Pneumovirus Biology And Vaccine Development

人呼吸道合胞病毒（human respiratory syncytial virus，HRSV）是世界范围内小儿呼吸道疾病最重要的病毒因子，在成人、老年人和骨髓移植受者中尤为重要。疫苗开发的障碍包括病毒在细胞培养物中生长不良，大多数动物模型中感染的半容许性质，难以在免疫原性和减毒之间达到适当的平衡，以及非常年幼的婴儿中免疫应答的效率低下。我们以前开发了一种完全从cDNA克隆（？反向遗传学？），由此可以通过cDNA中间体将确定的变化引入感染性病毒。我们已经广泛使用这种技术来绘制和表征病毒基因组中的RNA信号，并表征病毒基因和蛋白质。此外，反向遗传学是开发减毒活疫苗候选物的有力方法，主要重点是开发鼻内给药的儿科疫苗，该疫苗将与LID/NIAID中也在开发的人偏肺病毒和人副流感病毒1、2和3的疫苗结合使用。 减毒HRSV的一种方法是基于我们通过常规生物学方法开发的一组现有候选疫苗的序列分析鉴定的减毒点突变。其中一些涉及温度敏感（ts）点突变，每一个都是独立的衰减。Ts突变有可能增加较温暖下呼吸道的生长限制，因此可能提供更高的安全性。第二个衰减元件是一组五个非ts点突变，当一起使用时会衰减。第三种减毒方法是基于我们的发现，即NS 1、NS 2、SH和M2-2基因是非必需的，可以单独删除或以某些组合删除，以产生在体外复制良好但在体内减毒的病毒。基因缺失突变体的一个优点是它们应该对回复突变具有抗性。 已经构建了许多含有点突变和/或基因缺失的减毒病毒。其中之一，称为rA 2cp 248/404/1030/delSH，含有四个减毒元件，涉及点突变与SH基因缺失的组合，并被发现在婴儿和幼儿中具有理想的减毒和免疫原性特征。然而，也有涉及两个ts点突变之一的单一点突变的逆转发生率。这表明需要增加遗传稳定性的ts点突变 我们目前正在采取一项战略，以？稳定？这些TS氨基酸点突变对抗减毒表型的丧失。该策略涉及（i）确定与点突变位点的每个氨基酸分配相关的表型，以及（ii）使用遗传密码的简并性来选择密码子？衰减？氨基酸分配，不同的核苷酸尽可能多的所有密码子的任何可能？非衰减性？分配任务。因此，减毒分配的丧失将需要两个或三个核苷酸取代，并且将相应地较不频繁。 发现其中NS 1和/或NS 2基因已被删除的病毒在A549 pnemocyte细胞系和人单核细胞衍生的巨噬细胞中诱导大量干扰素α、β和λ的产生。NS 1和NS 2蛋白显示独立地和协同地干扰干扰素调节因子3的活化，干扰素调节因子3是参与诱导干扰素α和β合成的转录因子之一。这确定了这些突变的减毒表型的基础，其涉及减轻病毒诱导的对宿主先天免疫应答的干扰。由于干扰素α和β上调先天性和适应性免疫，因此缺乏NS 1和/或NS 2基因的HRSV具有增加免疫原性的潜力。 相反，上述ts点突变涉及病毒聚合酶蛋白或转录基因起始信号，因此可能通过减少病毒RNA合成来发挥其减毒作用。非ts突变涉及N、F和L蛋白，并且其减弱效应的基础尚不清楚。减毒的另一个基础是M2-2基因的缺失，其改变了病毒RNA合成程序并导致基因组复制的下调和基因转录的上调以及病毒蛋白质（包括主要保护性抗原）的合成。涉及病毒抗原合成增加的减毒表型对于活疫苗应该特别有利。 另一种策略，已被调查减毒HRSV是取代一个或多个基因编码？内部？蛋白质与来自牛RSV（BRSV）的对应物。替代基因的实例包括NS 1、NS 2、N和P基因。减毒的基础是BRSV在灵长类动物中复制的天然宿主范围限制。这表明P基因的取代产生了有希望的减毒水平，而NS 1、NS 2和N基因的取代具有小的减毒效果。这提供了用于基于又一原理（即，主机范围限制）来衰减HRSV的附加方法。

Human respiratory syncytial virus (HRSV) is the most important viral agent of pediatric respiratory tract disease worldwide and also is important in adults in general and in the elderly and bone marrow transplant recipients in particular. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi-permissive nature of infection in most animal models, the difficulty of achieving an appropriate balance between immunogenicity and attenuation, and the inefficiency of the immune response in the very young infant. We previously developed a method for producing infectious RSV entirely from cDNA clones (?reverse genetics?), whereby defined changes can be introduced into infectious virus via the cDNA intermediate. We have used this technique extensively to map and characterize RNA signals in the viral genome and to characterize the viral genes and proteins. In addition, reverse genetics is a powerful method for developing live-attenuated vaccine candidates, with the primary focus being the development of an intranasally-administered pediatric vaccine that would be used in conjunction with vaccines for human metapneumovirus and human parainfluenza viruses 1, 2 and 3 that also are under development in the LID/NIAID. One means for attenuating HRSV is based on attenuating point mutations that we identified by sequence analysis of a panel of existing vaccine candidates developed by conventional biological methods. Some of these involve temperature-sensitive (ts) point mutations, each of which is independently attenuating. Ts mutations have the potential for increased growth restriction in the warmer lower respiratory tract and thus might provide increased safety. A second attenuating element is a set of five non-ts point mutations that is attenuating when used together. A third means of attenuation is based on our finding that the NS1, NS2, SH, and M2-2 genes are nonessential and can be deleted individually and in certain combinations to yield viruses that replicate well in vitro but are attenuated in vivo. An advantage of gene deletion mutants is that they should be refractory to reversion. A number of attenuated viruses containing point mutations and/or gene deletions have been constructed. One of these, called rA2cp248/404/1030/delSH, contained four attenuating elements involving point mutations combined with deletion of the SH gene, and was found to have desirable characteristics of attenuation and immunogenicity in infants and young children. However, there was incidence of reversion of a single point mutation involving either of two ts point mutations. This indicated a need for ts point mutations that have increased genetic stability We presently are following a strategy to ?stabilize? these ts amino acid point mutations against loss of the attenuation phenotype. This strategy involves (i) determining the phenotypes associated with every amino acid assignment at the point mutation locus, and (ii) using the degeneracy of the genetic code to choose a codon for an ?attenuating? amino acid assignment that differs by as many nucleotides as possible from all codons for any possible ?non-attenuating? assignments. Thus, the loss of the attenuating assignment would require two or three nucleotide substitutions and will be correspondingly less frequent. Viruses in which the NS1 and/or NS2 genes had been deleted were found to induce the production of large amounts of interferon alpha, beta and lambda in the A549 pnemocyte cell line and in human monocyte-derived macrophages. The NS1 and NS2 proteins were shown to independently and cooperatively interfere with the activation of interferon regulatory factor 3, which is one of the transcription factors involved in inducing the synthesis of interferon alpha and beta. This identified a basis for the attenuation phenotype of these mutations, one that involves alleviating virus-induced interference with the host innate immune response. Because interferons alpha and beta up-regulate both innate and adaptive immunity, HRSV lacking the NS1 and/or NS2 gene has the potential for increased immunogenicity. In contrast, the ts point mutations described above involve the viiral polymerase protein or a transcription gene-start signal, and hence likely exert their attenuating effect by reducing viral RNA synthesis. The non-ts mutations involve the N, F and L proteins, and the basis for their attenuating effect is not known. Yet another basis of attenuation is exemplified by deletion of the M2-2 gene, which alters the viral RNA synthetic program and results in a down-regulation of genome replication and an up-regulation of gene transcription and the synthesis of the viral proteins, including the major protective antigens. An attenuated phenotype that involves increased synthesis of the viral antigens should be particularly advantageous for a live vaccine. Another strategy that has been investigated for attenuating HRSV is to replace one or more genes encoding ?internal? proteins with its counterpart from bovine RSV (BRSV). Examples of replaced genes include the NS1, NS2, N and P genes. The basis for attenuation is the natural host range restriction of BRSV for replication in primates. This showed that substitution of the P gene yielded a promising level of attenuation, whereas substitution of the NS1, NS2 and N genes had small attenuating effects. This provides an additional method for attenuating HRSV based on yet another principle, namely host range restriction.