Enhanced Live Attenuated Influenza Virus With Improved Safety and Immunogenicity

描述(由申请人提供)：减毒活流感疫苗(LAIV)已被证明比灭活疫苗对儿童有更好的疗效。然而，由于不可接受的安全性，它没有被批准用于哮喘患者或两岁以下的儿童。如果可以在保持免疫原性的同时进一步减弱LAIV，这些安全问题就可以减轻，LAIV的使用可以扩大到覆盖这些群体。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突变病毒相比，这种病毒的基因组(命名为SGR-ts)的特征是病毒聚合酶中有五个点突变。这些突变中的三个(在PB1和PB2中)足以在37摄氏度几乎完全消除病毒聚合酶的活性，同时没有任何影响 在34oC的活性上；重要的是，这些突变都不与目前的LAIV共享。这些发现表明，通过减少LAIV的关闭，应该有可能增加LAIV的安全性 温度(聚合酶活性丧失的温度)到37摄氏度。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提高：(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(提高免疫原性)。目标1将重点介绍增强型LAIV的构建和体外特性。为了做到这一点，我们将在选定的温度(33、37和39摄氏度)下使用病毒复制分析来确定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的关闭温度，同时保持33oC的复制--从而创建“ts增强型”H1N1 LAIV。最后，我们将在H3N2LAIV的背景下确认我们的发现的可重复性，并将测试是否可以通过使用密码子优化策略来增加HA表达来进一步改进“TS增强的”H1N1 LAIV。目的2比较TS增强型H1N1 LAIV(+/-a密码子优化HA)与传统H1N1 LAIV(+/-a密码子优化HA)的安全性和有效性。安全性将通过测定小鼠的MLD50，以及感染过程中的体重下降，以及大剂量感染后肺和鼻道中的病毒滴度来评估。将通过测量免疫小鼠中HA结合抗体和病毒中和抗体的滴度，以及测试免疫小鼠免受致命H1N1病毒攻击的保护，来评估免疫原性和保护效果。最后，我们将在第二个动物模型-雪貂身上检验我们的LAIV的安全性和有效性，因为这个模型是在导致LAIV最初获得许可的研究中使用的。

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.