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: 7732437
• 负责人: Brian Murphy
• 金额: $ 122.42万
• 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/7732437
• 关键词: A549; Accounting; Affect; Amino Acid Sequence; Amino Acids; Antigens; Apical; Apoptosis; Apoptosis Inhibitor; Apoptotic; Attenuated; Attenuated Live Virus Vaccine; Bronchiolitis; C protein, human parainfluenza virus type 1; C-terminal; Cells; Cercopithecus pygerythrus; Child; Childhood; Clinical; Complement; Complementary DNA; Croup; Cysteine; Cytopathology; Disabled Persons; Disease; Epithelial Cells; Equilibrium; Facility Construction Funding Category; Family; Fingers; Gene Expression; Gene Mutation; Gene-Modified; Genes; Genetic; Genome; Glycine decarboxylase; Goals; Hemagglutinin; Hospitalization; Human; Human Activities; Human Parainfluenza Virus 2; IRF3 gene; Immune response; In Vitro; Infant; Infection; Interferons; Kinetics; Laboratories; Licensing; Lung diseases; Membrane Glycoproteins; Messenger RNA; Microarray Analysis; Molecular Profiling; Molecular Virology; Mutation; NF-kappa B; Neuraminidase; Nuclear; Nucleocapsid; Nucleocapsid Proteins; Open Reading Frames; Otitis Media; Para-Influenza Virus Type 1; Paramyxoviridae; Paramyxovirinae; Pathway interactions; Pharyngitis; Phenotype; Phosphoproteins; Play; Pneumonia; Polymerase; Primates; Protein C; Proteins; RNA Viruses; Range; Recombinants; Respiratory Tract Diseases; Respirovirus; Rhinitis; Role; Rubulavirus; STAT2 gene; Serine; Serotyping; Signal Transduction; Site; Specific qualifier value; Surface; Vaccines; Vero Cells; Viral; Virulence; Virulence Factors; Virus; Virus Diseases; Virus Replication; airway epithelium; attenuation; caspase-3; citrate carrier; immunogenic; immunogenicity; in vitro Model; in vivo; multiple myeloma M Protein; mutant; parainfluenza virus; positional cloning; preclinical study; recombinant virus; respiratory; respiratory virus; response; tool; vaccine development; virus host interaction

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. HPIV1 vaccine development: We further sought to understand the role of the C proteins in HPIV1 replication in vitro and in vivo. Recombinant HPIV1 (rHPIV1) was modified to create rHPIV1-P(C-), a virus in which expression of the C proteins (C′, C, Y1 and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C-) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. rHPIV1-P(C-) replicated in vitro as efficiently as HPIV1 wt early during infection, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect (cpe) not observed with rHPIV1 wt. rHPIV1-P(C-) infection, but not rHPIV1 wt infection, resulted in activation of caspase 3 and caused nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to rHPIV1 wt, rHPIV1-P(C-) and rHPIV1-CF170S each induced IFN and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C-) induced apoptosis. Thus, the anti-IFN and anti-apoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C-) whereas only the anti-IFN activity is disabled in rHPIV1-CF170S. In AGMs, rHPIV1-P(C-) was considerably more attenuated than rHPIV1-CF170S, suggesting that disabling the anti-IFN and anti-apoptotic activities of HPIV1 had additive effects on attenuation in vivo. Thus, the C proteins of HPIV1 are non-essential but have anti-IFN and anti-apoptosis activities required for virulence in primates. In an in vitro model of human ciliated airway epithelium (HAE), a useful tool for studying respiratory virus-host interactions, HPIV1 wt selectively infected ciliated cells within the HAE and progeny virus was released from the apical surface with little apparent gross cytopathology. In HAE, type I IFN is induced following infection with rHPIV1-CF170S but not following infection with HPIV1 wt. IFN induction coincided with a 100 to 1000-fold reduction in virus titer, supporting the hypothesis that the HPIV1 C proteins are critical for inhibition of the innate immune response. A microarray-based analysis of the kinetics of gene expression of respiratory epithelial cells infected with wt or mutant HPIV1 or treated with IFNβ was performed to examine: 1) how wt HPIV1 infection alters human respiratory epithelial cell gene expression; 2) what role IFNβ plays in this response; 3) how the response to infection with the C mutant viruses, rHPIV1-CF170S and rHPIV1-P(C-), compares to infection with wt HPIV1; and 4) whether the phenotypic differences between the two C mutant viruses (level of attenuation and apoptosis phenotype) can be explained at the transcriptional level. mRNA levels in A549 cells treated with IFNβ or infected with HPIV1 wt, rHPIV1-CF170S, or rHPIV1-P(C-) were compared using a microarray that represented the full complement of known human genes. By 48 h post-infection, the CF170S mutant significantly induced 1,632 genes and suppressed 690 genes, whereas P(C-) induced 1,255 genes and suppressed 168 genes by 48 h p.i. Wt HPIV1 significantly induced 274 genes and suppressed 3 genes, and IFNβ treatment induced 176 genes and suppressed 1 gene. Therefore, rHPIV1s encoding C gene mutations modify the expression of up to 2,322 genes, whereas HPIV1 encoding the wt C gene modifies the expression of only 277 genes, indicating the profound effect the C proteins have on suppression of the host cell response to HPIV1 infection. The gene expression profiles of rHPIV1-P(C-) and rHPIV1-CF170S infected cells did not differ. In summary, the HPIV1 C proteins exert remarkable control over the cellular transcriptional response to viral infection, indicating that the C proteins are important virulence factors for HPIV1. Mutations within the C gene permit the activation of a broad array of cellular genes involved in the type I IFN, IRF3 and NF-kB pathways that would otherwise be repressed by HPIV1 infection, and these mutations specify an attenuation phenotype in vivo. 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. Last year we described the construction of two highly attenuated viruses, rHPIV2-V94(15C)/460A948L and rHPIV2-V94(15C)/948L/Δ1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys. A clinical lot of rHPIV2-V94(15C)/948L/Δ1724 has been manufactured and will be evaluated in humans in 2008 and 2009. The V gene of HPIV2, which has been described as an IFN antagonist promoting STAT2 degradation, is another target for the introduction of attenuating mutations. Since the P and V ORFs in the P/V gene of HPIV2 overlap, we separated the P and V ORFs into two gene units to permit the introduction of genetically stable mutations into V without affecting P. A HPIV2 P+V virus was recovered that had wt phenotype in vitro and in vivo. The V protein contains a RING finger-like domain in its C terminal region that is involved in STAT2 degradation. Mutations of the seven cysteine amino acid residues of the RING domain to serine were either lethal (could not be recovered) or highly debilitating, i.e., virus replication was over 1000-fold reduced in vitro, even in Vero cells that do not produce IFN. This indicated that the V protein is required for efficient replication of HPIV2 in vitro and that the RING domain is not a useful site for the introduction of att mutations intended for use in HPIV2 vaccines. We are seeking to identify mutations that modulate STAT2 degradation but that permit efficient replication in vitro.

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。 HPIV1疫苗的开发：我们进一步试图了解C蛋白在体外和体内HPIV1复制中的作用。 重组HPIV1（RHPIV1）被修饰以创建RHPIV1-P（C-），RHPIV1-P（C-）是一种病毒，其中C蛋白的表达（C'，C，Y1和Y2）沉默而不影响P蛋白的氨基酸序列。 感染性RHPIV1-P（C-）很容易从cDNA中回收，表明四种C蛋白对于病毒复制不是必需的。 RHPIV1-P（C-）在感染之前早期与HPIV1 WT一样有效地复制了体外，但随后其效率随着与Rhpiv1 WT观察到的广泛的细胞病作用（CPE）的发作相吻合。 RHPIV1-P（C-）感染，但没有RHPIV1 WT感染，导致caspase 3的激活，并在LLC-MK2细胞中引起核破坏，从而将HPIV1 C蛋白鉴定为凋亡的抑制剂。 与RHPIV1 WT相反，RHPIV1-P（C-）和RHPIV1-CF170诱导IFN，并且在体外没有抑制IFN信号传导。 但是，只有RHPIV1-P（C-）诱导的凋亡。 因此，HPIV1的抗IFN和抗凋亡活性都是可分离的：在RHPIV1-P（C-）中，两种活性都是残疾的，而在RHPIV1-CF170中，仅抗IFN活性是禁用的。 在AGMS中，RHPIV1-P（C-）比RHPIV1-CF170S更为衰减，这表明HPIV1的抗IFN和抗凋亡活性使Vivo的衰减具有添加剂。因此，HPIV1的C蛋白是非必需的，但具有抗fn和抗凋亡活性。 在人类纤毛气道上皮（HAE）的体外模型中，这是一种用于研究呼吸道病毒宿主相互作用的有用工具，HPIV1 WT在HAE中选择性感染了HAE和后代病毒中的纤毛细胞，而后代病毒是从根尖表面释放的，几乎没有明显的细胞病理学。 在HAE中，在RHPIV1-CF170s感染后，I型IFN被诱导，但不感染HPIV1 WT。 IFN诱导与病毒滴度的降低100至1000倍，支持HPIV1 C蛋白对于抑制先天免疫反应至关重要的假设。 对感染WT或突变体HPIV1感染或用IFNβ治疗的呼吸道上皮细胞基因表达的动力学的基于微阵列的分析进行了检查：1）WT HPIV1感染如何改变人类呼吸道上皮细胞基因表达； 2）IFNβ在此反应中起着什么作用； 3）与WT HPIV1感染相比，RHPIV1-CF170S和RHPIV1-P（C-）对感染的反应如何； 4）是否可以在转录水平上解释两个C突变病毒（衰减和凋亡表型水平）之间的表型差异。使用代表已知人类基因的完整补体的微阵列比较了用IFNβ或感染HPIV1 WT，RHPIV1-CF170S或RHPIV1-P（C-）的A549细胞中的mRNA水平。感染后48小时，CF170S突变体显着诱导了1,632个基因，并抑制了690个基因，而P（c-）诱导了1,255个基因，并在48 h p.i抑制168个基因。 WT HPIV1显着诱导了274个基因并抑制了3个基因，IFNβ处理诱导了176个基因并抑制了1个基因。因此，编码C基因突变的RHPIV1会改变多达2,322个基因的表达，而编码WT C基因的HPIV1仅修饰了277个基因的表达，表明C蛋白对HPIV1感染的宿主细胞反应抑制具有深远影响。 RHPIV1-P（C-）和RHPIV1-CF170S感染细胞的基因表达谱没有差异。总而言之，HPIV1 C蛋白对病毒感染的细胞转录反应具有明显的控制，表明C蛋白是HPIV1的重要毒力因子。 C基因内的突变允许激活I IFN，IFN，IRF3和NF-KB途径中涉及的一系列细胞基因，否则该途径将被HPIV1感染抑制，这些突变指定了体内衰减表型。 HPIV2疫苗发育：以前使用反向遗传学来产生衰减的突变，该突变是人parainfluenza病毒2型（HPIV2）的L聚合酶蛋白，并增强其遗传稳定性。 去年，我们描述了两种高度减毒病毒RHPIV2-V94（15C）/460A948L和RHPIV2-V94（15C）/948L/δ1724，它们是免疫原性的，它们是针对非洲绿色猴子野生型HPIV2的挑战的免疫原性和保护性的。 已经制造了RHPIV2-V94（15C）/948L/δ1724的临床批次，并将在2008年和2009年在人类中进行评估。 HPIV2的V基因被描述为促进STAT2降解的IFN拮抗剂，是引入衰减突变的另一个靶标。 由于HPIV2的P/V基因中的P和V ORF重叠，我们将P和V ORF分离为两个基因单元，以允许将遗传稳定的突变引入V中，而不会影响P。A HPIV2 P+V病毒被恢复为WT在体外和体内具有WT表型。 V蛋白在其C末端区域中包含一个与STAT2降解有关的环形域。 环域与丝氨酸的七个半胱氨酸氨基酸残基的突变要么是致命的（无法回收），要么高度衰弱，即，即使在未产生IFN的Vero细胞中，病毒复制也降低了1000倍以上。 这表明V蛋白在体外有效复制需要V蛋白，并且环域不是用于引入旨在用于HPIV2疫苗的ATT突变的有用位点。 我们正在寻求确定调节STAT2降解但可以在体外有效复制的突变。

项目成果

Live-attenuated virus vaccines for respiratory syncytial and parainfluenza viruses: applications of reverse genetics.

用于呼吸道合胞病毒和副流感病毒的减毒活病毒疫苗：反向遗传学的应用。

• DOI: 10.1172/jci16077
• 发表时间: 2002
• 期刊: The Journal of clinical investigation
• 作者: Murphy,BrianR;Collins,PeterL
• 通讯作者: Collins,PeterL

Construction of a live-attenuated bivalent vaccine virus against human parainfluenza virus (PIV) types 1 and 2 using a recombinant PIV3 backbone.

使用重组 PIV3 主链构建针对 1 型和 2 型人副流感病毒 (PIV) 的减毒活二价疫苗病毒。

• DOI: 10.1016/s0264-410x(01)00101-3
• 发表时间: 2001
• 期刊: Vaccine
• 影响因子: 5.5
• 作者: Tao,T;Skiadopoulos,MH;Davoodi,F;Surman,SR;Collins,PL;Murphy,BR
• 通讯作者: Murphy,BR

Replacement of the ectodomains of the hemagglutinin-neuraminidase and fusion glycoproteins of recombinant parainfluenza virus type 3 (PIV3) with their counterparts from PIV2 yields attenuated PIV2 vaccine candidates.

将重组副流感病毒 3 型 (PIV3) 的血凝素神经氨酸酶和融合糖蛋白的胞外域替换为 PIV2 的对应物，可产生减毒的 PIV2 候选疫苗。

• DOI: 10.1128/jvi.74.14.6448-6458.2000
• 发表时间: 2000
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Tao,T;Skiadopoulos,MH;Davoodi,F;Riggs,JM;Collins,PL;Murphy,BR
• 通讯作者: Murphy,BR

Long nucleotide insertions between the HN and L protein coding regions of human parainfluenza virus type 3 yield viruses with temperature-sensitive and attenuation phenotypes.

3 型人副流感病毒 HN 和 L 蛋白编码区之间的长核苷酸插入产生具有温度敏感和减毒表型的病毒。

• DOI: 10.1006/viro.2000.0372
• 发表时间: 2000
• 期刊: Virology
• 影响因子: 3.7
• 作者: Skiadopoulos,MH;Surman,SR;Durbin,AP;Collins,PL;Murphy,BR
• 通讯作者: Murphy,BR

Live-attenuated intranasal parainfluenza virus type 2 vaccine candidates developed by reverse genetics containing L polymerase protein mutations imported from heterologous paramyxoviruses.

通过反向遗传学开发的减毒活鼻内副流感病毒 2 型候选疫苗，含有从异源副粘病毒导入的 L 聚合酶蛋白突变。

• DOI: 10.1016/j.vaccine.2005.04.043
• 发表时间: 2005
• 期刊: Vaccine
• 影响因子: 5.5
• 作者: Nolan,SheilaM;Surman,SonjaR;Amaro-Carambot,Emerito;Collins,PeterL;Murphy,BrianR;Skiadopoulos,MarioH
• 通讯作者: Skiadopoulos,MarioH