新型乙型肝炎病毒核心区核酸疫苗的免疫原性研究

目的观察新型乙型肝炎病毒(HBV)核心抗原(HBcAg)核酸疫苗的免疫原性。方法应用新型人体应用载体质粒pSW389l构建HBcAg核酸疫苗(pSW3891／HBc)，对照组和实验组Balb／c小鼠分别以基因枪法免疫对照载体质粒(PSW3891)和HBcAg核酸疫苗，采用酶联免疫吸附试验检测抗HBc，乳酸脱氢酶(LDH)释放测定法检测小鼠HBcAg特异性cTL杀伤活性。结果HBcAg核酸疫苗可在体外293T细胞中高效表达，免疫小鼠后可产生高滴度抗HBc(1：97200)，免疫鼠脾细胞HBc特异性CTL杀伤活性达73．25％。结论新型HBcAg核酸疫苗在Balb／c小鼠实验中表现出良好的体液和细胞免疫原性。     

HBcAg核酸疫苗与白细胞介素表达质粒联合接种小鼠的免疫应答观察

目的观察HBcAg核酸疫苗与鼠白细胞介素12(IL12)和白细胞介素18(IL18)表达质粒联合免疫小鼠所诱导的特异性免疫应答。方法小鼠随机分为载体质粒组、HBcAg核酸疫苗组(核酸疫苗组)、HBcAg核酸疫苗 IL12组(C IL12组)、HBcAg核酸疫苗 IL18组(C IL18组)和HBcAg核酸疫苗 IL12/IL18组(C IL12/IL18组)。载体质粒、HBcAg核酸疫苗、IL12及IL18表达质粒经肌内注射法免疫各组小鼠。采用酶联免疫吸附试验检测小鼠血清HBcAg特异性抗体、IgG亚类(IgG1,IgG2a)以及小鼠脾淋巴细胞培养上清液干扰素(IFN)γ含量。采用乳酸脱氢酶(LDH)释放法检测免疫小鼠特异杀伤性T淋巴细胞(CTL)活性。结果除对照质粒组外,核酸疫苗免疫的各组小鼠均能检出血清抗HBc,C IL12组、C IL18组和C IL12/IL18组的抗HBc终点滴度与C组相比均明显增高(P<0.05)。各组小鼠抗HBcIgG亚类均以IgG2a占优。核酸疫苗免疫组除C IL12 IL18组外,小鼠脾细胞培养上清液IFNγ水平均显著高于对照质粒组(P<0.01)。C IL18组和C IL12/IL18组小鼠脾细胞HBcAg特异性CTL活性强于其他各组。结论IL12和(或)IL18表达质粒与HBcAg核酸疫苗联合免疫,具有增强HBcAg核酸疫苗所激发的免疫应答特别是细胞免疫应答的作用。     

IL-12及HBV基因疫苗共同免疫小鼠的效果

目的观察编码鼠IL-12的真核表达载体对HBV DNA疫苗诱导Balb/c小鼠免疫应答的影响.方法肌肉注射DNA疫苗,ELISA法检测小鼠血清抗HBs,4h51Cr释放法检测小鼠脾细胞CTL杀伤活性.结果免疫8wk后,注射pCR3.1组、注射pCR.1-S组及共注射IL-12真核表达载体的血清A(OD值)分别为0.04±0.01,0.87±0.1及1.67±0.15.CTL细胞杀伤活性分别为10.1%±6.4%,50.5%±6.4%及73.3%±8.8%,后两组与pCR3.1组比较均有显著差异(P＜0.01),后两组比较均有显著差异(P＜0.01).脾细胞悬液经抗CD4+单克隆抗体处理后分别为48.3%±5.9%,75.6%±9.1%,抗CD8+单克隆抗体处理后分别为10.6%±1.4%,16.9%±2.3%.结论 IL-12的真核表达载体能够提高小鼠对DNA疫苗的免疫应答,CTL细胞杀伤活性主要由CD8+执行.基因疫苗可能用于预防及治疗HBV感染.     

恶性疟原虫复合抗原DNA疫苗诱导小鼠的细胞免疫及体液免疫应答

目的 观察用恶性疟原虫复合抗原基因HGFSP构建的DNA疫苗pc-HGFSP免疫小鼠诱导的细胞及体液免疫应答。方法 用pc-HGFSP肌注免疫C57BL／6小鼠并加强免疫2次，取小鼠脾淋巴细胞及血清测定；脾淋巴细胞增殖活性（MTT）法，NK细胞活性和CTL活性（LDH）；脾脏CD4^ 及CD8^ T细胞亚群（免疫荧光法）：血清pc-HGFSP批原特异性抗体（ELISA法）及一氧化氮（NO）含量。结果 与pcDNA3对照比较，pc-HGFSP免疫小鼠脾淋巴细胞增殖活性增高24％－37％；NK细胞活性增高38％－90％；CTL活性增高65％－153％；CD8^ T细胞亚群增加。免疫血清产生HGFSP抗原特异性IgG抗体；NO含量也有所增高。结论 恶性疟DNA疫 pc-HGFSP有一定的诱导小鼠细胞免疫和体液免疫应答的作用。     

幽门螺杆菌ureI核酸疫苗免疫活性的初步研究

目的 观察幽门螺杆菌ureI核酸疫苗诱导C57BL/6小鼠产生体液免疫应答及细胞免疫应答水平.方法 重组质粒pcDNA3.1(+)/ureI、空质粒pcDNA3.1(+)及PBS分别肌注免疫C57BL/6小鼠.ELISA法检测小鼠血清中特异性IgG抗体水平以及脾淋巴细胞培养上清中IL-4和IFN-γ含量,MTT比色法检测脾淋巴细胞增殖反应,PCR和免疫荧光组化法检测ureI基因和蛋白表达.结果 pcDNA3.1(+)/ureI能够诱导小鼠产生特异性IgG抗体,该组小鼠脾淋巴细胞经UreI重组蛋白刺激后,其刺激指数和培养上清中IL-4、IFN-γ含量明显升高; ureI基因可在小鼠肌细胞中有效表达.结论 pcDNA3.1(+)/ureI核酸疫苗能刺激C57BL/6小鼠产生较强的体液免疫应答和细胞免疫应答,为进一步研究该疫苗的免疫保护作用奠定了基础.     

乙型肝炎病毒核心抗原核酸疫苗对猕猴的体液和细胞免疫原性观察

目的：观察乙型肝炎病毒核心抗原（HBcAg）核酸疫苗对猕猴的体液和细胞免疫原性。方法：实验组和对照组猕猴分别肌内注射HBcAg核酸疫苗（pJW4303/HBc)和对照质粒（pJW4303)。采用酶联免疫试验检测接种前后猕猴血清中抗-HBc、抗-HBc IgG亚类和猕猴外周血单个核细胞（PBMC）培养上清中干扰素-γ（IFN-γ）及白介素-4（IL-4）水平。采用^3H-TdR掺入法检测猕猴PBMC HBcAg特异性增殖反应。结果：实验组猕猴接种该核酸疫苗后均产生了较强的抗-HBc反应。实验组猕猴抗-HBc IgG亚类以IgG2为主,PBMC培养上清中IFN-γ水平显著增高,提示为Th1型免疫应答。实验组猕猴的PBMC抗原特异性增殖反应明显高于对照组。结论：HBcAg核酸疫苗对猕猴具有良好的体液和细胞免疫原性。     

DNA疫苗诱导小鼠特异性细胞免疫及抗乙型肝炎病毒皮下移植瘤的研究

目的观察乙型肝炎病毒(HBV)DNA疫苗(pCR3 1-S)诱导Balb/c小鼠(H-2d)的特异性细胞免疫应答及其对稳定表达HBsAg的小鼠肥大细胞瘤P815细胞(P815 HBV-S)(H-2d)成瘤性的影响.方法肌肉注射DNA疫苗,背部皮下接种P815-HBV S细胞,观察成瘤情况,4h 51Cr释放法检测小鼠脾细胞细胞毒T细胞(CTL)杀伤活性.结果DNA疫苗可以降低成瘤率,抑制肿瘤生长,延长小鼠平均存活期,提高小鼠存活率.CTL细胞杀伤活性明显增加(P＜0.001).结论DNA疫苗可以诱导细胞免疫应答,对体内HBV感染可能具有预防及治疗作用.     

不同载体及靶基因对乙型肝炎病毒DNA疫苗免疫效果的影响

目的探讨不同DNA载体及靶基因对DNA疫苗免疫效果的影响.方法用已构建的不同载体HBV S基因疫苗(pCR3.1-S,pcDNA3-S)和HBVC基因疫苗(pCR3.1-C)分别给Balb/c小鼠多点肌肉注射,2 wk后追加免疫一次,用ELISA法及MTT法分别检测小鼠血清抗-HBs及抗HBc及脾细胞对HBsAg或HBcAg的特异性增殖反应.结果免疫接种2 wk后小鼠血清抗-HBs及抗HBc抗体均明显高于对照组,载体pCR3.1的表达效力稍高于pcDNA3,但同一基因不同载体间无显著差异.不同靶基因血清抗体滴度及脾细胞对HBsAg或HBcAg的刺激指数均明显高于对照组,刺激指数pCR3.1-C组明显高于单纯pCR3.1-S注射组.结论两种载体均可以诱导较强的体液免疫应答;但同一基因不同载体间无显著差异.HBV S和C基因疫苗均可诱导较强的体液和细胞免疫应答强度;C基因以细胞免疫增高为主.     

乙型及丙型肝炎DNA疫苗联合重复接种小鼠的免疫应答

目的 :观察由编码 HBs Ag与 HCV- CE2 抗原的两种重组真核细胞表达质粒制备的 DNA疫苗重复联合接种 BAL B/c小鼠后 ,其诱生的特异性免疫应答反应。方法 :应用上述两种 NDA疫苗重复联合免疫小鼠 ,动态观察血中特异性抗体水平、特异性细胞毒性 T淋巴细胞 (CTL )体外杀伤活性 ,并进行 CTL杀伤活性活体诱生实验。结果 :两种 DNA疫苗联合重复免疫小鼠 ,能够诱生机体特异性体液免疫及细胞免疫完全应答。其小鼠荷瘤表达目的抗原的靶细胞后 ,生存率明显高于未免疫鼠 (P <0 .0 5 )。结论 :乙型及丙型肝炎联合 DNA疫苗的重复接种 ,可有效地诱生小鼠机体特异性免疫应答反应。 DNA疫苗诱生的 CTL应答可与体液免疫应答分离存在 ,可能是 DNA疫苗免疫保护的更重要方面。     

恶性疟原虫复合抗原DNA疫苗诱导小鼠的细胞免疫及体液免疫应答

目的　观察用恶性疟原虫复合抗原基因 HGFSP构建的 DNA疫苗 pc- HGFSP免疫小鼠诱导的细胞及体液免疫应答。　方法　用 pc- HGFSP肌注免疫 C5 7BL/6小鼠并加强免疫 2次 ,取小鼠脾淋巴细胞及血清测定 :脾淋巴细胞增殖活性 (MTT法 ) ;NK细胞活性和 CTL活性 (L DH法 ) ;脾脏 CD4 +及 CD8+ T细胞亚群 (免疫荧光法 ) ;血清 pc- HGFSP抗原特异性抗体 (EL ISA法 )及一氧化氮 (NO)含量。　　结果　与 pc DNA3对照比较 ,pc- HGFSP免疫小鼠脾淋巴细胞增殖活性增高 2 4 %～ 37% ;NK细胞活性增高 38%～ 90 % ;CTL 活性增高 6 5 %～ 15 3% ;CD8+ T细胞亚群增加。免疫血清产生HGFSP抗原特异性 Ig G抗体 ;NO含量也有所增高。　结论　恶性疟 DNA疫苗 pc- HGFSP有一定的诱导小鼠细胞免疫和体液免疫应答的作用。     

Novel DNA vaccine based on hepatitis B virus core gene induces specific immune responses in Balb/c mice

AIM:To investigate the immunogenicity of a novel DNA vaccine,pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS:A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS:HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW389l/HBc DNA vaccine. CONCLUSION:pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.     

乙型肝炎病毒核心区DNA疫苗接种后H-2d小鼠的特异性免疫应答

目的观察乙型肝炎病毒（ＨＢＶ）核心区ＤＮＡ疫苗接种后ＢＡＬＢ／ｃ（－２ｄ）小鼠的特异性免疫应答。方法构建ＨＢＶ核心区ＤＮＡ疫苗（ＰＪＷ４３０３／ＨＢｃ）;用基因枪法和肌肉注射祛将该ＤＮＡ疫苗接种ＢＡＬＢ／ｃ小鼠;ＥＬＩＳＡ法检测小鼠血清抗－ＨＢＣ（ＩｇＧ）及ＩｇＧ亚类（ＩｇＧ１,ＩｇＧ２ａ）;５１铬释放法检测小鼠脾细胞ＨＢｃＡｇ特异性ＣＴＬ活性。结果该ＤＮＡ疫苗在体外转染细胞中可良好表达ＨＢｃＡｇ。血清抗－ＨＢｃ终点滴度在ＤＮＡ疫苗基因枪组和肌肉注射接种组小鼠分别为１：３２８０５０和１：１０９３５０．两组小鼠的抗－ＨＢｃＩｇＧ亚类均以ＩｇＧ２ａ略占优势。两组小鼠的ＨＢＣＡｔｈｅ异性ＣＴＬ杀伤活性分别达到５１．１％和５５．２％。结论ＨＢＶ核心区ＤＮＡ疫苗在小鼠实验中具有良好的细胞免疫和体液免疫原性。     

Ubiquitin conjugation of hepatitis B virus core antigen DNA vaccine leads to enhanced cell-mediated immune response in BALB/c mice

Background

Nearly 350 million persons worldwide are chronically infected with hepatitis B virus (HBV). Ubiquitin (Ub) is a highly conserved small regulatory protein, ubiquitous in eukaryotes, that usually serves as a signal for the target protein that is recognised and degraded in proteasomes . The Ub-mediated processing of antigens is rapid and efficient and stimulates cell-mediated immune responses. Accordingly, Ub-mediated processing of antigens has been widely used in chronic-infection and cancer studies to improve immune response.

Objectives

Many clinical trials have shown that DNA vaccine potency needs to be greatly enhanced. Here, we report a new strategy for designing an HBV DNA vaccine using the ubiquitin (Ub) sequence. The aim of this study was to investigate a novel DNA vaccination, based on the expression of HBV core antigen (HBcAg), fused to Ub to enhance DNA vaccine potency.

Materials and Methods

Mouse ubiquitin fused to the HBcAg gene and cloned into the eukaryotic vector pcDNA3.1 (-). BALB/c mice were immunized with recombinant pUb-HBcAg or pHBcAg DNA vaccine. Lymphocyte proliferation assay, intracellular IFN-γ assay, CTL cytotoxicity assay, and antibody assay were performed to analyze the cellular and humoral immune responses to our DNA constructs.

Results

HBcAg was expressed effectively in the COS-7 cells that were transiently transfected with pUb-HBcAg. Strong anti-HBc IgG responses were elicited in mice that were immunized with pUb-HBcAg. The endpoint titers of anti-HBc peaked at 1:656100 on the 42nd day after the third immunization. pUb-HBcAg stimulated greater lymphocyte proliferation and induced higher levels of IL-2 and IFN-γ and a greater percentage of HBcAg-specific CD8+ T cells in mice than pHBcAg. In the CTL assay, the specific lysis rate reached 56.5% at an effector:target ratio of 50:1 in mice that were immunized with pUb-HBcAg.

Conclusions

pUb-HBcAg elicits specific anti-HBc responses and induces HBc-specific CTL responses in immunized BALB/c mice. Our results imply that Ub can be used as a molecular adjuvant that enhances the potency of DNA vaccines.     

CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice

Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1–14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0–10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.     

结核分枝杆菌MPT64抗原DNA疫苗在小鼠体内诱导的免疫应答

目的研究结核分枝杆菌MPT64抗原DNA疫苗在小鼠体内诱导的免疫应答。方法用表达MPT64的真核表达质粒pcDNA-M免疫BALB/c小鼠,ELISA法检测免疫小鼠的特异性抗体滴度和抗体亚类。分离免疫小鼠的脾淋巴细胞,检测淋巴细胞增殖、IFN-γ和IL-12产生水平、流式细胞仪计CD4+细胞和CD8+细胞数、脾淋巴细胞特异性CTL杀伤效应。结果MPT64基因免疫可诱导小鼠高水平的体液免疫应答,免疫小鼠脾淋巴增殖显著,IFN-γ和IL-12含量增加,CD4+细胞和CD8+细胞百分比明显增加,CTL杀伤效应明显。结论MPT64 DNA疫苗可诱导小鼠有效的体液和细胞免疫应答,有可能作为新型TB疫苗的组分。     

多聚赖氨酸预处理花生DNA疫苗对花生蛋白诱导变态反应的作用

目的探讨经多聚赖氨酸预处理的花生DNA疫苗对花生蛋白诱导变态反应的预防作用。方法以编码花生蛋白Arah2的质粒DNA(pArah2)、经多聚赖氨酸(PLL)预处理的质粒DNA(PLL-pArah2)、磷酸盐缓冲液(PBS)和PLL对照皮内注射免疫Balb/c小鼠,1次/周,连续3周,再经腹腔注射纯化的Arah2蛋白免疫小鼠。在不同时间点收集各组小鼠血清,用酶联免疫吸附法检测血清中Arah2特异性IgG1、IgG2a抗体以及血清总IgE抗体水平,分析DNA疫苗免疫对蛋白诱导小鼠变态反应的影响。结果 pArah2及PLL-pArah2质粒DNA免疫后,小鼠血清Arah2特异性IgG1和IgG2a升高,但血清总IgE抗体未见升高。Arah2蛋白免疫后,不同处理组血清总IgE、Arah2特异性IgG1和IgG2a抗体水平均升高,但pArah2组血清总IgE和Arah2特异性IgG1抗体水平明显低于PBS对照;PLL-pArah2组血清总IgE和Arah2特异性IgG1、IgG2a抗体水平不仅明显低于PBS和PLL对照,且明显低于单纯pAra h2组。结论经多聚赖氨酸预处理的Arah2 DNA免疫能够更好地抑制之后蛋白免疫产生的变态反应应答,为DNA疫苗治疗过敏性疾病提供了新的思路。     

乙型肝炎表面抗原与粒细胞巨噬细胞集落刺激因子融合表达质粒诱导小鼠特异性免疫应答

目的 观察乙型肝炎表面抗原(HBsAg)与粒细胞巨噬细胞集落刺激因子(GM CSF)融合表达质粒诱导特异性免疫应答的效果。方法 以HBsAg与GM-CSF融合表达质粒DNA免疫BALB/C小鼠,用酶链免疫吸附方法检测质粒诱导BALB/C小鼠产生抗HBs及脾细胞经HBsAg体外刺激后分泌白细胞介素(IL)-2、4及γ干扰素(IFNγ)的水平;并用~(21)Cr释放法检测HHsAg特异性细胞毒性T淋巴细胞(CTL)反应。结果 各组小鼠加强免疫后血清抗体水平(P/N值)A组(PcDNA3.1-S)16.1 20.3,B组(PcDNA3.1 GM-CSF-S)28.3±19.7,F组(重组酵母乙型肝炎疫苗)29.5±21.5,C组(PcDNA3.1S-GM-CSF)0.3±0.0,D组(PcDNA3.1—S PcDNA3.1-GM-CSF)4.5 5.9,E组(PcDNA3.1—GM-CSF)0.9±1.2,G组(PcDNA3.1)及H组(PBS)为阴性(F-4.176,P<0.01);IL-2分泌水平A组(26.6±1.9)、B组(56.0±2.2)、C组(4.3±1.2)、D组(33.5±1.7)、F组(3.5 1.4)、F组(7.5±2.0)、G组(1.5±0.7),H组未检测到IL-2分泌(F=31.188,P<0.01);IFN-γ分泌水平A组(64.0±7.5)、B组(139.0±17.0)、C组(11.0±5.0)、D组(53.0±9.5)、E组(9.0±3.0)、F组(13.0±2.0)、G组(8.0±2.0,P(0.05),H组未检测到IFN—γ泌(F=31.796,P<0.01);HBsAg特异性CTL活性,效靶比为30:1及10:1时各组音有差异有显著性(F值分别为26.891、12     

HIV-1CN54合成gp120基因(syngp120)的DNA疫苗鼻内免疫小鼠诱导全身的和粘膜的免疫应答

目的 初步探讨HIV-1CN54合成gp120基因的DNA疫苗（pcDNA3.1-syngp120）鼻内接种小鼠是否诱发免疫应答。方法　DNA疫苗免疫后,制备脾和肠系膜淋巴结（MLN）淋巴细胞,在体外测其增殖应答和CD8+CTL应答。间接ELISA法测血清和粘膜洗液抗原-特异的IgG和IgA抗体滴度。中和实验测免疫血清和阴道洗液是否中和 HIV-1SF33。结果 在 DNA疫苗未次免疫后,小鼠第 1周检测到脾和 MLN CD8+CTL应答较弱,而第 5周未检测到。另外,在末次免疫后的第1、5周检测到MLN而未检测到脾淋巴细胞发生增殖应答。并且检测到特异的血清IgG抗体和粘膜的（包括粪便和阴道洗液）IgA抗体,但未检测到血清的IgA抗体和粘膜的（包括粪便和阴道洗液）IgG抗体。中和实验发现末次免疫后第5周的血清能中和实验室毒株HIV-1SF33（B亚型）,而阴道洗液则没有。结论 该DNA疫苗鼻内免疫小鼠可诱导粘膜免疫应答,包括 MLN淋巴细胞增殖应答和 CD8+CTL应答,同时诱导较弱的粘膜IgA抗体应答。此外,能诱导脾CD8+CTL应答和血清IgG抗体应答。免疫血清中和HIV-1S F33,而阴道洗液不能。