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首页> 外文学位 >Novel DNA vaccine development against porcine circovirus type 2 and swine influenza virus H1N1 subtype.
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Novel DNA vaccine development against porcine circovirus type 2 and swine influenza virus H1N1 subtype.

机译：针对猪圆环病毒2型和猪流感病毒H1N1亚型的新型DNA疫苗开发。

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摘要

Porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are important viral pathogens in pig populations. Current vaccines against these viruses are based on inactivated viruses or recombinant proteins. They are effective in inducing humoral immunity only, while cellular immunity is essential to clear intracellular pathogens. DNA vaccines are capable of stimulating both humoral and cellular immunity. Therefore, developing DNA vaccines against these two viruses is the main goal in these studies.;To evaluate the vaccine efficacy in the natural host pig of these two viruses, a virus challenge model was established successfully to reproduce diseases caused by these viruses via experimentally infecting pigs. Also, the challenge model was utilized to investigate the pathogenesis of PCV2-SIV co-infection. SIV H1N1 did not influence PCV2 replication in dually infected pigs under the conditions in this study.;To develop DNA vaccines, a commercial vector pcDNA3.1 was modified by inserting several components in the plasmid backbone. A chimeric intron and a Kozak sequence were inserted downstream of the CMV promoter and upstream of the starting codon to enhance the expression of the target gene insert. The C-terminal region of VP22 gene (VP22c), encoding one of the major tegument proteins of bovine herpesvirus-1, was fused to the target gene at the N- or C-terminus. VP22c can transport the protein fused with it from the transfected cells to surrounding untransfected cells, thus enhancing the transfection and presentation efficiency.;ORF2, encoding the most immunogenic capsid protein of PCV2, was the target gene in the effort to develop a DNA vaccine against PCV2. The plasmid with VP22c fused to the N-terminal of the ORF2 gene (pVP22cORF2) reached the highest percentage of ORF2-positive cells in an in vitro transfection experiment. A plasmid pGM-CSF, encoding the secreted porcine GM-CSF protein was constructed and co-delivered with pVP22cORF2 to pigs intramuscularly. Vaccinated pigs showed increased growth performance, reduced duration of clinical signs, as well as reduced viral load in serum, lung, and lymph nodes. The DNA vaccine protected pigs against virus infection and disease development in this study.;SIVs are fast-evolving viruses and highly variable due to frequent mutation and reassortment. A modified pcDNA3.1 plasmid incorporating conservative epitopes among SIVs was constructed with the goal to protect animals against influenza virus infection. Firstly, a platform for a DNA-epitope vaccine was established in mice. In this study, two B-cell epitopes (HA91-108 and M2e), and two T-cell epitopes (NP366-374 and NP380-393), were quadruplicated and fused separately to the C-terminal of the VP22c gene in the modified pcDNA3.1 plasmid. Linker -KK- was used to space between each copy of the two B-cell epitopes and -RVKR- was used for the two T-cell epitopes in order to enhance the presentation of the epitopes to the immune system. After the DNA plasmids were administered to mice intramuscularly in combination or separately, or boosted with recombinant proteins of quadruplicated epitopes fused to VP22c, the vaccine stimulated the desired epitope-specific humoral immunity to the two B-cell epitopes and cellular immunity to the epitope NP380-393. Our results indicate that the vaccine design is a successful platform for developing DNA-epitope vaccines. The efficacy of this platform was evaluated in the following mice vaccination/challenge study. One B-cell epitope (HA2.30-130), a quadruplicated Th-cell epitope (NP55-69), and a quadruplicated CTL epitope (NP147-158) were fused separately to the C-terminal of VP22c gene in the modified pcDNA3.1 plasmid. Chitosan was used as an adjuvant to deliver plasmids intranasally. DNA plasmids were also administered to mice intramuscularly. In the intramuscularly vaccinated group, the vaccine stimulated epitope-specific immunity that provided 100% protection upon a lethal dose of SIV H1N1 challenge. However, the chitosan/plasmid vaccinated group only had 20% survival upon virus infection, compared to 0% in the challenge control group. Our results indicate that DNA plasmids administered intramuscularly are effective in mice.;To conclude, the developed DNA vaccines in these studies provided efficient protection to animals against virus infections and the modified DNA plasmid can be used as a general DNA vehicle to deliver proteins or peptides against various targets.
机译：猪圆环病毒2型（PCV2）和猪流感病毒（SIV）是猪群中重要的病毒病原体。当前针对这些病毒的疫苗是基于灭活的病毒或重组蛋白。它们仅在诱导体液免疫方面有效，而细胞免疫对清除细胞内病原体至关重要。 DNA疫苗能够刺激体液和细胞免疫。因此，开发针对这两种病毒的DNA疫苗是这些研究的主要目标。为了评估这两种病毒在天然宿主猪中的疫苗效力，成功建立了病毒攻击模型，通过实验感染来复制由这两种病毒引起的疾病猪。而且，利用攻击模型来研究PCV2-SIV共感染的发病机理。在本研究条件下，SIV H1N1不会影响双重感染猪的PCV2复制。为了开发DNA疫苗，通过在质粒主链中插入几种成分来修饰商业载体pcDNA3.1。将嵌合内含子和Kozak序列插入到CMV启动子的下游和起始密码子的上游，以增强靶基因插入物的表达。编码牛疱疹病毒1的主要外皮蛋白之一的VP22基因（VP22c）的C端区域在N端或C端与靶基因融合。 VP22c可以将融合了它的蛋白质从转染的细胞转运到周围的未转染的细胞，从而提高转染和提呈效率。ORF2编码PCV2最具免疫原性的衣壳蛋白，是开发针对DNA疫苗的目标基因。 PCV2。在体外转染实验中，具有与ORF2基因N端融合的VP22c的质粒（pVP22cORF2）达到了ORF2阳性细胞的最高百分比。构建了编码分泌的猪GM-CSF蛋白的质粒pGM-CSF，并与pVP22cORF2肌肉内共递送给猪。接种疫苗的猪生长性能提高，临床体征持续时间缩短，血清，肺和淋巴结中的病毒载量降低。在这项研究中，DNA疫苗保护猪免受病毒感染和疾病发展。SIV是快速发展的病毒，由于频繁的突变和重配而高度可变。为了保护动物免受流感病毒感染，构建了一种在SIV中掺入保守表位的修饰pcDNA3.1质粒。首先，在小鼠中建立了DNA表位疫苗的平台。在这项研究中，将两个B细胞表位（HA91-108和M2e）和两个T细胞表位（NP366-374和NP380-393）一式四份，分别与修饰后的VP22c基因的C端融合pcDNA3.1质粒。为了增强表位向免疫系统的呈递，使用接头-KK-在两个B细胞表位的每个拷贝之间隔开，并且-RVKR-用于两个T细胞表位。在将DNA质粒组合或分别肌内注射给小鼠后，或用融合有VP22c的四联表位的重组蛋白加强免疫后，疫苗刺激了针对两个B细胞表位的所需表位特异性体液免疫和对表位NP380的细胞免疫-393。我们的结果表明，疫苗设计是开发DNA表位疫苗的成功平台。在以下小鼠疫苗接种/挑战研究中评估了该平台的功效。将一个B细胞表位（HA2.30-130），一个四倍的Th细胞表位（NP55-69）和一个四倍的CTL表位（NP147-158）分别融合到修饰的pcDNA3中VP22c基因的C端.1质粒。壳聚糖用作佐剂以鼻内递送质粒。还将DNA质粒肌内注射给小鼠。在肌肉内接种组中，疫苗刺激了表位特异性免疫，在致死剂量的SIV H1N1攻击后提供了100％的保护。但是，壳聚糖/质粒疫苗接种组在感染病毒后仅具有20％的存活率，而攻击对照组仅占0％。我们的结果表明，肌肉内施用的DNA质粒在小鼠中是有效的。总之，在这些研究中开发的DNA疫苗为动物提供了有效的保护，使其免受病毒感染，并且修饰的DNA质粒可用作一般的DNA载体来递送蛋白质或肽针对各种目标。

著录项

  • 作者

    Wei, Huiling.;

  • 作者单位

    Purdue University.;

  • 授予单位 Purdue University.;
  • 学科 Biology Virology.;Health Sciences Immunology.;Agriculture Animal Pathology.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 203 p.
  • 总页数 203
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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