Flt3L与CCL5细胞因子佐剂对HBc抗原DNA疫苗特异性免疫应答的促进作用

观察真核重组表达质粒pFlt3L及pCCL5对携带HBc抗原的DNA疫苗诱导的抗原特异性免疫应答的促进作用。将pFlt3L及pCCL5分别用脂质体的方法转染Hep G2细胞,然后采用骨髓细胞增殖及趋化小室实验检测细胞上清Flt3L及CCL5两种细胞因子的生物学活性。将pFlt3L和pCCL5两种重组质粒单独或联合使用与携带HBc抗原的DNA疫苗经肌内注射法免疫小鼠,采用MTT法检测脾淋巴细胞增殖、流式细胞仪检测脾CD8+T淋巴细胞中IFN-γ表达、ELISA法检测脾淋巴细胞培养上清IL-4含量及乳酸脱氢酶(LDH)释放法检测特异性CTL杀伤活性。结果:骨髓细胞增殖及趋化实验证实了Flt3L及CCL5均有生物学活性。pFlt3L和pCCL5单独或联合使用均可促进特异性淋巴细胞增殖反应(P<0.05),提高小鼠脾脏CD8+T淋巴细胞中IFN-γ表达量(P<0.05或P<0.01),IL-4表达水平在各组无显著区别(P>0.05),Flt3L+CCL5组小鼠脾细胞特异性CTL活性显著高于其他各组(P<0.05)。pFlt3L和pCCL5表达质粒联用可显著促进小鼠Th1型细胞因子的表达,并对带HBc抗原的DNA疫苗的免疫应答具有促进作用。     

pcDNA3.1/MAGE-3-HSP70融合蛋白诱导特异性抗肿瘤免疫应答的研究

目的构建重组表达质粒pcDNA3．1／MAGE-3-HSP70，观察其诱导免疫应答的能力和抗肿瘤免疫治疗的作用。方法通过RT-PCR扩增MAGE-3和HSP70 cDNA，以pcDNA3．1为载体，构建pcDNA3．1／MAGE-3-HSP70重组质粒，肌肉注射接种小鼠，间隔7d，共3次，以pcDNA3．1／MAGE-3、pcD—NA3．1／HSP70、pcDNA3．1和PBS为对照，检测脾淋巴细胞CTL杀伤活性、脾细胞培养上清IL-2、IFN-γ浓度、外周血淋巴细胞亚群变化及鼠血清中抗MAGE-3抗体水平。pcDNA3．1／MAGE-3-HSP70重组质粒免疫已负荷B16／MAGE-3的小鼠，观察小鼠成瘤时间、生存时间。结果pcDNA3．1／MAGE-3-HSP70质粒免疫的小鼠，其脾淋巴细胞对MAGE-3阳性靶细胞表现出明显的杀伤活性，与各对照组相比，P〈0．01，差异有统计学意义；外周血中CD4^＋、CD8^＋T细胞和脾细胞培养上清中TH1类细胞因子IFN-γ、IL-2水平明显升高。pcDNA3．1／MAGE-3-HSP70重组质粒免疫已负荷B16／MAGE-3的小鼠后，小鼠成瘤时间明显延迟，生存期明显延长。结论pcDNA3．1／MAGE-3-HSP70DNA疫苗在体内能诱导出显著的MAGE-3特异性抗肿瘤免疫应答，且能抑制体内已经存在的少量肿瘤细胞的成瘤。     

不同免疫途径对chitosan-DNA疫苗诱导CVB3特异性免疫应答的影响及其意义

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。     

pcDNA3.1+/MAGE-3 DNA疫苗诱导特异性抗肿瘤免疫应答的实验研究

目的：构建pcDNA3.1+/MAGE-3 DNA疫苗，观察其在小鼠体内诱导特异性抗肿瘤免疫应答的能力。方法： 通过RT-PCR构建重组表达质粒pcDNA3.1+/MAGE-3；以pcDNA3.1+/MAGE-3 DNA疫苗免疫已接种肿瘤细胞的小鼠，每10 d重复免疫1次，共3次，以pcDNA3.1+、PBS为对照。末次免疫后5 d检测血清中MAGE-3抗体滴度、小鼠脾淋巴细胞的细胞毒T细胞（cytotoxic T lymphocytes，CTL）杀伤活性、细胞因子IL-2和IFN-γ的浓度，同时计算抑瘤率。结果： 成功构建了pcDNA3.1+/MAGE-3 DNA疫苗，用此疫苗免疫已接种B16/MAGE-3细胞的小鼠后，能诱导小鼠脾淋巴细胞MAGE-3特异性的杀伤活性，脾细胞培养上清中细胞因子IL-2和IFN-γ的浓度明显增高，血清中抗MAGE-3抗体在1∶20滴度时阳性，肿瘤生长被显著抑制，与pcDNA3.1+组、PBS组相比，差异显著（P＜0.01）。结论： 成功构建了pcDNA3.1+/MAGE-3 DNA疫苗，该疫苗在小鼠体内既能激活CTL杀伤活性和CD4+ T细胞活性，又能激活体液免疫反应，从而诱导出特异性的抗肿瘤免疫应答。     

HCV核心-包膜E2抗原融合基因DNA疫苗的构建及对小鼠的免疫应答试验

目的：观察一种结构新颖的HCV融合抗原DNA疫苗在BALB/c小鼠的免疫效果，探讨其用于防治丙型肝炎的可行性。方法：用重叠延伸PCR拼接编码小鼠IgG kappa链信号肽和通用型辅助性T细胞表位PADRE的DNA片段，PCR分别扩增HCV核心抗原基因和包膜E2抗原基因，将3段基因插入真核表达载体pcDNA3.1，构成重组表达质粒pST-CE2t，转染COS7细胞，免疫组化检测HCV抗原的表达。将pST-CE2t和HCV核心抗DNA疫苗pcDNA3.1core分别肌肉注射接种BALB/c小鼠，检测小鼠的血清抗体、T细胞增殖和CTL反应。结果：pST-CE2t可在COS7细胞内表达HCV核心抗原和E2抗原，接种于BALB/c小鼠能有效诱导体液和细胞免疫应答，其中抗HCV核心抗原免疫应答的强度明显超过pcDNA3.1core，且更趋向于TH1型免疫应答。结论：pST-CE2t对于丙型肝炎的防治有潜在的应用价值。     

OX40/OX40L在免疫应答中的作用及与疾病关系的研究

随着对OX40OX40L的深入研究,发现这对重要的协同刺激分子除了能增强CD4+T细胞、B细胞、DC细胞的扩增、成熟和产生效应功能外,还能影响CD8+T细胞的杀伤效应,能使T细胞免于凋亡,并促进更多记忆T细胞的生成。OX40OX40L在许多疾病的发生发展中也起着关键的作用,通过干预其相互作用所进行的免疫治疗已在许多动物模型上取得非常好的疗效,这为今后的临床治疗提供了理论和实验基础。     

Flt3L对小鼠胸腺树突状细胞在FTOC体系中分化发育的作用

为了借助FTOC体系探讨Flt3L对小鼠胸腺树突状细胞分化发育的影响。摘取15～16d龄胎鼠胸腺进行体外器官培养(胎鼠胸腺器官培养-FTOC),根据所使用培养基的不同实验分为两组:对照组(基础培养基)和Flt3L组(培养基中含有细胞因子Flt3L),在体外进行FTOC常规培养,12d后分别收集两实验组的胸腺细胞,流式细胞仪检测细胞表面分子CD4、CD8、CD11c、Ia等的表达,通过光学显微镜观察细胞形态。骨髓来源的c-kit+造血干细胞通过悬滴培养方法种植入2-脱氧鸟苷处理过的胸腺,随后将胸腺放置于组织器官培养皿中所使用的培养基为基础培养基或加入细胞因子Flt3L的培养基,进行为期12d的FTOC常规培养。12d后收集不同条件下FTOC培养的胸腺细胞,通过流式细胞仪对胸腺细胞的表型进行分析;将在不同条件下FTOC培养获得的胸腺细胞进行MACS分选,从而获得胸腺树突状细胞(CD11c+DC),再与异源的CD4+T细胞进行混合淋巴细胞反应,通过MTT法检测T细胞的增殖情况。结果:在正常FTOC体系中,流式细胞仪检测结果和细胞形态学结果显示:Flt3L组胸腺DC有明显的增加,且FTOC联合悬滴培养体系中Flt3L组胸腺DC的生成率明显高于对照组;MTT检测结果也显示:没有CpG2006刺激时,胸腺DC刺激T细胞增殖的能力比较弱,但添加CpG2006刺激后,胸腺DC趋向于成熟表型,刺激T细胞增殖的能力有所增强。提示,Flt3L在小鼠胸腺DC的分化发育中发挥重要的调节作用,明显促进小鼠骨髓来源的c-kit+造血干细胞向胸腺DC的分化。     

丙型肝炎病毒与恶性疟原虫复合多表位抗原基因在小鼠及家兔中免疫应答的研究

目的 :探讨丙型肝炎病毒 (HCV)及恶性疟原虫 (Pf)复合DNA疫苗的可行性。方法 :把HCV复合多表位抗原基因PCX与Pf复合基因AB克隆到带CMV启动子的真核表达载体pcDNA3中 ,构建真核表达载体pcDNA3 CAB ,肌肉注射免疫小鼠及家兔 ,检测其诱发特异性免疫应答水平及安全性。结果 :小鼠及家兔分别于免疫后第 6周及第 8周可检测到抗GZ PCX抗体 ,于第 10周达最高 ,滴度分别为 1:40 0及 1:3 2 0 0 ,但持续时间较短 ;免疫血清可识别Pf抗原 ;免疫动物还可产生针对GZ PCX融合蛋白的迟发性超敏反应 ;免疫后小鼠体重正常 ,肝脾脏未见明显肿大 ,具有良好的安全性。结论 :HCV Pf双价多表位DNA抗原基因在小鼠及家兔中可诱发特异性免疫应答 ,但抗体的持续时间较短。     

B7-2表达质粒对HBV DNA疫苗诱导的特异性免疫应答的影响

目的：探讨B7－2分子是否能够增强乙型肝炎病毒（HBV）DNA疫苗诱导的特异性免疫应答。方法：将B7－2表达质粒与HBV DNA疫苗共接种于小鼠腓肠肌内，检测细胞毒性T淋巴细胞（CTL）活性，迟发性超敏反应（DTH）及抗－HBs滴度。结果：B7－2表达质粒与HBV DNA疫苗共接种组的DTH反应和CTL活性，明显强于单独接种HBV DNA疫苗组（P＜0．01）。两组的抗－HBs滴度差异无显著性（P＞0．05）。结论：B7－2表达质粒与HBV DNA疫苗共接种可显著增强抗－HBV特异性细胞免疫应答（CMI）。     

人β防御素-2与前列腺癌特异性膜抗原嵌合蛋白真核表达质粒构建及其诱导的小鼠特异性免疫应答

探索人β防御素2(hBF)-2)和前列腺特异性膜抗原(PSMA)共表达重组核酸疫苗对前列腺癌的免疫治疗。研究以pcDNA3．1为载体,构建重组质粒pcDNA3．1／PSMA和pcDNA3．1／hBD-2-PSMA．通过RT-PCR和免疫组化检测其表达。并免疫小鼠,进行血清中抗体检测．CD4^ 、CD8^ T淋巴细胞数目测定及CTL特异性杀伤作用检测。结果显示构建的质粒转染COS-7细胞后能表达目的基因,     

Pre-clinical evaluation of a CEA DNA prime/protein boost vaccination strategy against colorectal cancer

In preparation for a clinical trial in patients diagnosed with colorectal cancer, a vaccination strategy targeting the carcinoembryonic antigen (CEA) was evaluated in mice using a GMP-produced plasmid DNA vaccine, CEA66, encoding a truncated form of the tumour-associated antigen, CEA. The GMP-produced CEA DNA vaccine was also evaluated for toxicity. Repeated intradermal administration of the GMP-produced vaccine using a novel needle-free jet injection device (Biojector) induced robust CD4 and CD8 T-cell responses in mice, and did not result in any vaccine-related toxicity. In a heterologous DNA prime/protein boost setting, cellular immune responses were of higher magnitude in animals primed with CEA66 DNA than in animals receiving repeated doses of recombinant CEA protein. These responses were further enhanced if recombinant murine granulocyte-macrophage colony-stimulating factor was given as an adjuvant prior to vaccination. In contrast to repeated administration of recombinant CEA protein as a single modality vaccine, the heterologous CEA66 DNA prime/rCEA boost vaccination strategy resulted in a qualitatively broader immune response, and supports clinical testing of this vaccination regimen in humans.     

Preclinical evaluation of cellular immune responses elicited by a polyvalent DNA prime/protein boost HIV-1 vaccine

While DNA vaccines have been shown to prime cellular immune responses, levels are often low in nonhuman primates or humans. Hence, efforts have been directed toward boosting responses by combining DNA with different vaccination modalities. To this end, a polyvalent DNA prime/protein boost vaccine, consisting of codon optimized HIV-1 env (A, B, C, E) and gag (C) and homologous gp120 proteins in QS-21, was evaluated in rhesus macaques and BALB/c mice. Humoral and cellular responses, detected following DNA immunization, were increased following protein boost in macaques and mice. In dissecting cellular immune responses in mice, protein-enhanced responses were found to be mediated by CD4+ and CD8+ T cells with a Th1 cytokine bias. Our study reveals that, in addition to augmenting humoral responses, protein boosting of DNA-primed animals augments cellular immune responses mediated by CD8+ CTL, CD4+ T-helper cells and Th1 cytokines; thus, offering much promise in controlling HIV-1 in vaccinees.     

Pre-clinical development of a multi-CTL epitope-based DNA prime MVA boost vaccine for AIDS

Reliable and effective methods for induction of cytotoxic T-lymphocytes (CTL) are constantly persued. Central to this search is work in animal models, which allow to test novel vaccine strategies and ultimately lead to a more efficient planning of clinical trials. Here, human immunodeficiency virus (HIV) vaccine candidates were constructed as a string of partially overlapping CTL epitopes (20 human, 3 macaque and 1 mouse) delivered and expressed using plasmid DNA and modified virus Ankara (MVA; an attenuated vaccinia virus), which are both vaccine vehicles acceptable for use in humans. In mice, these vaccines were shown to induce virus-specific interferon-gamma-producing and cytolytic CD8+ T-cells after a single intramuscular needle injection. When immunization protocols were sought which would improve the level of induced HIV-specific T-cells, DNA priming-MVA boosting was found to be the most potent protocol. The multi-epitope DNA also elicited CTL when delivered intradermally using the Accell gene delivery device (gene gun). Finally, a combined intradermal gene gun DNA-MVA vaccination regimen induced in macaques high frequencies of circulating CTL, which were comparable to those observed in simian immunodeficiency virus (SIV)-infected monkeys. Further optimization of this method in non-human primates is under way. Thus, a vaccination regimen for an effective elicitation of CTL has been developed which might facilitate evaluation of the role(s) that these lymphocytes play in the control of SIV and HIV infections.     

ELISPOT analysis of a new CTL based DNA vaccine for HIV-1 using GM-CSF in DNA prime/peptide boost strategy: GM-CSF induced long-lived memory responses

Genetic adjuvants have potential role in improvement of immune responses against DNA vaccines. GM-CSF as a genetic adjuvant can recruit and augment dendritic cell numbers in the site of immune responses and thereby induce cellular and humoral immune responses. Here we show that co-immunization of a DNA vaccine from HIV-1P24-Nef with GM-CSF in DNA priming and peptide boost strategy increases the immunogenicity of our candidate vaccine. Analysis of immune response shows that co-immunization with GM-CSF boosts cellular immune responses through increasing proliferation activity and CTL function. Results of cytokine profile studies show that both IL-4 and IFN-γ levels were augmented. Also, co-immunization with GM-CSF resulted in a higher level of total IgG, comprising approximately equal levels of both specific IgG1 and IgG2a subtypes. Monitoring of cellular and humoral immune responses for 20 weeks after final immunization revealed the aptitude of GM-CSF for inducing long-lived humoral and cell mediated immune responses. Overall, our results suggest that GM-CSF is able to induce long term memory for the HIV-1 P24-Nef vaccine candidate while the exact mechanisms involved remained to be clarified.     

HIV-1 prophylactic vaccine comprised of topical DermaVir prime and protein boost elicits cellular immune responses and controls pathogenic R5 SHIV162P3

Topical DNA vaccination (DermaVir) facilitates antigen presentation to naive T cells. DermaVir immunization in mice, using HIV-1 Env and Gag, elicited cellular immune responses. Boosting with HIV-1 gp120 Env and p41 Gag augmented Th1 cytokine levels. Intramuscular DNA administration was less efficient in priming antigen-specific cytokine production and memory T cells. In rhesus macaques, DermaVir immunization induced Gag- and Env-specific Th1 and Th2 cytokines and generation of memory T cells. Boosting of DermaVir-primed serum antibody levels was noted following gp140(SHIV89.6P)/p27(SIV) immunization. Rectal challenge with pathogenic R5-tropic SHIV162P3 resulted in control of plasma viremia (4/5 animals) that was reflected in jejunum, colon and mesenteric lymph nodes. An inverse correlation was found between Gag- and Env-specific central memory T cell responses on the day of challenge and plasma viremia at set point. Overall, the topical DermaVir/protein vaccination yields central memory T cell responses and facilitates control of pathogenic SHIV infection.     

Improved SIV DNA vaccine can be effectively used as a boost for Ad5 in prime-boost immunization strategy

In the study under evaluation, optimized SIV DNA were used to boost T-cell responses induced by a highly immunogenic SIV Ad5-prime in Chinese rhesus macaques. A regular prime-boost regimen (SIV DNA-prime and rAd boost) and naive macaques were used as the control. After vaccination, the animals were challenged intrarectally with SIVmac251, and partial protection was observed in the macaques immunized by the Ad5-prime DNA-boost regimen. SIV-specific T-cell responses in the enzyme-linked immunospot assay were significantly higher in the Ad5-prime DNA-boost, compared with the responses in the control macaques. Viral control correlated with the generation of HLA-DR+ T cells 2 weeks after the viral challenge. Further studies using prime and boost strategies and alternative routes of vaccination (including a simultaneous approach) are warranted to fully explore the potential of prime and boost regimens for HIV-1 vaccine development.     

Production of antibodies to Little cherry virus 1 coat protein by DNA prime and protein boost immunization

Little cherry, an economically important disease of cherry is caused by at least two different viruses. One of these is Little cherry virus 1 (LChV-1) for the detection of which no efficient serological tools are available, so that diagnosis is based on molecular methods. In this study, different immunization strategies for producing antibodies against the viral coat protein of LChV-1 were tried, using either purified virus preparations, or bacterially expressed protein, or a DNA vector that expressed the cloned coat protein (CP) gene in vivo. Effective induction of specific antibodies to LChV-1 CP was obtained using DNA intramuscular immunization followed by a single boost with the recombinant protein. The entire coat protein sequence was cloned in a mammalian expression vector and, after being coated by an amphiphilic non-toxic reagent was delivered into rabbit. A protein boost increased the specific immune response against the virus protein. The sensitivity of this antiserum is lower if compared with that of antisera raised conventionally against other viruses, thus it requires improvements for use for diagnostic purposes.     

Immunization of rhesus macaques with a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine elicits protective antibody response against simian human immunodeficiency virus of R5 phenotype

The immunogenicity of a poylvalent HIV-1 vaccine comprised of Env antigens from primary R5 isolates was evaluated in rhesus macaques. DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 from multiple HIV-1 isolates that were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization. These sera neutralized homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV encoding Env from HIV-1(Ba-L), whereas the virus load was reduced in the remaining animals compared to na?ve controls. Hence priming with DNA encoding Env antigens from multiple HIV-1 clades followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of R5 tropic SHIV isolate.     

Robust immune response elicited by a novel and unique Mycobacterium tuberculosis protein using an optimized DNA/protein heterologous prime/boost protocol

An efficacious tuberculosis (TB) vaccine will probably need to induce both CD4 and CD8 T‐cell responses specific to a protective Mycobacterium tuberculosis antigen(s). To achieve this broad cellular immune response we tested a heterologous DNA/protein combination vaccine strategy. We used a purified recombinant protein preparation of a unique M. tuberculosis antigen (rMT1721) found in the urine of TB patients, an optimized plasmid DNA expressing this protein (DNA‐MT1721), and a Toll‐like receptor 4 agonist adjuvant. We found that priming mice with DNA‐MT1721 and subsequently boosting with rMT1721 elicited high titres of specific IgG1 and IgG2a antibodies as well as high magnitude and polyfunctional CD4⁺ T‐cell responses. However, no detectable CD8⁺ T‐cell response was observed using this regimen of immunization. In contrast, both CD4⁺ and CD8⁺ T‐cell responses were detected after a prime/boost vaccination regimen using rMT1721 as the priming antigen and DNA‐MT1721 as the boosting immunogen. These findings support the exploration of heterologous DNA/protein immunization strategies in vaccine development against TB and possibly other infectious diseases.     

DNA prime followed by protein boost enhances neutralization and Th1 type immunity against FMDV

Prime-boost strategy has been exhibited its potency to enhance immune responses, which would be important to the success to develop a vaccine against the foot-and-mouth disease virus (FMDV). An eukaryotic expression construct encoding the FMDV capsid VP1 protein with a recombinant VP1 protein or a commercial FMDV vaccine were tested in the prime-boost strategy in mice and cattle trials. The levels of induced specific antibodies, T cell proliferations, and DTH activities were significantly higher in the prime-boost groups than in those vaccinated with DNA, protein or FMDV vaccine alone. More importantly, the levels of neutralizing antibodies in the former groups were significantly higher than others and could last for at least four months in cattle trials. This study suggests that the prime-boost strategy significantly improves the effective immunity and may provide a longer protection against FMDV infection.