Wressnigg Nina, Voss Daniel, Wolff Thorsten, Romanova Julia, Ruthsatz Tanja, Mayerhofer Ines, Reiter Manfred, Nakowitsch Sabine, Humer Johannes, Morokutti Alexander, Muster Thomas, Egorov Andrej, Kittel Christian
Avir Green Hills Biotechnology, Gersthoferstrasse 29-31, 1180 Vienna, Austria.
Vaccine. 2009 May 11;27(21):2851-7. doi: 10.1016/j.vaccine.2009.02.087. Epub 2009 Mar 11.
We discovered a unique, single amino acid mutation in the influenza B M1 protein promoting viral growth of NS1 truncation mutants in Vero cells. Due to this mutation, we were able to generate an influenza B virus lacking the complete NS1 open reading frame (DeltaNS1-B virus) by reverse genetics, which was growing to titers of 8log(10)TCID(50)/ml in a Vero cell culture-based micro-carrier fermenter. The DeltaNS1-B vaccine candidate was attenuated in IFN-competent hosts such as human alveolar epithelial cells (A549) similar to influenza A DeltaNS1 viruses. In ferrets, the DeltaNS1-B virus was replication-deficient and did not provoke any clinical symptoms. Importantly, a single intranasal immunization of ferrets at a dose as low as 6 log(10)TCID(50)/animal induced a significant HAI response and provided protection against challenge with wild-type influenza B virus. So far, the lack of a DeltaNS1-B virus component growing to high titers in cell culture has been limiting the possibility to formulate a trivalent vaccine based on deletion of the NS1 gene. Our study closes this gap and paves the way for the clinical evaluation of a seasonal, trivalent, live replication-deficient DeltaNS1 intranasal influenza vaccine.
我们在乙型流感病毒M1蛋白中发现了一种独特的单氨基酸突变，该突变促进了NS1截短突变体在Vero细胞中的病毒生长。由于这种突变，我们能够通过反向遗传学方法构建出一种缺失完整NS1开放阅读框的乙型流感病毒（ΔNS1-B病毒），其在基于Vero细胞培养的微载体发酵罐中的滴度可达8log(10)TCID(50)/ml。与甲型流感ΔNS1病毒类似，候选疫苗ΔNS1-B在具有IFN活性的宿主如人肺泡上皮细胞（A549）中表现出减毒特性。在雪貂中，ΔNS1-B病毒复制缺陷，未引发任何临床症状。重要的是，以低至6 log(10)TCID(50)/动物的剂量对雪貂进行单次鼻内免疫可诱导显著的血凝抑制（HAI）反应，并提供针对野生型乙型流感病毒攻击的保护。到目前为止，由于缺乏能在细胞培养中生长至高效滴度的ΔNS1-B病毒成分，限制了基于NS1基因缺失来制备三价疫苗的可能性。我们的研究填补了这一空白，为季节性、三价、活的复制缺陷型ΔNS1鼻内流感疫苗的临床评估铺平了道路。
