Pneumovirus Biology And Vaccine Development

肺炎病毒生物学和疫苗开发

• 批准号: 7192842
• 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/7192842
• 关键词: Pan; attenuated microorganism; biotechnology; communicable disease control; confocal scanning microscopy; enzyme linked immunosorbent assay; 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. There is no approved vaccine or effective antiviral therapy. 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 attenuated viruses for use as a live intranasal pediatric vaccine in conjunction with vaccines for human metapneumovirus and human parainfluenza viruses 1, 2 and 3 that also are under development in LID/NIAID. Obstacles to vaccine development include the difficulty of achieving an appropriate balance between immunogenicity and attenuation and the inefficiency of the immune response in the very young infant. As described in an accompanying report (B. R. Murphy et al), we developed several recombinant vaccine candidates are in clinical trials. One virus, called rA2cp248/404/1030/delSH, was safe and immunogenic in young infants. We presently are constructing additional candidates with point mutations that have been stabilized genetically or which have deletions of genes encoding non-essential accessory proteins. We previously showed that HRSV nonstructural proteins 1 and 2 (NS1 and NS2) inhibit the induction of interferon (IFN)-a/b in A549 cells and human macrophages. Two principal transcription factors for the early IFN-b and -a1 response are interferon regulatory factor (IRF)-3 and nuclear factor-kB (NF-kB). At early times post-infection, wild type HRSV and the NS1/NS2 deletion mutants were very similar in the ability to activate IRF-3. However, once NS1 and NS2 were expressed significantly, they act independently and cooperatively to suppress activation and nuclear translocation of IRF-3. 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. We also found that deletion of the NS2 gene sharply reduced the ability of HRSV to induce activation of NF-kB. Since recombinant HRSVs from which the NS1 or NS2 genes have been deleted are being developed as vaccine candidates, we investigated whether the changes in activation of host transcription factors and increased IFN-a/b production had an effect on the epithelial production of proinflammatory factors. Viruses lacking NS1 and/or NS2 stimulated modestly lower production of RANTES (Regulated on Activation Normal T-cell Expressed and Secreted), interleukin (IL)-8 and tumor necrosis factor (TNF)-a compared to wt-rRSV, supporting their use as attenuated vaccine candidates. The attachment G protein of HRSV has a conserved cysteine-rich region whose function is unknown and which we previously showed is not necessary for efficient viral replication. In studies with collaborators at Johns Hopkins University School of Medicine, this cysteine domain was shown to be a powerful inhibitor of the innate immune response against RSV, and that early secretion of G is critical to modulate inflammation after RSV infection. Importantly, the cysteine domain is also a potent inhibitor of cytokine production mediated by several TLR agonists, indicating that this peptide sequence displays broad anti-inflammatory properties. This indicates that this domain has the potential to alter HRSV pathogenesis and immunogenicity, although further study will be needed before we understand its impact to a live vaccine. We investigated the effect of increased local expression of the Th2 cytokine interleukin (IL)-4 from the genome of a recombinant HRSV. This was done because Th2 cytokines like IL-4 induced by certain experimental HRSV vaccines are associated with enhanced disease upon subsequent HRSV exposure. Mice infected with HRSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with the wt HRSV, although the wt HRSV group caught up by day 8. Infection with HRSV/IL-4 also induced a CTL response that was nonfunctional. Following challenge with wt HRSV, there was essentially no increase in the amount of pulmonary inflammation compared to animals that had previously been infected with wild type HRSV, and the secondary CTL response was not significantly affected. Thus, a strong Th2 environment during primary pulmonary immunization with live HRSV resulted in enhanced inflammation and a nonfunctional CTL response, but had a minimal effect on disease and the host immune response following secondary infection. This supports that idea that live HRSV vaccines are not associated with enhanced disease. We previously used helper-dependent mini-replicon surrogates of the HRSV genome to show that the 44 nt leader (Le) region is sufficient to initiate RNA replication, producing antigenome RNA, and that the Le and adjoining gene start (GS) signal of the first gene are sufficient to initiate transcription, producing mRNA. A cis-acting element necessary for both transcription and replication was mapped within the first 11 nts at the 3' end of Le. In collaboration with a previous lab member now at the University of Dundee, this analysis was extended to shown that, although the GS signal in conjunction with the 3' terminal 11-nt sequence was sufficient to direct accurate mRNA synthesis initiation, nts 36-43 located immediately upstream of the GS signal were found to be necessary for optimal levels of mRNA synthesis. With regards replication, the first 15 nts of Le were found to be sufficient to direct initiation of antigenome synthesis, but efficient encapsidation and production of full-length antigenome were dependent additionally on nts 16-34. This provides a detailed picture of the HRSV promoter. Pneumonia virus of mice (PVM) is a pneumovirus that causes respiratory tract disease in the mouse, and thus can be used as a model for pneumovirus infection in a convenient animal model. In order to develop a reverse genetics system for PVM, we determined a complete consensus sequence for the genome of strain 15. A complete sequence had not been previously available. In addition, the incomplete sequence that was available was for a cell culture-adapted derivative that had sustained attenuating mutations including loss of part of the G glycoprotein. Thus, this represents the first complete sequence of a virulent strain of PMV.

人呼吸道合胞病毒（HRSV）是世界范围内儿童呼吸道疾病最重要的病毒病原体，在成人、老年人和骨髓移植受者中也很重要。目前还没有批准的疫苗或有效的抗病毒治疗方法。我们之前开发了一种完全从cDNA克隆(？反向遗传学？)，由此定义的变化可以通过cDNA中间体引入感染性病毒。我们已经广泛使用这项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病毒的有力方法，可与LID/NIAID正在开发的人偏肺病毒和人副流感病毒1、2和3的疫苗一起作为儿童鼻内活疫苗使用。疫苗开发的障碍包括难以在免疫原性和衰减之间取得适当的平衡，以及幼龄婴儿的免疫反应效率低下。