Pneumovirus Biology And Vaccine Development

肺炎病毒生物学和疫苗开发

• 批准号: 6985558
• 负责人: PETER LEON COLLINS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6985558
• 关键词: Pan; attenuated microorganism; biotechnology; communicable disease control; gene expression; gene mutation; host organism interaction; immunoregulation; laboratory mouse; live vaccine; microorganism immunology; molecular genetics; pediatrics; recombinant virus; respiratory infections; respiratory syncytial virus; tissue /cell culture; vaccine development; vaccine evaluation; viral vaccines; virus antigen; virus genetics; virus infection mechanism; virus protein

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.

人类呼吸道合胞病毒(HRSV)是世界范围内引起儿童呼吸道疾病的最重要的病原体，对一般成人，特别是老年人和骨髓移植受者也很重要。疫苗开发的障碍包括病毒在细胞培养中生长不良，在大多数动物模型中感染的半允许性，难以在免疫原性和衰减性之间实现适当的平衡，以及幼儿免疫反应的低效。我们以前开发了一种完全从cDNA克隆(反向遗传学)产生传染性RSV的方法，通过这种方法，可以将特定的变化通过cDNA中间体引入到传染性病毒中。我们已经广泛地使用这项技术来定位和表征病毒基因组中的RNA信号，以及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疫苗的有效方法，主要重点是开发一种鼻腔给药的儿科疫苗，该疫苗将与人类偏肺病毒和人类副流感病毒1、2和3型疫苗一起使用，这些疫苗也在LID/NIAID中开发。 减毒HRSV的一种方法是基于减弱点突变，我们通过对传统生物学方法开发的一组现有候选疫苗的序列分析确定了这些点突变。其中一些涉及温度敏感(Ts)点突变，每一种突变都是独立衰减的。TS突变有可能增加较温暖的下呼吸道的生长限制，因此可能提供更高的安全性。第二个衰减性元件是一组五个非ts点突变，当它们一起使用时会减弱。第三种减毒方法是基于我们的发现，即NS1、NS2、SH和M2-2基因不是必需的，可以单独删除，也可以在某些组合中删除，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是不可逆转的。 已经构建了一些含有点突变和/或基因缺失的减毒病毒。其中一个称为rA2cp248/404/1030/delSH，含有4个涉及点突变和SH基因缺失的减毒元件，在婴幼儿中被发现具有理想的减毒和免疫原性特征。然而，存在涉及两个ts点突变之一的单点突变逆转的发生率。这表明需要增加遗传稳定性的ts点突变。 我们目前正在遵循一项稳定？稳定？的战略。这些ts氨基酸的点突变可对抗衰减表型的丢失。这一策略包括：(I)确定与点突变位点上的每个氨基酸分配相关的表型；(Ii)利用遗传密码的简并性来选择用于减毒的密码子。氨基酸分配与所有密码子尽可能多的核苷酸不同，任何可能的、非衰减性的？任务。因此，衰减性分配的丢失将需要两到三个核苷酸替换，并且将相应地较少发生。 缺失NS1和/或NS2基因的病毒可诱导A549单核细胞系和人单核细胞来源的巨噬细胞产生大量的干扰素。NS1和NS2蛋白被证明独立和协同地干扰干扰素调节因子3的激活，干扰素调节因子3是参与诱导合成干扰素α和β的转录因子之一。这为这些突变的衰减表型奠定了基础，包括减轻病毒诱导的对宿主先天免疫反应的干扰。由于干扰素α和β上调先天免疫和获得性免疫，缺乏NS1和/或NS2基因的HRSV具有增强免疫原性的潜力。 相反，上述ts点突变涉及病毒多聚酶蛋白或转录基因启动信号，因此可能通过减少病毒RNA合成来发挥其减弱作用。非ts突变涉及N、F和L蛋白，其减弱作用的基础尚不清楚。另一个衰弱的基础是M2-2基因的缺失，它改变了病毒RNA合成程序，导致基因组复制下调，基因转录和病毒蛋白合成(包括主要保护性抗原)上调。对于活疫苗来说，包括增加病毒抗原合成的减毒表型应该特别有利。 另一种已被研究的减弱HRSV的策略是替换一个或多个编码内部？与牛呼吸道合胞病毒(BRSV)的同源蛋白。替换基因的例子包括NS1、NS2、N和P基因。BRSV在灵长类动物体内复制的自然宿主范围限制是其衰减的基础。这表明，P基因的替换产生了一个有希望的衰减水平，而NS1、NS2和N基因的替换对衰减的影响很小。这基于另一个原则，即宿主范围限制，提供了一种用于衰减HRSV的附加方法。