Department of Veterinary and Animal Sciences, University of Copenhagen, Stigbøjlen 4, 1870 Frb. C., 1-20, Building: 301, Copenhagen, Denmark.
Department of International Health, Immunology and Microbiology ISIM, University of Copenhagen, Øster Farigmagsgade 5, Bldg 22/23, København K, 1014, Copenhagen, Denmark.
Vet Res. 2017 Nov 9;48(1):74. doi: 10.1186/s13567-017-0479-5.
Despite numerous actions to prevent disease, Actinobacillus pleuropneumoniae (A. pleuropneumoniae) remains a major cause of porcine pleuropneumonia, resulting in economic losses to the swine industry worldwide. In this paper, we describe the utilization of a reverse vaccinology approach for the selection and in vitro testing of serovar-independent A. pleuropneumoniae immunogens. Potential immunogens were identified in the complete genomes of three A. pleuropneumoniae strains belonging to different serovars using the following parameters: predicted outer-membrane subcellular localization; ≤ 1 trans-membrane helices; presence of a signal peptide in the protein sequence; presence in all known A. pleuropneumoniae genomes; homology with other well characterized factors with relevant data regarding immunogenicity/protective potential. Using this approach, we selected the proteins ApfA and VacJ to be expressed and further characterized, both in silico and in vitro. Additionally, we analysed outer membrane vesicles (OMVs) of A. pleuropneumoniae MIDG2331 as potential immunogens, and compared deletions in degS and nlpI for increasing yields of OMVs compared to the parental strain. Our results indicated that ApfA and VacJ are highly conserved proteins, naturally expressed during infection by all A. pleuropneumoniae serovars tested. Furthermore, OMVs, ApfA and VacJ were shown to possess a high immunogenic potential in vitro. These findings favour the immunogen selection protocol used, and suggest that OMVs, along with ApfA and VacJ, could represent effective immunogens for the prevention of A. pleuropneumoniae infections in a serovar-independent manner. This hypothesis is nonetheless predictive in nature, and in vivo testing in a relevant animal model will be necessary to verify its validity.
尽管采取了许多措施来预防疾病，但胸膜肺炎放线杆菌（A. pleuropneumoniae）仍然是猪传染性胸膜肺炎的主要原因，给全球养猪业造成了经济损失。在本文中，我们描述了利用反向疫苗学方法选择和体外测试血清型独立的 A. pleuropneumoniae 免疫原。使用以下参数在属于不同血清型的三种 A. pleuropneumoniae 菌株的完整基因组中鉴定潜在的免疫原：预测的外膜亚细胞定位；≤1 个跨膜螺旋；蛋白质序列中存在信号肽；存在于所有已知的 A. pleuropneumoniae 基因组中；与其他具有关于免疫原性/保护潜力的相关数据的特征良好的因子具有同源性。使用这种方法，我们选择了 ApfA 和 VacJ 蛋白进行表达，并进一步进行了计算机模拟和体外分析。此外，我们分析了 A. pleuropneumoniae MIDG2331 的外膜囊泡（OMVs）作为潜在的免疫原，并比较了 degS 和 nlpI 的缺失，以增加与亲本菌株相比 OMVs 的产量。结果表明，ApfA 和 VacJ 是高度保守的蛋白，在所有测试的 A. pleuropneumoniae 血清型感染过程中自然表达。此外，OMVs、ApfA 和 VacJ 表现出很高的体外免疫原性。这些发现支持所使用的免疫原选择方案，并表明 OMVs 以及 ApfA 和 VacJ 可能代表针对血清型独立的 A. pleuropneumoniae 感染的有效免疫原。然而，这种假设是预测性的，需要在相关动物模型中进行体内测试来验证其有效性。
