Laboratory And Pre-clinical Studies Of Parainfluenza Viruses

副流感病毒的实验室和临床前研究

• 批准号: 7592133
• 负责人: Brian Murphy
• 金额: $ 150.97万
• 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/7592133
• 关键词: A549; Accounting; Address; Affect; African; Amino Acid Substitution; Amino Acids; Animals; Antigens; Attenuated; Attenuated Live Virus Vaccine; Bronchiolitis; Cells; Cercopithecus pygerythrus; Child; Childhood; Cistrons; Clinical; Clinical Trials; Complementary DNA; Croup; Data; Deletion Mutation; Disease; Electroporation; Equilibrium; Evaluation; Family; Future; Generations; Genes; Genetic; Genome; Genomics; Goals; Heat-Shock Response; Hemagglutinin; Hospitalization; Human; Human Parainfluenza Virus 2; Immunization; In Vitro; Infant; Infection; Interferon Activation; Interferon Type I; Interferons; Laboratories; Licensing; Life; Lipids; Lower respiratory tract structure; Lung diseases; Membrane Glycoproteins; Methods; Molecular Virology; Monkeys; Mutation; Neuraminidase; Nucleocapsid; Nucleocapsid Proteins; Nucleotides; Otitis Media; Para-Influenza Virus Type 1; Paramyxoviridae; Paramyxovirinae; Pharyngitis; Phenotype; Phosphoproteins; Plasmids; Pneumonia; Pneumovirus vaccine; Point Mutation; Polymerase; Positioning Attribute; Preparation; Procedures; Production; Property; Proteins; RNA Viruses; Range; Recombinants; Recovery; Respiratory Tract Diseases; Respirovirus; Rhinitis; Rubulavirus; Safety; Serotyping; Signal Induction; Signal Transduction; System; Techniques; Temperature; Testing; Transfection; Vaccines; Vero Cells; Vertebral column; Viral; Virus; attenuation; base; citrate carrier; design; human IRF3 protein; immunogenic; immunogenicity; in vivo; interferon regulatory factor-3; multiple myeloma M Protein; mutant; novel; parainfluenza virus; positional cloning; preclinical study; promoter; response; vaccine development

Human parainfluenza virus type 1 (HPIV1) is a significant cause of severe respiratory tract disease in infants and young children. HPIV1 is an enveloped, non-segmented, single-stranded, negative-sense RNA virus belonging to the subfamily Paramyxovirinae within the Paramyxoviridae family, which also includes the HPIV2 and HPIV3 serotypes. These serotypes can be further classified as belonging to either the Respirovirus (HPIV1 and HPIV3) or Rubulavirus (HPIV2) genus and are immunologically distinct in that primary infection does not result in cross-neutralization or cross-protection. The HPIV1 genome encodes three nucleocapsid-associated proteins including the nucleocapsid protein (N), the phosphoprotein (P) and the large polymerase (L) and three envelope-associated proteins including the internal matrix protein (M) and the fusion (F) and hemagglutinin-neuraminidase (HN) transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and are the major viral protective antigens. The HPIVs cause respiratory tract disease ranging from mild illness, including rhinitis, pharyngitis, and otitis media, to severe disease, including croup, bronchiolitis, and pneumonia. HPIV1, HPIV2 and HPIV3 have been identified as the etiologic agents responsible for 6.0%, 3.3% and 11.5%, respectively, of hospitalizations of infants and young children for respiratory tract disease. Together these viruses account for approximately 20% of all pediatric hospitalizations due to respiratory disease. A licensed vaccine is currently not available for any of the HPIVs. HPIV2 vaccine development Reverse genetics was used previously to generate attenuating mutations in the L polymerase protein of human parainfluenza virus type 2 (HPIV2) and to enhance their genetic stability. An attenuating mutation at nucleotide 15 (15TC ) in the 3 genomic promoter was identified and was found to be present in the previously characterized mutants. We evaluated the properties of this promoter mutation alone and in various combinations with the L polymerase mutations. Amino acid substitutions at L protein positions 460 (460A or 460P) or 948 (948L), or deletion of amino acids 1724 and 1725 (1724), each conferred a temperature sensitivity (ts) phenotype whereas the 15TC mutation did not. The 460A and 948L mutations each contributed to restricted replication in the lower respiratory tract of African green monkeys, but the 1724 mutation increased attenuation only in certain combinations with other mutations. We constructed two highly attenuated viruses, rV94(15C)460A948L and rV94(15C)948L1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys and, therefore, appear to be suitable for evaluation in humans. Studies have been initiated to generate HPIV2 backbones in with the P and V genes have been separated in two cistrons to permit introductions of mutations in V that do not affect P. HPIV1 vaccine development Two recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) mutant viruses have been developed, using a reverse genetics system, for evaluation as potential intranasal vaccine candidates. These rHPIV1 vaccine candidates have two non-temperature sensitive (non-ts) attenuating (att) mutations primarily in the PC gene, namely CR84GHNT553A (two point mutations used together as a set) and C170 (a short deletion mutation), and two ts att mutations in the L gene, namely LY942A (a point mutation), and L1710-11 (a short deletion), the last of which has not been previously described. The latter three mutations were specifically designed for increased genetic and phenotypic stability. These mutations were evaluated on the HPIV1 backbone, both individually and in combination, for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs). The rHPIV1 mutant bearing the novel L1710-11 mutation was highly ts and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-CR84G170HNT553ALY942A and rHPIV1-CR84G170HNT553AL1710-11 vaccine candidates were highly ts, with shut-off temperatures of 38C and 35C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-CR84G170HNT553ALY942A protected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-CR84G170HNT553AL1710-11 was not protective in AGMs due to over-attenuation, but it is expected to replicate more efficiently and be more immunogenic in the natural human host. The rHPIV1-CR84G170HNT553ALY942A and rHPIV1-CR84G170HNT553AL1710-11 vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans. Studies have been initiated to generate HPIV1 backbones in with the P and C genes have been separated in two cistrons to permit introductions of mutations in C that do not affect P. HPIV1 mutations in C and the interferon response. Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion () mutations in the PC gene (r-C10-15HNT553A, r-CR84G, r-CF170S and r-C170), which have previously been evaluated as HPIV1 vaccine candidates, were evaluated for their effect on the type I interferon (IFN) response in vitro. HPIV1 wt infection inhibited the IFN response by inhibiting IFN regulatory factor-3 (IRF-3) activation and IFN production in A549 cells and IFN signaling in Vero cells. In contrast, r-CR84G, r-CF170S and r-C170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-C170 did not inhibit IFN signaling. Thus, HPIV1 antagonizes the IFN response at both the level of induction and signaling, and antagonism at both levels was disrupted by mutations in the PC gene. Since CF170S affects C and not P, the anti-IFN function can be attributed to the C proteins. These data, in the context of previous in vivo studies, suggest that the loss of antagonism of the IFN response at both the level of induction and signaling, observed with the PC mutants, r-CF170S and r-C170, was necessary for significant attenuation in African green monkeys (AGMs). Plasmid only system for recovery of PIV vaccine candidates A method for the generation of clinical grade, live-attenuated vaccines in Vero cells entirely from cDNA plasmids was developed. The entire electroporation procedure can be completed in less than 15 minutes and this is a significant improvement over previous lipid or electroporation based transfection techniques that also involve a heat-shock step. Importantly, the virus preparations can be generated with a minimal use of animal product derived materials, an important consideration for a vaccine candidate that is to be tested in humans. Since it is likely that all live-attenuated parainfluenza virus and pneumovirus vaccines in the future will be generated using reverse genetics, this simplified method provides guidance on how this can be achieved.

人类副磷氟糖1型（HPIV1）是婴儿和幼儿严重呼吸道疾病的重要原因。 HPIV1是一种包裹，非细分，单链，负义的RNA病毒，属于paramyxoviridae家族中的paramyxovirinae，其中还包括HPIV2和HPIV3血清型。这些血清型可以进一步归类为属于呼吸病毒（HPIV1和HPIV3）或Rubulavirus（HPIV2）属，并且在免疫学上是不同的，因为原发性感染不会导致交叉中性化或交叉保护。 HPIV1基因组编码三种与核苷酸相关的蛋白质，包括核苷酸蛋白（N），磷酸蛋白（P）和大型聚合酶（L）和三个包膜相关蛋白，包括内部基质蛋白（M）以及融合蛋白（F）和血小蛋白酶（Hemagglutin-neramin-neramin-neramin-necomemencase）。 F和HN是两个病毒中和抗原，是主要的病毒保护抗原。 HPIV会引起呼吸道疾病，包括轻度疾病，包括鼻炎，咽炎和中耳炎，到严重疾病，包括臀部，支气管炎和肺炎。 HPIV1，HPIV2和HPIV3已被确定为病因学剂，分别为呼吸道疾病的婴儿和幼儿住院治疗分别为6.0％，3.3％和11.5％。由于呼吸道疾病，这些病毒约占所有儿科住院的20％。目前，任何HPIVS都无法使用持牌疫苗。 HPIV2疫苗开发 反向遗传学以前被用来在人parainfluenza病毒2型（HPIV2）的L聚合酶蛋白中产生衰减突变，并增强其遗传稳定性。 鉴定了3个基因组启动子中核苷酸15（15TC）处的衰减突变，发现在先前表征的突变体中存在。我们单独评估了该启动子突变的特性，并与L聚合酶突变进行了各种组合。 L蛋白位置的氨基酸取代460（460a或460p）或948（948L），或氨基酸1724和1725（1724）的缺失，每个氨基酸均赋予温度敏感性（TS）表型，而15TC突变则没有。 460a和948l突变都导致非洲绿猴下呼吸道中的复制受限，但仅在某些与其他突变的组合中，1724年的突变仅增加了衰减。 我们构建了两种高度减毒病毒RV94（15C）460A948L和RV94（15C）948L1724，它们具有免疫原性和保护性，可抵抗非洲绿猴中野生型HPIV2的挑战，因此，在人类中似乎适合于人类评估。 已经启动了研究以与P和V基因分离的两个Cistron中的P和V基因的HPIV2骨架，以允许在V中引入突变的引入。 HPIV1疫苗开发 使用反向遗传学系统，已经开发了两种重组的活体parainfluenza病毒1型（RHPIV1）突变病毒，以评估作为潜在的鼻内疫苗候选物。这些RHPIV1疫苗候选物具有两个非温度敏感（非TS）衰减（ATT）突变，主要是PC基因中的突变，即CR84GHNT553A（一起使用的两个点突变）和C170和C170和C170和C170（一个短的缺失突变），以及两个TS ATT突变，两个TS ATT突变在l基因中，l1111101011 cut and ly942a（namy942a）简短删除），以前尚未描述最后一个。 后三个突变是专门设计用于提高遗传和表型稳定性的。 这些突变在非洲绿猴（AGMS）中的衰减，免疫原性和保护性疗效（AGMS）上对HPIV1主链进行了评估。 带有新型L1710-11突变的RHPIV1突变体高度TS，并在AGM中衰减，对HPIV1 WT挑战具有免疫原性和有效性。 RHPIV1-CR84G170HNT553ALY942A和RHPIV1-CR84G170HNT553AL1710-11710-11候选疫苗候选物高度TS，分别关闭温度为38C和35C，并且在AGMS中高度减弱。 在上呼吸道和下呼吸道中，用RHPIV1-CR84G170HNT553ALY942A免疫受到HPIV1 WT挑战的免疫接种。相比之下，由于过度衰减，RHPIV1-CR84G170HNT553AL1710-11在AGM中没有保护性，但预计它将更有效地复制并在自然人类宿主中更加免疫原性。 RHPIV1-CR84G170HNT553ALY942A和RHPIV1-CR84G170HNT553AL1710-11候选疫苗在AGMS中显然受到高度减弱，临床试验计划对人类的安全性和免疫发育来解决。 已经启动了研究以与P和C基因一起产生HPIV1骨架，在两个cistron中分离出来，以允许在不影响P的C中引入突变。 C和干扰素反应中的HPIV1突变。 重组人副帕氟二氮病毒1型（HPIV1）和包含点和缺失（）突变的突变体（R-C10-15HNT553A，R-CR84G，R-CR84G，R-CF170S和R-C170）先前对AS HPIV1疫苗进行了评估（如果在typern中进行了评估），则该类型均在Internor中进行了评估。 HPIV1 WT感染通过抑制A549细胞中的IFN调节因子-3（IRF-3）激活和IFN产生来抑制IFN反应，而VERO细胞中的IFN信号传导。 相反，R-CR84G，R-CF170S和R-C170因抑制IRF-3激活而有缺陷，IFN产生和R-CF170S和R-C170并未抑制IFN信号传导。 因此，HPIV1在诱导和信号传导水平上拮抗IFN响应，两种水平的拮抗作用都被PC基因突变所破坏。 由于CF170会影响C而不是P，因此抗IFN功能可以归因于C蛋白。 在先前的体内研究的背景下，这些数据表明，在PC突变体，R-CF170S和R-C170上观察到IFN反应水平上IFN反应的拮抗作用丧失对于非洲绿猴（AGMS）的大量衰减是必不可少的。 仅质粒的系统恢复PIV疫苗候选物 完全从cDNA质粒中开发了一种生成Vero细胞中临床等级的活体疫苗的方法。整个电穿孔过程可以在不到15分钟的时间内完成，这比以前的脂质或基于电穿孔的转染技术有了显着改善，该技术也涉及热冲步。重要的是，可以通过最少使用动物产品衍生的材料来产生病毒制剂，这是候选疫苗的重要考虑因素，该疫苗应在人类中进行测试。由于将来可能会使用反向遗传学生成所有活体衰减的Parainfluenza病毒和肺炎病毒疫苗，因此这种简化的方法提供了有关如何实现这一目标的指导。