Waring Barbara M, Sjaastad Louisa E, Fiege Jessica K, Fay Elizabeth J, Reyes Ismarc, Moriarity Branden, Langlois Ryan A
Department of Microbiology and Center for Immunology, University of Minnesota, Minneapolis, Minnesota, USA.
Biochemistry, Molecular Biology and Biophysics Graduate Program, University of Minnesota, Minneapolis, Minnesota, USA.
J Virol. 2018 Jan 2;92(2). doi: 10.1128/JVI.01741-17. Print 2018 Jan 15.
Influenza A virus drives significant morbidity and mortality in humans and livestock. Annual circulation of the virus in livestock and waterfowl contributes to severe economic disruption and increases the risk of zoonotic transmission of novel strains into the human population, where there is no preexisting immunity. Seasonal vaccinations in humans help prevent infection and can reduce symptoms when infection does occur. However, current vaccination regimens available for livestock are limited in part due to safety concerns regarding reassortment/recombination with circulating strains. Therefore, inactivated vaccines are used instead of the more immunostimulatory live attenuated vaccines. MicroRNAs (miRNAs) have been used previously to generate attenuated influenza A viruses for use as a vaccine. Here, we systematically targeted individual influenza gene mRNAs using the same miRNA to determine the segment(s) that yields maximal attenuation potential. This analysis demonstrated that targeting of NP mRNA most efficiently ablates replication. We further increased the plasticity of miRNA-mediated attenuation of influenza A virus by exploiting a miRNA, miR-21, that is ubiquitously expressed across influenza-susceptible hosts. In order to construct this targeted virus, we used CRISPR/Cas9 to eliminate the universally expressed miR-21 from MDCK cells. miR-21-targeted viruses were attenuated in human, mouse, canine, and avian cells and drove protective immunity in mice. This strategy has the potential to enhance the safety of live attenuated vaccines in humans and zoonotic reservoirs. Influenza A virus circulates annually in both avian and human populations, causing significant morbidity, mortality, and economic burden. High incidence of zoonotic infections greatly increases the potential for transmission to humans, where no preexisting immunity or vaccine exists. There is a critical need for new vaccine strategies to combat emerging influenza outbreaks. MicroRNAs were used previously to attenuate influenza A viruses. We propose the development of a novel platform to produce live attenuated vaccines that are highly customizable, efficacious across a broad species range, and exhibit enhanced safety over traditional vaccination methods. This strategy exploits a microRNA that is expressed abundantly in influenza virus-susceptible hosts. By eliminating this ubiquitous microRNA from a cell line, targeted viruses that are attenuated across susceptible strains can be generated. This approach greatly increases the plasticity of the microRNA targeting approach and enhances vaccine safety.
甲型流感病毒在人类和牲畜中引发严重的发病和死亡。该病毒在牲畜和水禽中的年度传播导致严重的经济破坏，并增加了新毒株人畜共患传播到人群中的风险，而人群中不存在预先存在的免疫力。人类的季节性疫苗接种有助于预防感染，并在感染发生时减轻症状。然而，目前可用于牲畜的疫苗接种方案有限，部分原因是担心与流行毒株发生重配/重组。因此，使用的是灭活疫苗，而非免疫刺激性更强的减毒活疫苗。微小RNA（miRNA）此前已被用于生产减毒甲型流感病毒用作疫苗。在此，我们使用相同的miRNA系统地靶向单个流感基因mRNA，以确定产生最大减毒潜力的片段。该分析表明，靶向NP mRNA最有效地消除了病毒复制。我们通过利用在对流感易感的宿主中普遍表达的一种miRNA——miR-21，进一步提高了miRNA介导的甲型流感病毒减毒的可塑性。为构建这种靶向病毒，我们使用CRISPR/Cas9从MDCK细胞中消除普遍表达的miR-21。靶向miR-21的病毒在人、小鼠、犬和禽类细胞中均表现出减毒，并在小鼠中激发了保护性免疫。这种策略有可能提高人类和人畜共患病宿主中减毒活疫苗的安全性。甲型流感病毒每年在禽类和人类群体中传播，导致严重的发病、死亡和经济负担。人畜共患感染的高发生率大大增加了传播给人类的可能性，而人类不存在预先存在的免疫力或疫苗。迫切需要新的疫苗策略来应对新出现的流感疫情。微小RNA此前已被用于使甲型流感病毒减毒。我们提议开发一种新型平台，以生产高度可定制、在广泛物种范围内有效的减毒活疫苗，并比传统疫苗接种方法具有更高的安全性。这种策略利用了一种在对流感病毒易感的宿主中大量表达的微小RNA。通过从细胞系中消除这种普遍存在的微小RNA，可以产生对易感毒株均有减毒作用的靶向病毒。这种方法大大增加了微小RNA靶向方法的可塑性，并提高了疫苗安全性。
