Enhanced Live Attenuated Influenza Virus With Improved Safety and Immunogenicity

增强型减毒活流感病毒，具有更高的安全性和免疫原性

• 批准号: 8883370
• 负责人: Stephen Dewhurst
• 金额: $ 24.84万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8883370
• 关键词: A549; Age; Animal Model; Attenuated; Binding; Biological Assay; Body Weight decreased; Cells; Cessation of life; Child; Codon Nucleotides; Defect; Development; Disease; Dose; Effectiveness; Elderly; Epithelial Cells; Ferrets; Hemagglutinin; Human; Immune response; Immunoblotting; In Vitro; Infection; Influenza A Virus, H1N1 Subtype; Influenza A virus; Kinetics; Lethal Dose 50; Licensing; Licensure; Life; Lung; MDCK cell; Measurement; Measures; Messenger RNA; Modeling; Mus; Mutation; Nose; Open Reading Frames; Phenotype; Point Mutation; Polymerase; Populations at Risk; Primer Extension; Property; RNA; Reproducibility; Risk; Safety; Structure of parenchyma of lung; Temperature; Testing; Vaccines; Viral; Viral Antigens; Viral Genome; Virus; Virus Diseases; asthmatic; base; cohort; comparative; immunogenic; immunogenicity; improved; influenza virus vaccine; influenzavirus; mutant; neutralizing antibody; novel; protective efficacy; protein expression; public health relevance; vaccine safety

DESCRIPTION (provided by applicant): The Live Attenuated Influenza Vaccine (LAIV) has been shown to have greater efficacy in children than its inactivated counterpart. However, due to an unacceptable safety profile, it is not licensed in either asthmatics or children under the ag of two. If one could further attenuate LAIV while retaining immunogenicity, these safety concerns could be alleviated and LAIV usage could be extended to cover these groups. We recently discovered a novel influenza virus mutant that has increased temperature sensitivity, compared to conventional LAIV. The genome of this virus (designated SGR-ts) is characterized by five point mutations within the viral polymerase, when compared to a phenotypic revertant virus with normal temperature sensitivity. Three of these mutations (in PB1 and PB2) are sufficient to almost completely eliminate viral polymerase activity at 37oC, while having no effect on activity at 34oC; importantly, none of these mutations is shared with the current LAIV. These findings suggest that it should be possible to increase the safety of LAIV, by reducing its shutoff temperature (the temperature at which polymerase activity is abolished) to 37oC. Our central hypothesis is that the safety and effectiveness of LAIV can be increased by: (1) using newly discovered polymerase mutations to decrease the shutoff temperature for viral replication (increasing safety) and (2) using selective codon optimization to increase expression of the major virus antigen, hemagglutinin [HA] (increasing immunogenicity). Aim 1 will focus on the construction and in vitro characterization of an enhanced LAIV. To do this, we will identify the minimal essential mutations necessary for the ts polymerase phenotype of SGR-ts, using viral replication assays at selected temperatures (33, 37, and 39oC). We will test whether introduction of these mutations into a conventional H1N1 LAIV can further reduce the viral shutoff temperature, while preserving replication at 33oC - thus creating a "ts enhanced" H1N1 LAIV. Finally, we will confirm the reproducibility of our findings in the context of an H3N2 LAIV, and we will test whether the "ts-enhanced" H1N1 LAIV can be further improved by using a codon optimization strategy to increase HA expression. Aim 2 will compare the safety and efficacy of a ts-enhanced H1N1 LAIV (+/- a codon optimized HA) to that of a conventional H1N1 LAIV (also +/- a codon optimized HA). Safety will be assessed by determining MLD50 in mice, as well as weight loss over the course of infection, and virus titers in lung and nasal tract following high-dose infection. Immunogenicity and protective efficacy will be assessed by measuring titers of HA-binding and virus-neutralizing antibodies in immunized mice, and by testing the protection of immunized mice from a lethal H1N1 virus challenge. Finally, we will examine the safety and efficacy of our LAIVs in a second animal model - ferrets - since this model was used during studies that led to the original licensure of LAIV.

描述（由申请方提供）：已证明流感减毒活疫苗（LAIV）在儿童中的有效性高于灭活疫苗。然而，由于不可接受的安全性，它没有被许可用于哮喘患者或两岁以下的儿童。如果可以进一步减弱LAIV，同时保留免疫原性，这些安全性问题可以得到缓解，LAIV的使用可以扩展到覆盖这些群体。 我们最近发现了一种新的流感病毒突变体，与传统的LAIV相比，它具有更高的温度敏感性。与具有正常温度敏感性的表型回复突变病毒相比，该病毒的基因组（命名为SGR-ts）的特征在于病毒聚合酶内的五个点突变。其中三个突变（在PB 1和PB 2中）足以在37 ℃下几乎完全消除病毒聚合酶活性，而没有影响 重要的是，这些突变都与当前的LAIV没有共同之处。这些发现表明，通过减少LAIV的关闭时间， 温度（聚合酶活性消失的温度）至37 ℃。 我们的中心假设是LAIV的安全性和有效性可以通过以下方式增加：（1）使用新发现的聚合酶突变来降低病毒复制的关闭温度（增加安全性）和（2）使用选择性密码子优化来增加主要病毒抗原血凝素[HA]的表达（增加免疫原性）。 目的1将集中于增强LAIV的构建和体外表征。为此，我们将在选定的温度（33、37和39 ° C）下使用病毒复制试验，鉴定SGR-ts的ts聚合酶表型所需的最小必要突变。我们将测试将这些突变引入传统的H1N1 LAIV是否可以进一步降低病毒关闭温度，同时在33 ℃下保持复制-从而产生“ts增强”的H1N1 LAIV。最后，我们将确认我们的研究结果在H3 N2 LAIV的背景下的再现性，我们将测试是否可以通过使用密码子优化策略来增加HA表达来进一步改善“TS增强的”H1N1 LAIV。 目标2将比较ts增强的H1N1 LAIV（+/-密码子优化的HA）与传统H1N1 LAIV（也是+/-密码子优化的HA）的安全性和有效性。将通过测定小鼠中的MLD 50以及感染过程中的体重减轻以及高剂量感染后肺和鼻道中的病毒滴度来评估安全性。将通过测量免疫小鼠中HA结合和病毒中和抗体的滴度，以及通过测试免疫小鼠对致死性H1N1病毒攻击的保护来评估免疫原性和保护效力。最后，我们将在第二种动物模型-雪貂-中检查LAIV的安全性和有效性，因为该模型在导致LAIV最初许可的研究中使用。