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首页> 外文会议>International Colloquium on Paratuberculosis >Prime boost vaccination with pSG2 plasmid and MVA each recombinant for a MAP fusion protein attenuates pre-existing MAP infection in C57/BL6 mice
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Prime boost vaccination with pSG2 plasmid and MVA each recombinant for a MAP fusion protein attenuates pre-existing MAP infection in C57/BL6 mice

机译：用PSG2质粒和MVA的初始促进疫苗接种，每种重组地图融合蛋白在C57 / BL6小鼠中衰减预先存在的地图感染

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To develop a therapeutic vaccine for use in animals and humans infected with MAP, we performed a targeted bioinformatic analysis of the MAP genome and selected two secreted and two membrane bound components each related to the pathogenic phenotype. A fusion construct including these four MAP antigens was assembled from overlapping 40mer oligonucleotides incorporating a sequence adjusted for optimal mammalian codon usage. Functional domains including potential cross-reacting human epitopes and hydrophobic transmembrane regions were excluded. A monoclonal antibody recognition peptide was added to the C-terminus and a short human ubiquitin leader sequence to the N-terminus. The construct was cloned into the pSG2 expression vector and inserted by homologous recombination into Modified Vaccinia Ankara (MVA). Expression of the 95 kDa polyprotein in cell culture was confirmed. Prime boost vaccination using single dose pSG2.rec (i.m.) followed by single dose MVA.rec (i.v.) resulted in significant antigen-specific IFN-gammaT-cell responses in ELISPOT assays compared with control immunization using wild type vector or buffer only. No adverse effects occurred in the hyper-immune mice. In two subsequent studies, a modified protocol incorporating two priming doses of pSG2.rec (i.m.) followed by a single boost of MVA.rec (i.v.) was tested for its ability to attenuate pre-existing MAP infection and to protect against subsequent MAP infection compared with control groups. The burden of MAP infection was measured using IS900-specific quantitative real-time PCR on liver/spleen DNA extracts and by culture on solid medium for future determination of cfu. Prime boost vaccination four weeks after MAP infection resulted in a significant attenuation of infective load (qRT-PCR) in spleen and liver after 26 weeks compared with controls.
机译：为了开发用于动物和人类感染的动物和人类的治疗疫苗，我们对地图基因组进行了靶向生物信息分析，并选择了与致病表型相关的两个分泌的和两个膜结合组分。包括将包括这四种地图抗原的融合构建体从重叠的40mer寡核苷酸组装，其掺入用于最佳哺乳动物密码子使用的序列。包括潜在的交叉反应人表位和疏水跨膜区域的功能结构域被排除在外。将单克隆抗体识别肽加入到C-末端和N-末端的短的人遍布蛋白前导序列中。将构建体克隆到PSG2表达载体中，并通过同源重组插入修饰的痘苗病毒Ankara（MVA）中。确认了95kDa多蛋白在细胞培养中的表达。使用单剂量psg2.rec（即）促进疫苗接种疫苗接种（即）单剂量mva.rec（I.v.）导致ELISPOT测定中的显着抗原特异性IFN-γ-细胞反应与使用野生型载体或缓冲液的对照免疫相比。 Hyper-Immune小鼠没有发生不良反应。在两个后续研究中，测试了一种改性方案，其包含两个引发剂量的PSG2.rec（IM），然后进行了单一的MVA.REC（IV），以便其衰减预先存在的地图感染和防止后续地图感染的能力与对照组相比。使用肝脏/脾脏DNA提取物上的IS900特异性定量实时PCR测量地图感染的负担，并通过固体培养基上的培养物来测量CFU的未来测定。在地图感染后四周素促疫苗接种导致26周与对照相比26周后脾脏和肝脏中感染载荷（QRT-PCR）的显着衰减。

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