaccine with CpG motif were observed. The strategy that priming with the lower dose DNA vaccine coadministered with adjuvant and boosting with the lower dose DNA vaccine only is the most potent vaccination regimen known to date for DNA vaccine (pVAX1-CpG-Loop) to induce antiviral response.
CpG寡聚脱氧核苷酸(CpG-ODN)中的CpG基序能够刺激B细胞,T细胞,自然杀伤细胞和抗原提呈细胞发生免疫级联放大反应。CpG-ODN可以诱发机体产生Th1型免疫反应并能诱导致炎因子的产生。在体内,骨髓来源的免疫细胞包括单核细胞,巨噬细胞和骨髓来源的树突细胞均表达TLR9受体,TLR9受体对CpG基序作出反应。溴化二甲基双十八铵(DDA)是一种阳离子脂质,作为一种佐剂它可以吸附抗原到它的表面,能增强IFN-γ的分泌和诱发免疫保护反应,并能增强抗原在体内诱导的体液和细胞免疫反应。DDA的作用类似于微胶囊,缓慢释放包裹的质粒载体,从而达到长时间表达抗原的作用,因此在机体抵抗多种疾病中发挥重要作用。Lipofectamine TM 2000是一种阳离子聚合物,带净正电荷的聚合物与带负电荷的DNA之间相互作用,自发形成了脂质-DNA复合物。脂质体-DNA复合物能与细胞表膜和细胞内膜直接融合或通过内吞作用进入细胞。脂质扩散入靶细胞膜的过程,破坏了膜与DNA间的相互作用,从而使DNA进入细胞浆。作为佐剂Lipofectamine TM 2000可以诱导机体产生Th1反应,已被广泛应用于增强DNA疫苗免疫效果的研究。本室构建的犬传染性肝炎病毒核酸疫苗pVAX1-CpG-Loop免疫小鼠诱导了体液免疫和细胞免疫的产生。
为进一步提高pVAX1-CpG-Loop免疫效果,寻求最佳适配佐剂,本次实验是在本室对核酸疫苗pVAX1-CpG-Loop的研究基础上,用佐剂CpG-ODN,DDA和Lipofectamine TM 2000与质粒pVAX1-CpG-Loop共同免疫小鼠,并检测其诱导小鼠产生的细胞及体液免疫效果。
方法:
1质粒的大量提取
采用SDS-碱裂解法大量提取重组质粒pVAX1–CpG -Loop,采用聚乙二醇沉淀法纯化质粒。并对提取的重组质粒pVAX1-CpG-Loop经分光光度计进行纯度和浓度测定。将原核表达质粒pET28a(+)Loop转化大肠杆菌BL21(DE3),摇菌并诱导重组Loop蛋白的表达, Western blot鉴定。用尿素溶解沉淀,采用镍离子亲和层析柱纯化重组蛋白,透析并复性后浓缩目的蛋白。
2免疫佐剂的准备
CpG-ODN 1826序列为5′TCCATGACGTTC CTGACG TT3′非甲基化,全硫代修饰,PAGE纯化。- 20℃保存备用。PBS溶解并与质粒pVAX1-CpG-Loop混匀后备用。
DDA溶于pH7.4的10mM Tris-Cl,终浓度为1.25 mg/ml,加热80℃,20分钟,并反复颠倒混匀,冷却到室温与质粒pVAX1-CpG-Loop混合后备用。
Lipofectamine TM 2000 40μl/只与质粒pVAX1-CpG-Loop混合后备用。
3小鼠的免疫方案:
方案1:6-8周龄雌性BALB/c小鼠随机分为6组各5只:
①对照组:pVAX1-CpG-Loop 25μg/只。
②CpG-ODN高剂量组: CpG-ODN 50μg/只+质粒pVAX1-CpG-Loop 25μg/只。
③CpG-ODN低剂量组: CpG-ODN 20μg/只+质粒pVAX1-Cp G-Loop 25μg/只。
④LipofectamineTM 2000组: Lipofectamine TM 2000 40ul/只+质粒pVAX1-CpG-Loop 25μg/只。
⑤DDA高剂量组:DDA 250μg/只+质粒pVAX1-CpG-Loop 25μg/只。
⑥DDA低剂量组:DDA 25μg/只+质粒pVAX1-CpG-Loop 25μg/只。
小鼠左右股四头肌注射重组质粒pVAX1-CpG-Loop并混有相应佐剂,两周免疫一次,共免疫三次,末次免疫两周后,无菌取脾,进行T淋巴细胞特异性增殖分析,流式细胞术检测IFN-γ,蛋白特异性CTL活性分析。
方案2:分组同方案1。小鼠左右股四头肌注射重组质粒pVAX1-CpG-Loop并混有相应佐剂,两周免疫一次,免疫三次后,每组小鼠分别于第十三,十五,十八周用pVAX1-CpG-Loop (25μg/只)加强免疫三次,末次免疫后两周,无菌取脾,进行T淋巴细胞特异性增殖分析,流式细胞术IFN-γ检测,蛋白特异性CTL活性分析检测。免疫后每周尾尖采血,分离血清,ELISA测定血清抗体效价。
方案3:6-8周龄雌性BALB/c小鼠随机分为5组每组各5只:
①对照组:pVAX1-CpG-Loop 100μg/只。
②CpG-ODN高剂量组: CpG-ODN 50μg/只+质粒pVAX1-CpG-Loop 100μg/只。
③CpG-ODN低剂量组:CpG-ODN 20μg/只+质粒pVAX1 -CpG-Loop 100μg/只。
④Lipofectamine TM 2000组:Lipofectamine TM 2000 40ul/只+质粒pVAX1-CpG-Loop 100μg/只。
⑤DDA组:DDA 250μg/只+质粒pVAX1-CpG-Loop 100μg/只。
小鼠左右股四头肌注射重组质粒pVAX1-CpG-Loop (100μg/只)并混有相应佐剂,两周免疫一次,共免疫三次,末次免疫两周后,无菌取脾,进行T淋巴细胞特异性增殖分析,流式细胞术检测IFN-γ,蛋白特异性CTL活性分析。免疫后每周尾尖采血,分离血清,ELISA测定血清抗体效价。
4免疫效果评价
4.1 ELISA测定血清抗体效价免疫后每周尾尖采血,分离血清,以犬传染性肝炎病毒抗原每孔100μg/ml包被酶标板,4℃过夜。洗涤封闭后加倍比稀释的小鼠血清100μl,孵育60min后洗涤加HRP标记的二抗孵育60min,OPD显色。酶标仪492nm波长处测吸光度值
4.2 T淋巴细胞特异性增殖分析采用MTT方法检测免疫小鼠的T淋巴细胞增殖活性。
4.3流式细胞术检测IFN-γ的分泌。加入Loop蛋白30μg/ml和10ng/ml interleukin-2孵育72h。收集细胞,加入荧光标记的抗体CD8-PE,IFN-γ-FITC,固定过夜,用流式细胞仪检测。
4.4蛋白特异性CTL活性分析采用LDH释放法。
4.4.1效应细胞制备:调整脾淋巴细胞密度至1×107 /ml ,脾淋巴细胞悬液2ml/孔加入6孔板,加入Loop蛋白30μg/ml和10 ng/ml interleukin-2孵育7天,于第7d收集细胞作为效应细胞,进行特异性杀伤活性测定。
4.4.2靶细胞的制备:胰酶消化CT26细胞,传代于12孔细胞培养板中,次日进行转染。无血清DMEM稀释的质粒(pVAX1-CpG和pVAX1-CpG-loop)分别与无血清DMEM稀释的Lipofectamine TM 2000混匀并在室温下静置20分钟。每个孔加混合液200μl, CO2培养箱,37℃5﹪CO2培养48小时。
4.4.3效应细胞对靶细胞的杀伤活性的检测:上述转染细胞混合作为靶细胞,效应细胞:靶细胞混合比例为100:1, 50:1, 25:1。同时设靶细胞自发释放孔和靶细胞最大释放孔。测492nm波长的吸光值,按下面的公式计算CTL对靶细胞的杀伤活性:实验组释放量-自发释放量最大释放量-自发释放量
结果:
1碱裂解法大量提取的重组质粒pVAX1-CpG-Loop经双酶切和1.2﹪琼脂糖凝胶电泳鉴定,显示860bp左右的×100%电泳条带。所得重组质粒OD260/OD280在1.8–2.0之间。Western blot结果显示诱导表达蛋白与抗六聚组氨酸抗体特异性结合。诱导表达的Loop蛋白经镍离子亲和层析纯化,纯度可达95%以上。
2将重组质粒pVAX1-CpG-Loop与不同免疫佐剂混合后,免疫小鼠,间接ELISA检测显示:各实验组小鼠所产生的抗体效价明显高于pVAX1-CpG-Loop对照组(P<0.05),各组之间无统计学差异(P>0.05)。
3 T淋巴细胞特异性增殖分析,方案2和方案3的小鼠检测到T淋巴细胞的增殖活性明显高于同组的pVAX1-CpG-Loop对照组(P<0.05)。方案1的小鼠无明显增殖活性(P>0.05)。pVAX1-CpG-Loop与CpG-ODN(20μg/只)共免疫组的小鼠脾淋巴细胞的增殖活性明显高于其他实验组。
4免疫小鼠脾淋巴细胞经流式细胞分析,方案2和方案3的小鼠CD8+T细胞被标记IFN-γ的抗体数量高于同组的pVAX1-CpG-Loop对照组(P<0.05)。方案2免疫的小鼠CD8+T细胞被标记IFN-γ的抗体数量高于方案3免疫的小鼠。方案3的小鼠CD8+T细胞被标记IFN-γ的抗体数量高于方案1免疫小鼠。
5蛋白特异性CTL活性分析结果显示,各实验组小鼠LDH释放率高于pVAX1-CpG-Loop对照组(P<0.05)。方案2免疫小鼠LDH释放率高于方案3免疫小鼠。方案3免疫小鼠LDH释放率高于方案1免疫小鼠。方案2免疫小鼠(25μg/只) DDA与pVAX1-CpG-Loop共免疫组小鼠LDH释放率高于(250μg/只)DDA与pVAX1-CpG-Loop共免疫组小鼠。
结论:
1 ELISA检测:各免疫组小鼠均产生了针对抗原病毒的特异性IgG抗体,且pVAX1-CpG-Loop 100μg/只+ CpG -ODN 20μg/只,pVAX1-CpG-Loop 100μg/只+DDA 250μg/只免疫组的抗体水平明显高于其它实验组。以上结果提示重组质粒pVAX1-CpG-Loop诱导小鼠产生了体液免疫应答,免疫佐剂可有效地增强DNA疫苗诱导的体液免疫应答。
2流式细胞分析显示:各实验组小鼠的CD8+T细胞被标记IFN-γ的抗体数量高于pVAX1-CpG-Loop对照组;采用MTT方法检测小鼠脾脏的淋巴细胞增殖活性,实验组小鼠淋巴细胞增殖活性高于pVAX1-CpG-Loop对照组;实验组小鼠LDH释放率高于pVAX1-CpG-Loop对照组(P<0.05)。以上结果提示免疫佐剂能增强重组质粒pVAX1-CpG-Loop诱导小鼠产生特异性的细胞免疫应答。
3 DDA,CPG-ODN,Lipofectamine TM 2000三种免疫佐剂均能增强DNA疫苗pVAX1-CpG-Loop对小鼠的体液免疫和细胞免疫。方案2免疫的小鼠能够产生对Loop蛋白特异性免疫记忆。证明免疫佐剂与低剂量DNA疫苗共免疫结合低剂量DNA疫苗加强免疫的策略是一种非常有效和有潜力的免疫策略。
Objective Nucleic acid vaccine is a new vaccine technique following inactivated vaccines and attenuated vaccines in 1990s. Nucleic acid vaccine is capable of eliciting both cellular and humoral immune responses more complete than conventional vaccination and have the advantage of mass-production easily. It has been actively developed in the past few years against a variety of infectious agents including virus, bacteria and parasites. Beause of it’s small antigen molecule,higher level of purification, short of immunogenicity,the effective adjuvant was needed to strengthen the immune response that induced by DNA vaccine. Efficient adjuvant is beneficial for DNA vaccine to enhance sufficient level of protection and long-lasting immunity.
Synthetic oligodeoxynucleotides (ODNs) that contain immunostimulatory CpG motifs trigger an immunomodulatory cascade that involves B and T cells, natural killer cells and professional antigen-presenting cells. The response to CpG-ODNs skews the host’s immune milieu in favour of T helper 1 (Th1)-cell responses. In mice, immune cells of the myeloid lineage including monocytes, macrophages and myeloid DCs express TLR9 and respond to CpG stimulation.
The cationic surfactant dimethyl dioctadecyl ammonium bromide (DDA) is a novel adjuvant, DDA can adsorb most antigens on their surface and promote a strong cell mediated immune response and a humoral immune response in vivo, which is essential for the induction of protective immunity against most diseases.
Lipofectamine TM 2000 is a cationic lipid, liposome has been widely used to enhance DNA vaccines and preferentially activate Th1 responses. in vitro, the lipide and DNA compounds can fuse with the celluar membrance and help the DNA come into the cytolymph .
In previous studies, we have constructed the DNA vaccine (pVAX1-CpG-Loop) that encoding two major Loop DNA fragments from CAV-1 and it has induced significantly cell mediated and humoral immune responses. In order to get the better immune efficacy in mice, especially induce a more durable T-cell response, we compared the effect of the three adjuvants on pVAX1-CpG-Loop DNA vaccine. This study evaluated the adjuvant effects of CpG-ODN, DDA, and LipofectamineTM 2000 on a DNA vaccine pVAX1-CpG-Loop, which was against canine adenovirus type-1. Selecting the appropriate adjuvant to specific antigenic components will not only enhance the level of immune response but also determine the type of immune response induced.
Methods
1 Plasmid isolation and purification
The plasmid pVAX1-CpG-Loop was isolated by the method of alkaline lysis, and purified by polyethylene glycol precipitation. The fusion protein was purified by Ni-NTA affinity chromatography column.
2 preparation of adjuvants
The CpG-ODN (1826) sequence is 5'-TCCATGACGTTCCTGACGTT-3', which was produced with DNase-protected phosphorothioate bonds backbone. CpG-ODN were dissolved in a sterile phosphate buffered saline solution and stored at -20℃.
DDA adjuvant was prepared as described previously. Briefly, DDA adjuvant was mixed into 10 mM Tris-buffer at pH 7.4 to a concentration of 1.25 mg/ml, heated to 80℃for 20 min with intermittent shaking, then cooled to room temperature.
3 Mice
BALB/c mice were bred and cared in the Animal Facilities of Hebei Medical University, Shijiazhuang. Only female mice ages 6-8 weeks old were used for the experiments.
4 Immunization protocols
Experiment 1: female mice were randomly divided into 6 experimental groups (5 mice /group). The detailed vaccination scheme were as followed. Biweekly mice were injected intramuscularly with a volume of 100μl/mouse for three times. After two weeks, the splenocytes (5 mice / group) were harvested. The Intracellular staining of IFN-γ,In vitro CTL activity assay and The lymphocyte proliferation assay was performed.
Group1. pVAX1-CpG-Loop 25μg/mouse
Group2.pVAX1-CpG-Loop25μg/mouse+CpG-ODN50μg/mous-e
Group3.pVAX1-CpG-Loop25μg/mouse+CpG-ODN20μg/mous-e
Group4.pVAX1-CpG-Loop25μg/mouse+LipofectamineTM 2000 (Invitrogen USA)40μl/mouse
Group5.pVAX1-CpG-Loop 25μg/ mouse +DDA 250μg/mouse
Group6.pVAX1-CpG-Loop 25μg/ mouse +DDA 25μg/mouse
Experiment 2: The grouping project same as Experiment 1. Biweekly mice were injected intramuscularly with a volume of 100μl/mouse for three times. All the groups were re-injected with pVAX1-CpG-Loop (25μg/mouse) at thirteenth, fifteenth and eighteenth week respectively. two weeks later after the final immunization, the splenocytes were harvested. The Intracellular staining of IFN-γ, In vitro CTL activity assay and The lymphocyte proliferation assay was performed.
Experiment 3: The mice were injected intramuscularly (100μg/100μl/mouse, 5 mice /group) with the DNA vaccine and the adjuvants at a 2-week interval for three times. After two weeks, the splenocytes were harvested. The Intracellular staining of IFN-γ, In vitro CTL activity assay and The lymphocyte proliferation assay was performed.
Group1.pVAX1-CpG-Loop100μg/mouse+CpG-ODN50μg/mo- use
Group2.pVAX1-CpG-Loop100μg/mouse+CpG-ODN20μg/mo-use
Group3.pVAX1-CpG-Loop 100μg/ mouse + LipofectamineTM 2000 40μl/ mouse
Group4.pVAX1-CpG-Loop100μg/ mouse +DDA250μg/ mouse
Group5. pVAX1-CpG-Loop 100μg/ mouse
5 The evaluation of the immuned mice
5.1 Enzyme-linked immunosorbent assay
Sera of immunized mice were collected and specific IgG antibody was detected by the method of indirect enzyme linked immunosorbent assay (ELISA) as described previously . Briefly, the dilution of serum sample was 1:40, purifed CAV-1 was used as antigen and specific anti-virus IgG antibody was detected. Absorbance was determined at 492 nm.
5.2 The Loop protein specific lymphocyte proliferation assay
OD 570 was measured by a standard 3-(4,5dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) method. The stimulation index was calculated from the formula: stimulation index (SI) = (Arestimulated -Ablank)/(Aunrestimulated-Ablank)
5.3 Intracellular staining of IFN-γ
1×107 spleen cells from immunized mice were cultured in 0.5 ml of complete DMEM in the presence of the recombinant Loop protein 30μg/ml and 10 ng/ml of interleukin-2 for 72h. The stimulated lymphocytes were harvested and labeled for surface CD8 and intracellular IFN-γusing PE (Jingmei Biotech) or FITC (BioLegend) labeled specific monoclonal antibodies following the detailed instructions provided by the vendor. Spleen cells were analyzed by flow cytometry.
5.4 The Loop protein specific CTL activity assay
5.4.1 preparation of the effectors :The cell line CT26 was transfected with plasmid pVAX1-CpG-Loop or pVAX1-CpG vector with Lipofectamine 2000 according to the manufacturer’s direction as the method described previously. After 48 h incubation at 37℃, 5% CO2, CT26 cells were collected and the transfection products were analysed by RT-PCR and Western blotting respectively.
5.4.2 Preparation of the target cells: splenocytes from primed mice (1×107 /well) were cultured with the recombinant Loop protein (30μg/ml) and recombinant mouse IL-2 (10 ng/ml) in DMEM medium for 7days in 6 -well tissue culture plates.
5.4.3 CTL activity assay: The restimulated splenocytes were effectors. Transfectant CT26 cells (transfected with plasmid pVAX1-CpG-Loop) were used as target cells. The mean percentage of specific lysis in triplicate wells was calculated as follows: % specific lysis = [(experimental release-spontaneous release)/(maximum release-spontaneous release)]×100.
6 Statistical analysis
Unless noted, Comparisons between two groups were performed by two-tailed Student’s t-test. A p-value < 0.05 was considered as significant.
Results
1 The recombinant plasmid pVAX1-CpG-Loop extracted abundantly by alkaline lysis was identified by double enzyme digestion. The electrophoretic band about 860 bp could be observe under ultraviolet light about 1.2﹪agarose gel electrophoresis. The purity and concentration of the extracted recombinant plasmid detected by grating spectrophotometer demenstrated that the ratio of OD260/OD280 was between 1.8 and 2.0. Western blot analysis indicated that the recombinant protein could be recognized by His-specific McAb; The purity of Loop protein was about 95% after purification with Ni-NTA affinity chromatography column;
2 Higher levels of Ag-specific IgG were induced by coimmunization with adjuvants and DNA vaccine. In the experiment2 and 3, in contrast to the pVAX1-CpG-Loop (25 or 100μg/ mouse) group, higher levels of IgG were elicited in DNA vaccine plus adjuvants groups (all p<0.05). However, no significant differences of the levels of IgG in DNA vaccine plus different adjuvants were observed (p>0.05).
3 Splenocytes from immunized mice with pVAX1-CpG-Loop plus adjuvants showed markedly higher proliferative response than those from immunized with pVAX1-CpG-Loop DNA vaccine solely in experiment 2 and in experiment 3 (all p<0.05) . However, the mice showed no porliferative response in experiment 1(all p>0.05) (Fig.2C).
4 11-29% of the splenic CD8+ T cells from mice that in the experiment 2 produced IFN-γ. There were significant differences between control and coimmunization groups, all p < 0.05. Whereas only 1.86-2.87% of the splenic CD8+ T cells from mice vaccinated with the DNA vaccine plus adjuvant produced IFN-γin the experiment 3. Compared with control, all p < 0.05. But only 0.26-0.58℅ of the spleen CD8+ T cells from mice in the experiment 1 produced IFN-γ, all p < 0.05.
5 Significantly higher CTL responses to the Loop protein were generated in mice in the experiment 2. In experiment 3, the mice vaccinated with a higher dose (100μg) of DNA vaccine plus adjuvants induced weaker CTL responses than that in mice in the experiment 2. Significantly lower CTL responses were generated in mice in the experiment 1 than that in the experiment 3. These results further revealed that the lower dose (25μg/ mouse) of DDA plus pVAX1-CpG-Loop DNA vaccine generated significantly higher CTL response than the higher dose (250μg/ mouse) of DDA coinjecting DNA vaccine in mice in the experiment 2.
Conclusions:
1 The higher level of IgG was elicited by the lower dose of DNA vaccine and the adjuvants in experiment 2. when the vaccination frequency was added, our data indicated that the adjuvants had higher efficency in humoral immune responses even on immunization with the lower dose of pVAX1-CpG-Loop.
2 The DNA vaccine plus adjuvant eliciting significantly higher level of T-cell proliferation and CTL response than the control group in experiment2 and 3. The data indicated that higher frequency of CD8+ T cells and higher level IFN-γproduction appeared in experiment2 and 3.
3 These studies showed that DDA, CpG-ODN and LipofectamineTM 2000 had corresponding adjuvant effect and the augmented effect on DNA vaccine with CpG motif were observed. The strategy that priming with the lower dose DNA vaccine coadministered with adjuvant and boosting with the lower dose DNA vaccine only is the most potent vaccination regimen known to date for DNA vaccine (pVAX1-CpG-Loop) to induce antiviral response.
引文
1 Tomoaki Yoshikawa, Susumu Imazu, Jianqing Gao, et al Non-methylated CpG motif packaged into fusogenic liposomes enhance antigen-specific immunity in Mice. Biol. Pharm. Bull, 2006, 29:105-109
2 张悦,戚晓红,冯振卿 免疫佐剂的研究进展. 中国血吸虫病防治杂志2004, 年第16, 卷第3期
3 Hunter RL. Overview of vaccine adjuvants: present and future [J].Vaccine, 2002, 20:S7-S12
4 Dennis Christensen, Camilla Foged, Ida Rosenkrands, et alTrehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying. Biochimica et al Biophysica Acta, 2007, 1768:2120–2129
5 Ha Jung Roh, Haan Woo Sung, Hyuk Moo Kwon. Effects of DDA, CpG-ODN, and plasmid-encoded chicken FN-γ on protective immunity by a DNA vaccine against IBDV in chickens. J. Vet. Sci, 2006, 7(4): 361–368
6 Dennis M. Klinman. Immunotherapeutic uses of CpG oli godeoxynucleotides. Nature reviews Immunology 2004, 4:1–10
7 Krieg AM, Wagner H. Causing a commotion in the blood: Immuno-therapy progress from bacterial to bacterial DNA. Immunol Today, 2000, 21 (10): 521-526
8 Tazio Storni, Christiane Ruedl, Katrin Schwarz, et al Non -methylated CG Motifs Packaged into Virus-Like Particles Induce Protective Cytotoxic T Cell Responses in the Absence of Systemic Side Effects. The Journal of Immunology, 2004, 172: 1777–1785
9 Isabelle Miconnet, Sylvain Koenig, Daniel Speiser, et al CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide. The Journal of Immunology, 2002, 168:1212–1218
10 Lindblad E B, Elhay M J, Silva R, et al. Adjuvant modulation of immune responses to tuberculosis subunit vaccines.Infect Immun,1997,65: 623-629
11 Cai H, Tian X, Hu X D, et al. Combined DNA vaccinesformutated either in DDA or in saline enhances protectivenity against tuberculosis. DNA and cell biology, 2004, 23:450-456
12 Zhu X, Venkataprasad N, Thangaraj H S, et al Functions and specificity of T cells following nucleic acid vaccination of mice against Mycobacterin tuberculosis infection. J Immunol, 1997, 158:5921-5926
13 Vogel I R. Improving vaccine performance with adjuvants. Clin.Infect Dis, 2000, 30:s266-270
14 Ho-Jen Peng, Lai-Chen Tsai, Song-Nan Su, et al Comparison of different adjuvants of protein and DNA vaccination for the prophylaxis of IgE antibody formation. Vaccine,2004,22:755–761
15 Klovinsks L.S. Cao L, Barnfield C. Mucosal immunization with DNA-liposome complexes. 〔J〕Vaccine, 1997, 15:818
16 McCluskie M J, Chu Y, Xia J L. Direct gene transfer to the respiratory tract of mice with pure plasmid and liquid -formμlated DNA〔J〕. Antisense Nucleic Acid Drg, 1998, 8:401
17 Barron L G, Uyechi L S, Szoka F C Jr. Cationic lipids are essential forgene delivery mediated by intravenous admini -stration of lipoplexes.〔J〕.Gene Ther,1999,6:1179
18 Junxia Wang, Long Zheng, Shuxia Song, et al Augmented humoral and cellular immune responses induced by canine adenovirus type 1 DNA vaccine in BALB/c mice. Viral Immunology, 2007, 20:461–468
19 S′ebastien Cornet, Jeanne Menez-Jamet, Franc?ois Lemonnier. CpG oligodeoxynucleotides activate dendritic cells in vivo and induce a functional and protective vaccine immunity against a TERT derived modified cryptic MHC class I-restricted epitope. Vaccine, 2006, 24: 1880–1888
20 Jesper Davidsen, Ida Rosenkrands, Dennis Christensen, et al Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6V-dibehenate)—A novel adjuvant inducing both strong CMI and antibody responses. Biochimica et Biophysica Acta, 2005, 1718:22 – 31
21 D.N. Wedlock, D.L. Keen, A.R. McCarthy, et al Effect of different adjuvants on the immune responses of cattle vaccinated with Mycobacterium tuberculosis culture filtrate proteins. Veterinary Immunology and Immunopathology, 2002, 86:79–88
22 Anil Vangala, Vincent W. Bramwell, Sarah McNeil, et al Comparison of vesicle based antigen delivery systems for delivery of hepatitis B surface antigen. Journal of Controlled Release, 2007, 119:102–110
23 Shuxia Song, Yue Wang, Yan Zhang, et al. Augmented induction of CD8+ cytotoxic T-cell response and antitumor effect by DCs pulsed with virus-like particles packaging with CpG. Cancer Letters, 2007, 256:90–100
24 Shuxia Song, Fang Wang, Xiaowen He, et al. Evaluation ofantitumor immunity efficacy of epitope-based vaccine with B16 cell line coexpressing HLA-A2/H-2kb and CTL multiepitope in HLA transgenic mice. Vaccine, 2007, 25:4853–4860
25 He Xiao-wen, Sun Shu-han, Hu Zhen-lin, et al Augmen -ted humoral and cellular immune responses of a hepatitis B DNA vaccine encoding HBsAg by protein boosting. Vaccine, 2005, 23:1649-1656
26 Rico MA, Quiroga JA, Subira D, et al. Hepatitis B rus–specific T-cell proliferation and cytokine secretion in chronic hepatitis B e antibody–positive patients treated with ribavirin and interferon alfa. Hepatology, 2001, 33:295-300
27 Katrin Schwarz, Edwin Meijerink, Danie E. Speiser, et al. Efficient homologous prime-boost strategies for T cell vaccination based on virus-like particles. Eur. J. Immunol. 2005, 35: 816–821
28 Davila E, Kenedy R, cells E. Generation of antitumor immunity by cytotoxic T lymphocyte epitope peptide vaccination, CpG oligodeoxynucleotide adjuvant, and CTLA-4 blochade. Cancer Res, 2003, 63:3281–8
29 Christine Klinguer-Hamour, Christine Libon, Hélène Plotnicky-Gilquin, et al. DDA adjuvant induces a mixed Th1/Th2 immune response when associated with BBG2Na, a respiratory syncytial virus potential vaccine. Vaccine, 2002, 20:2743–2751
30 Mahmoud R. Jaafari, Ali Badiee, Ali Khamesipour, et al. The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome. Vaccine, 2007, 25: 6107–6117
31 孙树汉 主编,核酸疫苗。上海:第二军医大学出版社,2000.1
32 熊盛,王一飞 BALB/C小鼠对不同佐剂SARS灭活疫苗的早期免疫反应,中国免疫学杂志, 2004,年第20卷第6期
1 邢钊. 兽医生物制品实用技术[J ] . 北京:首都经济贸易大学出版社,1999. 57～63
2 Cox JC, Coulter AR. A djuvants2a classification and review of their modes of action [J]. V accine, 1997, 15 (3) : 248- 256
3 Lim Y S, Kang B Y, Kim E J, et al. Potentiation of antigen- specific, Th1 immune responses bymultiple DNA vaccination with an walbumin / interferon-γ hybrid construct [ J ]. Immunol, 1998, 94:135-141
4 Xiang Z Q, Erd H C J. Manipulation of the immune response to a p lasmid-encoded virul atigen by coinocultion with plas -mids expressing cytokines [J]. Immuntiy, 1995, 2: 129-135
5 Hu J, CladelN M, Wang Z, et al. GM-CSF enhances protective immunity to cottontail rabbit papillomavirus E8 genetic vaccination inrabbits [J]. Vaccine, 2004, 22 (9210): 1124-1130
6 Tsuji T, Fukushima J, Hamajima K, et al. HIV-l specific cell-medit-ed immunity is enhanced by coinoculation of TCA3 exp ression p lasmidwith DNA vaccine [J]. Immunol, 1997, 90: 126
7 Deliyannis G, Boyle J S, Brady J L, et al. A fusion DNA vaccine that targets antigen2p resenting cells increases p rotection from viral challenge [J]. Proc Acad Sci USA, 2000,97 (12): 6676-6680
8 DeposeyW, AllisonM E, Akkaraju S, et al. C3d of complement as molecular adjuvant: bridging inate and acquired immunity [J]. Sci2ence, 1996, 271: 348-350
9 Ross TM, Xu Y, Rick A, et al. C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge [J]. Nature Immunol,2000,1(2): 127-131
10 Zhang M, Ishii K, Hisaeda H, et al. Ubiquitin-fusion degradation pathway plays an indispensable role in naked DNA caccination with a chimeric gene encoding a syngeneic cytotxic T lymphocyte epitope of melanocyte and green fluorescent protein. Immunology, 2004,112(4):567-74
11 Andrew M. Lew, Jamie L. Brady, Je.erey S. Boyle Site-directed immune responses in DNA vaccines encoding ligand±antigen fusions Vaccine 18 (2000) 1681±1685
12 Gregoriadis G, Bacon A, Wilson CW, et al A role for liposomes in genetic vaccination [J]. Vaccine, 2002, 20 (Suppl 5) : 72-91
13 Singh M,Briones M,Ott C,et al. Cationic potent delivery system for DNA vaccines [J]. micmparticles:a Prnc Nat1 Acad Sci USA,2000,97 (2):811-81
14 Ott C,Si ngh M,Kazzaz J,et al. A cationic sulmicmn emulsion (MF59/DOTAP) is an effective delivery system for DNA vaccines[J].J Controlled Release ,2002 ,79 (1-3):1-5
15 Gregoriadis G, Saffie R, Souza J B. Liposome-mediated DNA vaccination [J]. FEBS Letters, 1997, 402 (223) : 107-1101
16 Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor Recognizes bacterial DNA [J]. Nature,2000,408(68 13):740-745
17 Krieg AM, Yi A, Matson S, et al. CpGmotifs in bacterial DNA trigger direct B-cell activation. Nature 199 5,374 :546-549
18 Balla ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpGmotifs in oligo –deoxynucleotides and bacterial DNA. Immuno l ,1996 ,157 :18-40
19 Penna A, Del Prete G, Cavalli A, et al. Predominant T-helper 1 cytokine profile of hepatitis B virus nucleocapsid -specific T cells in cacuteself-limited hepatitis B. Hepa -tology ,1997 ,25 :10-22
20 姜荣龙,卢桥生,侯金林,等. 辅助性T 细胞极化群体在慢性 乙 型 肝 炎 病 毒 感 染 中 的 作 用 . 中 华 医 学 杂志,2000 ,80 :741
2 张悦,戚晓红,冯振卿 免疫佐剂的研究进展. 中国血吸虫病防治杂志2004, 年第16, 卷第3期
3 Hunter RL. Overview of vaccine adjuvants: present and future [J].Vaccine, 2002, 20:S7-S12
4 Dennis Christensen, Camilla Foged, Ida Rosenkrands, et alTrehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying. Biochimica et al Biophysica Acta, 2007, 1768:2120–2129
5 Ha Jung Roh, Haan Woo Sung, Hyuk Moo Kwon. Effects of DDA, CpG-ODN, and plasmid-encoded chicken FN-γ on protective immunity by a DNA vaccine against IBDV in chickens. J. Vet. Sci, 2006, 7(4): 361–368
6 Dennis M. Klinman. Immunotherapeutic uses of CpG oli godeoxynucleotides. Nature reviews Immunology 2004, 4:1–10
7 Krieg AM, Wagner H. Causing a commotion in the blood: Immuno-therapy progress from bacterial to bacterial DNA. Immunol Today, 2000, 21 (10): 521-526
8 Tazio Storni, Christiane Ruedl, Katrin Schwarz, et al Non -methylated CG Motifs Packaged into Virus-Like Particles Induce Protective Cytotoxic T Cell Responses in the Absence of Systemic Side Effects. The Journal of Immunology, 2004, 172: 1777–1785
9 Isabelle Miconnet, Sylvain Koenig, Daniel Speiser, et al CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide. The Journal of Immunology, 2002, 168:1212–1218
10 Lindblad E B, Elhay M J, Silva R, et al. Adjuvant modulation of immune responses to tuberculosis subunit vaccines.Infect Immun,1997,65: 623-629
11 Cai H, Tian X, Hu X D, et al. Combined DNA vaccinesformutated either in DDA or in saline enhances protectivenity against tuberculosis. DNA and cell biology, 2004, 23:450-456
12 Zhu X, Venkataprasad N, Thangaraj H S, et al Functions and specificity of T cells following nucleic acid vaccination of mice against Mycobacterin tuberculosis infection. J Immunol, 1997, 158:5921-5926
13 Vogel I R. Improving vaccine performance with adjuvants. Clin.Infect Dis, 2000, 30:s266-270
14 Ho-Jen Peng, Lai-Chen Tsai, Song-Nan Su, et al Comparison of different adjuvants of protein and DNA vaccination for the prophylaxis of IgE antibody formation. Vaccine,2004,22:755–761
15 Klovinsks L.S. Cao L, Barnfield C. Mucosal immunization with DNA-liposome complexes. 〔J〕Vaccine, 1997, 15:818
16 McCluskie M J, Chu Y, Xia J L. Direct gene transfer to the respiratory tract of mice with pure plasmid and liquid -formμlated DNA〔J〕. Antisense Nucleic Acid Drg, 1998, 8:401
17 Barron L G, Uyechi L S, Szoka F C Jr. Cationic lipids are essential forgene delivery mediated by intravenous admini -stration of lipoplexes.〔J〕.Gene Ther,1999,6:1179
18 Junxia Wang, Long Zheng, Shuxia Song, et al Augmented humoral and cellular immune responses induced by canine adenovirus type 1 DNA vaccine in BALB/c mice. Viral Immunology, 2007, 20:461–468
19 S′ebastien Cornet, Jeanne Menez-Jamet, Franc?ois Lemonnier. CpG oligodeoxynucleotides activate dendritic cells in vivo and induce a functional and protective vaccine immunity against a TERT derived modified cryptic MHC class I-restricted epitope. Vaccine, 2006, 24: 1880–1888
20 Jesper Davidsen, Ida Rosenkrands, Dennis Christensen, et al Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6V-dibehenate)—A novel adjuvant inducing both strong CMI and antibody responses. Biochimica et Biophysica Acta, 2005, 1718:22 – 31
21 D.N. Wedlock, D.L. Keen, A.R. McCarthy, et al Effect of different adjuvants on the immune responses of cattle vaccinated with Mycobacterium tuberculosis culture filtrate proteins. Veterinary Immunology and Immunopathology, 2002, 86:79–88
22 Anil Vangala, Vincent W. Bramwell, Sarah McNeil, et al Comparison of vesicle based antigen delivery systems for delivery of hepatitis B surface antigen. Journal of Controlled Release, 2007, 119:102–110
23 Shuxia Song, Yue Wang, Yan Zhang, et al. Augmented induction of CD8+ cytotoxic T-cell response and antitumor effect by DCs pulsed with virus-like particles packaging with CpG. Cancer Letters, 2007, 256:90–100
24 Shuxia Song, Fang Wang, Xiaowen He, et al. Evaluation ofantitumor immunity efficacy of epitope-based vaccine with B16 cell line coexpressing HLA-A2/H-2kb and CTL multiepitope in HLA transgenic mice. Vaccine, 2007, 25:4853–4860
25 He Xiao-wen, Sun Shu-han, Hu Zhen-lin, et al Augmen -ted humoral and cellular immune responses of a hepatitis B DNA vaccine encoding HBsAg by protein boosting. Vaccine, 2005, 23:1649-1656
26 Rico MA, Quiroga JA, Subira D, et al. Hepatitis B rus–specific T-cell proliferation and cytokine secretion in chronic hepatitis B e antibody–positive patients treated with ribavirin and interferon alfa. Hepatology, 2001, 33:295-300
27 Katrin Schwarz, Edwin Meijerink, Danie E. Speiser, et al. Efficient homologous prime-boost strategies for T cell vaccination based on virus-like particles. Eur. J. Immunol. 2005, 35: 816–821
28 Davila E, Kenedy R, cells E. Generation of antitumor immunity by cytotoxic T lymphocyte epitope peptide vaccination, CpG oligodeoxynucleotide adjuvant, and CTLA-4 blochade. Cancer Res, 2003, 63:3281–8
29 Christine Klinguer-Hamour, Christine Libon, Hélène Plotnicky-Gilquin, et al. DDA adjuvant induces a mixed Th1/Th2 immune response when associated with BBG2Na, a respiratory syncytial virus potential vaccine. Vaccine, 2002, 20:2743–2751
30 Mahmoud R. Jaafari, Ali Badiee, Ali Khamesipour, et al. The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome. Vaccine, 2007, 25: 6107–6117
31 孙树汉 主编,核酸疫苗。上海:第二军医大学出版社,2000.1
32 熊盛,王一飞 BALB/C小鼠对不同佐剂SARS灭活疫苗的早期免疫反应,中国免疫学杂志, 2004,年第20卷第6期
1 邢钊. 兽医生物制品实用技术[J ] . 北京:首都经济贸易大学出版社,1999. 57～63
2 Cox JC, Coulter AR. A djuvants2a classification and review of their modes of action [J]. V accine, 1997, 15 (3) : 248- 256
3 Lim Y S, Kang B Y, Kim E J, et al. Potentiation of antigen- specific, Th1 immune responses bymultiple DNA vaccination with an walbumin / interferon-γ hybrid construct [ J ]. Immunol, 1998, 94:135-141
4 Xiang Z Q, Erd H C J. Manipulation of the immune response to a p lasmid-encoded virul atigen by coinocultion with plas -mids expressing cytokines [J]. Immuntiy, 1995, 2: 129-135
5 Hu J, CladelN M, Wang Z, et al. GM-CSF enhances protective immunity to cottontail rabbit papillomavirus E8 genetic vaccination inrabbits [J]. Vaccine, 2004, 22 (9210): 1124-1130
6 Tsuji T, Fukushima J, Hamajima K, et al. HIV-l specific cell-medit-ed immunity is enhanced by coinoculation of TCA3 exp ression p lasmidwith DNA vaccine [J]. Immunol, 1997, 90: 126
7 Deliyannis G, Boyle J S, Brady J L, et al. A fusion DNA vaccine that targets antigen2p resenting cells increases p rotection from viral challenge [J]. Proc Acad Sci USA, 2000,97 (12): 6676-6680
8 DeposeyW, AllisonM E, Akkaraju S, et al. C3d of complement as molecular adjuvant: bridging inate and acquired immunity [J]. Sci2ence, 1996, 271: 348-350
9 Ross TM, Xu Y, Rick A, et al. C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge [J]. Nature Immunol,2000,1(2): 127-131
10 Zhang M, Ishii K, Hisaeda H, et al. Ubiquitin-fusion degradation pathway plays an indispensable role in naked DNA caccination with a chimeric gene encoding a syngeneic cytotxic T lymphocyte epitope of melanocyte and green fluorescent protein. Immunology, 2004,112(4):567-74
11 Andrew M. Lew, Jamie L. Brady, Je.erey S. Boyle Site-directed immune responses in DNA vaccines encoding ligand±antigen fusions Vaccine 18 (2000) 1681±1685
12 Gregoriadis G, Bacon A, Wilson CW, et al A role for liposomes in genetic vaccination [J]. Vaccine, 2002, 20 (Suppl 5) : 72-91
13 Singh M,Briones M,Ott C,et al. Cationic potent delivery system for DNA vaccines [J]. micmparticles:a Prnc Nat1 Acad Sci USA,2000,97 (2):811-81
14 Ott C,Si ngh M,Kazzaz J,et al. A cationic sulmicmn emulsion (MF59/DOTAP) is an effective delivery system for DNA vaccines[J].J Controlled Release ,2002 ,79 (1-3):1-5
15 Gregoriadis G, Saffie R, Souza J B. Liposome-mediated DNA vaccination [J]. FEBS Letters, 1997, 402 (223) : 107-1101
16 Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor Recognizes bacterial DNA [J]. Nature,2000,408(68 13):740-745
17 Krieg AM, Yi A, Matson S, et al. CpGmotifs in bacterial DNA trigger direct B-cell activation. Nature 199 5,374 :546-549
18 Balla ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpGmotifs in oligo –deoxynucleotides and bacterial DNA. Immuno l ,1996 ,157 :18-40
19 Penna A, Del Prete G, Cavalli A, et al. Predominant T-helper 1 cytokine profile of hepatitis B virus nucleocapsid -specific T cells in cacuteself-limited hepatitis B. Hepa -tology ,1997 ,25 :10-22
20 姜荣龙,卢桥生,侯金林,等. 辅助性T 细胞极化群体在慢性 乙 型 肝 炎 病 毒 感 染 中 的 作 用 . 中 华 医 学 杂志,2000 ,80 :741
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