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（由申请人提供）：已证明活体流感疫苗（LAIV）在儿童中具有更大的疗效。但是，由于安全性不可接受，它均未在哮喘患者或AG下的儿童中获得许可。如果可以在保留免疫原性的同时进一步减弱LAIV，则可以缓解这些安全问题，并且可以扩展使用LAIV以覆盖这些群体。 与常规LAIV相比，我们最近发现了一种新型的流感病毒突变体，其温度敏感性提高了。与具有正常温度敏感性的表型再生病毒相比，该病毒（指定的SGR-TS）的基因组（指定的SGR-TS）的特征是病毒聚合酶内五个突变。这些突变中的三个（在PB1和PB2中）足以几乎完全消除37oC的病毒聚合酶活性，而没有影响 关于34oC的活动；重要的是，这些突变都没有与当前的LAIV共享。这些发现表明，应该通过减少关闭方式来提高LAIV的安全性 温度（废除聚合酶活性的温度）至37oC。 我们的中心假设是：（1）使用新发现的聚合酶突变可以提高LAIV的安全性和有效性，以降低病毒复制的关闭温度（增加安全性），（2）（2）使用选择性密码子优化以增加病毒抗原的主要表达，增加了脑血凝集素[HA]（增加免疫原性）。 AIM 1将集中于增强LAIV的构建和体外表征。为此，我们将使用在选定温度下使用病毒复制测定法（33、37和39oC）来确定SGR-TS TS聚合酶表型所必需的最小本质突变。我们将测试将这些突变引入常规的H1N1 LAIV是否可以进一步降低病毒关闭温度，同时保留在33oC处的复制 - 从而产生了“ TS增强” H1N1 LAIV。最后，我们将在H3N2 LAIV的背景下确认我们的发现的可重复性，我们将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” H1N1 LAIV。 AIM 2将比较TS增强的H1N1 LAIV（+/-一个密码子优化的HA）与常规H1N1 LAIV（也是+/- +/-密码子优化的HA）的安全性和功效。将通过确定小鼠的MLD50以及在感染过程中的体重减轻来评估安全性，以及高剂量感染后肺和鼻腔中的病毒滴度。通过测量免疫小鼠中HA结合和病毒中和抗体的滴度来评估免疫原性和保护效能，并通过测试免疫小鼠免受致命H1N1病毒挑战的保护。最后，我们将在第二种动物模型（雪貂）中检查LAIV的安全性和功效，因为在导致LAIV的原始许可的研究中使用了该模型。