蛇毒半胱氨酸蛋白酶抑制剂基因转染对小鼠黑色素瘤B16细胞蛋白质表达谱的影响

目的研究蛇毒半胱氨酸蛋白酶抑制剂(sv-cystatin)基因转染对小鼠黑素瘤细胞B16F1的蛋白表达谱的影响,探讨其抗肿瘤机制。方法采用双向电泳-质谱的蛋白质组学研究策略,分析稳定表达sv-cystatin的B16F1细胞系B16F1/cystatin与转染空载体的对照细胞B16F1/pcDNA3.1的蛋白表达谱的差别,筛选鉴定差异表达蛋白;用实时定量PCR和Western blot进一步确定实验结果;对蛋白组数据和前期基因组数据进行比较分析。结果 B16F1/cystatin和B16F1/pcDNA3.1细胞总蛋白的双向电泳图谱上共有匹配蛋白点(412±20)个,其中有11个蛋白的表达差异在2倍以上,5个上调,6个下调。这11个蛋白和1个仅在B16F1/cystatin中表达的蛋白经MALDI-TOF-MS分析,其中10个蛋白得到了鉴定,它们分属于代谢酶系、小G蛋白Ran调控分子、真核翻译因子、核苷酸激酶等。Western blot显示NM23和RhoGDI-beta的蛋白表达分别上调了(2.23±0.34)倍和(1.57±0.11)倍;实时定量PCR结果显示NM23、RhoGDI-beta、RANBP1的mRNA表达上调,eIF5A、eEF1-beta2、MDH1表达下调;与蛋白质组学研究结果一致。基于Gene Ontology的分析提示sv-cystatin作用的靶分子主要定位于细胞外周、质膜和细胞核,与转录、半胱氨酸蛋白酶活性、细胞凋亡等过程有关。结论 sv-cystatin可能通过调控抑癌基因NM23、RhoGDI等蛋白质发挥其抗肿瘤作用。sv-cystatin的主要靶分子可能是转录因子、分泌蛋白和质膜受体。     

Ⅲ价轮状病毒基因重配疫苗免疫原性及对造血系统的影响

目的:研究Ⅲ价轮状病毒基因重配疫苗对SD大鼠的免疫原性及对造血系统的长期毒性。方法:疫苗组大鼠(n=30)免疫3次,每次每只动物2 mL ig,第1次免疫后3周、第3次免疫后1和4周各处死大鼠10只,检测血清特异性抗体,血液学和骨髓。结果:在各时间点疫苗组血清抗体滴度均明显高于对照组。第1次免疫后3周时可测出针对G1,G2,G3,G4和G10型抗原的抗体,逐渐升高。针对G2,G3和G4型的抗体滴度明显高于G1和G10型(P<0.01或<0.05)。疫苗组大鼠LY计数和MCH出现了可逆性变化(P<0.05),但无明显毒性,大鼠骨髓象无明显改变。结论:Ⅲ价轮状病毒基因重配疫苗对大鼠有明显免疫原性,对造血系统无明显毒性。     

重组HBsAg／HCV颗粒在已免疫乙型肝炎疫苗小鼠中的免疫原性

HBsAg病毒样颗粒(VLP)的免疫原性多聚体表位提呈特性和已注册作为人用疫苗的特点使其成为理想的外源表位插入载体。但部分人群中已有的抗-HBs对这类嵌合抗原免疫效果的影响不明，作者将丙型肝炎病毒包膜蛋白E2的抗原高变区1(HVRl)插     

多表位疫苗的研究进展

综述了最近几年国内外在多表位疫苗方面的研究进展。     

基因疫苗的研究进展及临床应用

基因疫苗是近10年来发展起来的新型疫苗。自1990年Woff等发现直接肌肉注射裸DNA质粒,不经任何载体手段即可在肌细胞内获得有效表达蛋白以来,基因疫苗的研究不断深入。1993年Ulmer等率先应用流感病毒基因进行疫苗的研究,之后研究范围不断拓宽。目前基因疫苗的研究已涉及到病毒、衣原体、支原体、螺旋体、细菌、寄生虫以及癌症等诸多领域,一些基因疫苗的研究已处于Ⅰ/Ⅱ期临床研究阶段。基因疫苗与常规的灭活疫苗、减毒活疫苗、亚单位疫苗以及     

治疗黑色素瘤的DC疫苗研究新进展

黑色素瘤是最具侵袭性的皮肤恶性肿瘤,易发生早期转移和治疗后复发。治疗性肿瘤疫苗是新兴的免疫疗法,具有毒性低以及可抑制肿瘤转移的特点。目前已有多个针对黑色素瘤治疗性疫苗的研究,其中黑色素瘤治疗性树突状细胞（DC）疫苗引起了广泛关注。虽然肿瘤治疗性DC疫苗在黑色素瘤中的疗效已被多项研究证实,但该类疫苗存在免疫效应不足、单独使用疗效不佳等问题,仍具有较大的改进空间。本文对黑色素瘤的治疗性DC疫苗的研究现状进行了综述,并对肿瘤治疗性DC肿瘤的研究重点及优化策略进行展望。     

对NOD/SCID和SCID小鼠皮下接种的黑色素瘤生长特性比较

目的比较两种常用免疫缺陷小鼠皮下接种黑色素肿瘤细胞后肿瘤生长及小鼠生存情况。方法 7和17周龄的NOD/SCID及SCID小鼠左腋下接种小鼠黑色素瘤细胞,接种第8天开始隔1天测量肿瘤长径和短径,计算肿瘤体积,每天观察小鼠生存情况,记录生存时间,待1组小鼠完全死亡结束实验。结果 a.7周龄不同品系小鼠接种小鼠黑色素瘤后,NOD/SCID较SCID小鼠肿瘤生长速度更快、中位生存期更长;17周龄2种品系小鼠肿瘤生长情况无显著性差异。b.同品系NOD/SCID和SCID小鼠接种小鼠黑色素瘤后,小周龄较大周龄肿瘤生长速度更快,中位生存期更短。结论与7和17周龄SCID小鼠及17周龄NOD/SCID小鼠相比,7周龄NOD/SCID小鼠肿瘤生长情况更好,生存期较为适当,可能更适合作为黑色素瘤小鼠模型的实验动物。     

胃泌素释放肽DNA疫苗抑制EMT6乳腺癌生长的实验研究

目的：观察胃泌素释放肽（GRP）DNA疫苗对EMT6小鼠乳腺癌生长的抑制作用。方法：将构建的GRP DNA疫苗pCR3.1-VS-HSP65-TP-GRP6-M2肌肉注射免疫BALB/c雌性小鼠,2周1次,共5次。采用ELISA法对小鼠的血清中抗GRP-IgG类抗体进行检测。最后一次免疫后第2周,接种EMT6小鼠乳腺癌细胞。于肿瘤细胞接种后d14,处死全部动物,称量肿瘤的质量和测量肿瘤的体积。并对瘤组织进行常规HE染色。结果：pCR3.1-VS-HSP65-TP-GRP6-M2免疫BALB/c小鼠,可诱导抗GRP-IgG类抗体的产生。并对随后的EMT6肿瘤细胞攻击有显著的抑制作用（P〈0.01）,抑瘤率为46.53%。病理学检查结果显示,GRP DNA疫苗成功地激发了宿主的抗肿瘤免疫反应;与pCR3.1-VS-HSP65-TP对照组相比,GRP DNA疫苗免疫组小鼠EMT6肿瘤组织浸润性下降。结论：GRP DNA疫苗显著抑制EMT6乳腺癌生长,为此类疫苗应用研究奠定了基础。     

ntranasal co-delivery of IL-6 gene enhances the mmunogenicity of anti-caries DNA vaccine

Aim： To investigate the effects of co-delivering 11_-6 expressing plasmid pCI-IL-6 on the immunogenicity of the anti-caries DNA vaccine pClA-P, which encodes the surface protein antigen PAc of Streptococcus mutans. Methods： Plasmid pCI-IL-6 was constructed by inserting the murine IL-6 gene into the pCI vector. Expression of IL-6 in vitro was assessed using Western blot analysis. BALB/c mice were intranasally co-immunized with pClA-P plus pCI-IL-6 on d 0 and 14. Anti-PAc IgG and secretory IgA （slgA） were assessed by ELISA. Splenocytes from the mice were re-stimulated with the PAc protein, and IFN-y and IL-4 production was measured using ELISA. Splenocyte proliferation was analyzed with flow cytometry. Rats were similarly immunized, and dental caries scores were determined using the Keyes method. Results： Marked expression of IL-6 was found in C0S-7 cells transfected with pCI-IL-6. In the pCI-IL-6 co-immunized mice, the specific IgG antibodies in serum and slgA antibodies in saliva were significantly higher than those in the control mice at weeks 4 and 8. Moreover, the secretion of IFN-y from splenocytes in response to re-stimulation with PAc protein was significantly higher in the pCI-IL-6 co-immunized mice than that in the control mice, whereas the secretion of IL-4 had no significant difference. The proliferation of splenocytes from the pCI-IL-6 co-immunized mice was significantly higher than that from the mice immunized with pClA-P and pCl vector. In the rat caries model, the pCI-IL-6 co-immunization rats displayed lower caries scores than the control rats. Conclusion： Intranasal co-delivery of IL-6 gene significantly enhances the immunogenicity of the anti-caries DNA vaccine.     

A novel microparticulate vaccine for melanoma cancer using transdermal delivery

In this study, we formulated a microparticulate melanoma cancer vaccine via the transdermal route. The vaccine was delivered using microneedle-based Dermaroller® which is available for cosmetic purposes. Unlike subcutaneous injections, administration using microneedles is painless and in general can increase the permeability of many compounds ranging in size from small molecules to proteins and microparticles that do not normally penetrate the skin. The vaccine microparticles were taken up by the antigen presenting cells which demonstrated a strong IgG titre level of 930?ug/mL in serum samples. The formulation increased the immunogenicity of the vaccine by incorporating the antigen into an albumin matrix having a size range of around 0.63–1.4?µm which acted as a synthetic adjuvant. The animals were vaccinated with 1 prime and 4 booster doses administered every 14 days over 8 weeks duration, followed by challenge with live tumour cells which showed protection after transdermal vaccination.     

Long-term survival of high-risk melanoma patients immunized with a Hyper-IL-6-modified allogeneic whole-cell vaccine after complete resection

Objective: Two single arm, Phase II trials (3 and 5) were undertaken to determine the efficacy and toxicity of an adjuvant treatment using Hyper-IL-6 gene-modified whole-cell allogeneic melanoma vaccine in patients with stage IIIB–IV resected disease.

Research design and methods: Ninety-seven and 99 patients were enrolled into Trials 3 and 5, respectively. The primary endpoint was disease-free survival (DFS), and the secondary was overall survival (OS). Vaccine was administered eight times every 2 weeks (induction), every month (maintenance) until patient's death. At progression, maintenance was continued or induction was repeated followed by maintenance.

Results: Median follow-up was 10.5 and 6.2 years for Trials 3 and 5, respectively. No grade 3 or 4 toxicities were observed. An extension of DFS and OS was observed, when compared with historical non-treated controls. DFS probability at 5 years for Trials 3 and 5 was, respectively, 54.8% and 40.6% for stage IIIB, 25.0% and 24.0% for IIIC, and 8.5% and 17.7% for IV. OS probability at 5 years was, respectively, 66.7% and 56.3% for IIIB, 43.8% and 39.8% for IIIC, and 26.1% and 41.2% for IV.

Conclusions: Continuous vaccination, regardless of the disease progression, re-induction, and immunization of patients until death resulted in patients a long-term survival.     

DNA疫苗用于防治艾滋病的研究概况

目的:将能表达抗原的DNA作为疫苗接种。方法:直接注射(皮下,肌肉等)含有DNA的制剂。结果:接种后机体可产生体液免疫和杀伤性T淋巴细胞(CTL)反应,并使机体产生抗病保护。结论:DNA疫苗将有可能用于防治人类艾滋病的理想化疫苗。     

Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine

Human immunodeficiency virus (HIV) infections mainly occur through the vaginal and rectal mucosal membranes. In the present study, to develop a DNA vaginal vaccine against viral and bacterial infections, the effects of the menstrual cycle on DNA transfection through the vaginal mucosa in female mice and transfection enhancement by electroporation, a chelating agent, cell-penetrating peptides (CPP) and nuclear localizing signals (NLS) were investigated. The transfection efficiencies of a marker plasmid DNA (pDNA), pCMV-Luc, on the vaginal mucosal membrane in mice at the stages of metestrus and diestrus were significantly higher than those at the stages of proestrus and estrus. The gene expression was markedly enhanced by electroporation and by pretreatment with the chelating agent. The highest level of expression was obtained by 2h pretreatment with 5% citric acid solution combined with electroporation with 15 pulses at 250 V/cm for 5 milliseconds (ms). Furthermore, a synergistic promoting effect on pDNA transfection was obtained by co-administration of CPP, the Tat peptide analog, and NLS, the NF-kappaB analog. These results indicate that effective DNA vaccination administered through the vaginal tract is possible by selecting the menstrual stage and overcoming the mucosal barrier using a combination of methods that promotes uptake.     

Strategies for DNA vaccine delivery

Strategies for gene delivery comprise a diverse range of live and synthetic approaches; DNA delivery for the purposes of immunisation in turn comprises a large part of this research. This review mainly discusses synthetic systems for application in the delivery of plasmid DNA vaccines, outlining polylactide-co-glycolide, liposome, chitosan and complex combination delivery systems. Areas of promise for DNA vaccine candidates include immune modulation of allergic responses and veterinarian application. The potential for realistic consideration of DNA vaccines as an alternative to existing approaches is dependent on the development of efficient DNA vaccine vectors and improved systems for DNA vaccine delivery. DNA vaccine technology may yet prove to be an important asset in an environment where there is a critical need for therapeutic and prophylactic strategies to combat a wide range of disease states.     

A DNA vaccine encoding foot-and-mouth disease virus B and T-cell epitopes targeted to class II swine leukocyte antigens protects pigs against viral challenge

Development of efficient and safer vaccines against foot-and-mouth disease virus (FMDV) is a must. Previous results obtained in our laboratory have demonstrated that DNA vaccines encoding B and T cell epitopes from type C FMDV, efficiently controlled virus replication in mice, while they did not protect against FMDV challenge in pigs, one of the FMDV natural hosts. The main finding of this work is the ability to improve the protection afforded in swine using a new DNA-vaccine prototype (pCMV-APCH1BTT), encoding FMDV B and T-cell epitopes fused to the single-chain variable fragment of the 1F12 mouse monoclonal antibody that recognizes Class-II Swine Leukocyte antigens. Half of the DNA-immunized pigs were fully protected upon viral challenge, while the remaining animals were partially protected, showing a delayed, shorter and milder disease than control pigs. Full protection in a given vaccinated-pig correlated with the induction of specific IFNγ-secreting T-cells, detectable prior to FMDV-challenge, together with a rapid development of neutralizing antibodies after viral challenge, pointing towards the relevance that both arms of the immune response can play in protection. Our results open new avenues for developing future FMDV subunit vaccines.     

A safety and immunogenicity study of a novel subunit plague vaccine in cynomolgus macaques

Plague has led to millions of deaths in history and outbreaks continue to the present day. The efficacy limitations and safety concerns of the existing killed whole cell and live‐attenuated vaccines call for the development of new vaccines. In this study, we evaluated the immunogenicity and safety of a novel subunit plague vaccine, comprising native F1 antigen and recombinant V antigen. The cynomolgus macaques in low‐ and high‐dose vaccine groups were vaccinated at weeks 0, 2, 4 and 6, at dose levels of 15 μg F1 + 15 μg rV and 30 μg F1 + 30 μg rV respectively. Specific antibodies and interferon‐γ and interleukin‐2 expression in lymphocytes were measured. For safety, except for the general toxicity and local irritation, we made a systematic immunotoxicity study on the vaccine including immunostimulation, autoimmunity and anaphylactic reaction. The vaccine induced high levels of serum anti‐F1 and anti‐rV antibodies, and caused small increases of interferon‐γ and interleukin‐2 in monkeys. The vaccination led to a reversible increase in the number of peripheral blood eosinophils, the increases in serum IgE level in a few animals and histopathological change of granulomas at injection sites. The vaccine had no impact on general conditions, most clinical pathology parameters, percentages of T‐cell subsets, organ weights and gross pathology of treated monkeys and had passable local tolerance. The F1 + rV subunit plague vaccine can induce very strong humoral immunity and low level of cellular immunity in cynomolgus macaques and has a good safety profile.