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Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA.

机译:由内源性痘病毒早期启动子驱动的转基因抗原在MVA中的真实位点的表达和细胞免疫原性。

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摘要

CD8(+) T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8(+) T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens.
机译:CD8(+)T细胞对痘苗病毒的反应几乎完全针对早期基因产物。减毒株修饰牛痘病毒安卡拉(MVA)正在设计用于引发针对病原体(包括疟原虫,结核分枝杆菌和HIV-1)的细胞免疫应答的新疫苗的临床试验中进行评估。所有这些重组MVA(rMVA)都利用成熟的方法,将目标基因与克隆的痘病毒启动子连接,然后通过感染细胞中的同源重组插入合适位置的病毒基因组中。使用BAC重组,我们显示驱动无功能或非必需MVA开放阅读框(ORFs)表达的有效早期启动子可用于重组抗原的免疫原性表达。使用定位为使用相同翻译起始密码子的模型抗原精确替换C11R,F11L,A44L和B8R的MVA直系同源物,可使早期转基因表达与常用的p7.5或短合成启动子相似或稍高。与传统构建体相比,通过单次注射或腺病毒初次接种rMVA加强疫苗在小鼠中诱导的抗原特异性CD8(+)T细胞的频率在其真实基因组位点使用内源性启动子相似地相等或略微提高。与p7.5相比,使用C11R或F11L启动子观察到的免疫原性增强与与p7.5相比,使用mH5启动子获得的增强。此外,病毒的生长速度不受损害,插入片段在遗传上是稳定的。因此,通过BAC重组插入转基因ORF代替病毒ORF不仅可以提供有效的启动子,而且还可以提供合适的插入位点,从而潜在地促进表达多种重组抗原的MVA疫苗的开发。

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