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: 7964228
• 负责人: Brian Murphy
• 金额: $ 221.48万
• 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/7964228
• 关键词: Accounting; Affect; Amino Acid Sequence; Annual Reports; Antigens; Antiviral Agents; Apoptosis; Attenuated; Attenuated Live Virus Vaccine; Bronchiolitis; C-terminal; Cells; Cercopithecus pygerythrus; Child; Childhood; Clinical Trials; Complementary DNA; Croup; Cysteine; Cytopathology; Data; Disease; Epithelial; Equilibrium; Evaluation; Exhibits; Family; Gene Deletion; Genes; Genome; Giant Cells; Glycine decarboxylase; Goals; Growth; Hemagglutinin; Hospitalization; Human; Human Parainfluenza Virus 2; Immune response; In Vitro; Infant; Infection; Interferon Receptor; Interferon Type I; Interferons; Laboratories; Licensing; Lower respiratory tract structure; Lung diseases; Membrane Glycoproteins; Modeling; Molecular Virology; Mutation; N-terminal; Necrosis; Neuraminidase; Nucleocapsid; Nucleocapsid Proteins; Open Reading Frames; Otitis Media; Para-Influenza Virus Type 1; Paramyxoviridae; Paramyxovirinae; Paramyxovirus; Pathway interactions; Pharyngitis; Phenotype; Phosphoproteins; Pneumonia; Polymerase; Protein C; Proteins; RNA Editing; RNA Viruses; RNA chemical synthesis; Recombinants; Recovery; Reporting; Respiratory Tract Diseases; Respirovirus; Rhinitis; Role; Rubulavirus; Serotyping; Signal Transduction; System; Temperature; Testing; Tissues; Tropism; Vaccines; Vertebral column; Viral; Virus; Virus Replication; airway epithelium; attenuation; caspase-8; caspase-9; citrate carrier; cytotoxicity; immunogenic; immunogenicity; in vitro Model; in vivo; multiple myeloma M Protein; mutant; nonhuman primate; novel; parainfluenza virus; pre-clinical research; preclinical study; prevent; response; vaccine candidate; vaccine development; vaccine safety; viral RNA

Human parainfluenza viruses are significant causes 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 18% of all pediatric hospitalizations due to respiratory disease. A licensed vaccine is currently not available for any of the HPIVs. The major goals of this project are to develop live attenuated virus vaccines that are effective in infants and children against HPIV1, HPIV2, and HPIV3. Pre-clinical research is currently not being conducted with HPIV3 in LID since we have multiple HPIV3 vaccines in clinical trials and three additional HPIV3 vaccine candidates are being prepared for manufacture. Thus, this report focuses on HPIV1 and HPIV2. HPIV1 vaccine development: A novel recombinant human parainfluenza virus type 1 (rHPIV1), rHPIV1-C+P, was generated in which the overlapping open reading frames of the C and P genes were separated in order to introduce mutations into the C gene without affecting P. Infectious rHPIV1-C+P was readily recovered and replicated as efficiently as HPIV1 wild-type (wt) in vitro and in African green monkeys (AGMs). rHPIV1-C+P expressed increased levels of C protein and, surprisingly, activated the type I IFN and apoptosis responses more strongly than HPIV1 wt. rHPIV1-C+P provided a useful backbone for recovering an attenuated P/C gene deletion mutation, (del84-85), which was previously unrecoverable, likely due to detrimental effects of the deletion on the function of the P protein. rHPIV1-C(del84-85)+P and an additional mutant, rHPIV1-C(del169-170) +P, were found to replicate to similar titers in vitro and to activate the type I IFN and apoptosis responses to a similar degree as rHPIV1-C+P. rHPIV1-C(del84-85)+P was highly attenuated in AGMs, and all of the viruses were immunogenic and effective in protecting AGMs against challenge with HPIV1 wt. rHPIV1-C(del84-85)+P will be investigated as a potential live-attenuated vaccine candidate for HPIV1. We further sought to understand the role of the C proteins in apoptosis. 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 which was the result of the activation of apoptosis in rHPIV1-P(C-) infected cells. The apoptosis was initiated by activation of both the intrinsic (caspase 9) and extrinsic (caspase 8) pathways, but was found not to be the cause of the reduction in viral replication. HPIV2 vaccine development: In wild-type human parainfluenza virus type 2 (wt HPIV2), one gene (the P/V gene) encodes both the polymerase-associated phosphoprotein (P) and the accessory cysteine-rich V protein. The P and V proteins share the same N-terminal sequence but have different C-terminal domains due to RNA editing. V is an accessory protein that, in model paramyxoviruses, inhibits the cellular interferon (IFN) response and regulates viral RNA synthesis, in addition to performing other functions that remain less well defined. We generated an HPIV2 virus (rHPIV2-Vko) in which the P/V gene encodes only the P protein to examine the role of V in replication in vivo and for use as a potential live attenuated virus vaccine. Preventing expression of the V protein severely impaired virus recovery from cDNA and growth in vitro, particularly in IFN-competent cells. The rHPIV2-Vko virus, unlike wt HPIV2, strongly induced type I IFN and permitted signaling through the IFN receptor, leading to establishment of a robust antiviral state. rHPIV2-Vko infection induced extensive syncytia and caused dramatic cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-Vko was highly restricted in the upper and lower respiratory tract of African green monkeys and was not detected in differentiated primary human airway epithelial (HAE) cultures, suggesting that the V protein is essential for efficient replication of HPIV2 in vivo and in HAE cultures in vitro. The high degree of restriction of rHPIV2-Vko in non-human primates and in primary HAE cultures suggests that this mutant is over-attenuated and would not be suitable as a live attenuated virus vaccine, but it will be useful to study the function of V during HPIV2 infection. We next characterized wild-type HPIV2 (rHPIV2-WT) infection in a well-established in vitro model of human airway epithelium (HAE) and revealed that the virus replicates to high titer, is shed only apically, targets ciliated cells, and induces minimal cytopathology. Since HPIV2 mutants are currently being developed as live attenuated vaccine candidates, we next sought to determine if infection of HAE with the HPIV2 vaccine candidate, which was described in last years annual report and which is currently in clinical trials, reflects replication in non-human primates. An experimental HPIV2 vaccine strain, rHPIV2-V94(15C)/948L/del1724, that contains both temperature sensitive (ts) and non-ts attenuating mutations, was previously found to be restricted in replication in the upper (URT) and lower respiratory tract (LRT) of African green monkeys (AGMS) and to be protective against wild-type HPIV2 challenge. rHPIV2-V94(15C)/948L/del1724 was reduced in replication by more than 30-fold compared to rHPIV2-WT in HAE cultures at 32C and exhibited little productive replication in cultures at 37C, reflecting a similar restriction of replication in the cooler URT and warmer LRT of AGMs . These data indicate that the HAE model provides a convenient experimental system for examining the cell tropism, cytotoxicity, and attenuation phenotypes of HPIV2 vaccine candidates as well as for characterizing the innate host responses of human airway epithelial tissues to infection. Since clinical trials are the only true tests of vaccine safety and efficacy, the results from this study encourage continued evaluation of rHPIV2-VAC in clinical trials.

人类副磷酸根病毒是婴儿和幼儿严重呼吸道疾病的重大原因。 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18％。目前，任何HPIVS都无法使用持牌疫苗。 该项目的主要目标是开发针对婴儿和儿童针对HPIV1，HPIV2和HPIV3有效的活病毒疫苗。目前尚未使用HPIV3在LID中进行临床前研究，因为我们在临床试验中有多种HPIV3疫苗，并且还准备了另外三个HPIV3疫苗候选物进行制造。 因此，本报告着重于HPIV1和HPIV2。 HPIV1疫苗的开发：生成一种新型的重组人副粉状病毒1（RHPIV1），RHPIV1-C+P，在其中分离了C和P基因的重叠的开放式阅读框体外和非洲绿猴（AGM）。 RHPIV1-C+P表示C蛋白的水平增加，令人惊讶地比HPIV1 WT更强烈地激活I型IFN和凋亡反应。 RHPIV1-C+P提供了一种有用的主链，用于恢复衰减的P/C基因缺失突变（DEL84-85），该骨架以前是不可撤销的，这可能是由于缺失对P蛋白功能的有害影响所致。 发现RHPIV1-C（DEL84-85）+P和其他突变体RHPIV1-C（DEL169-170）+P在体外重复到类似的滴度，并激活I型IFN和凋亡响应，其程度与RHPIV1-C+P相似。 RHPIV1-C（DEL84-85）+P在AGM中高度减弱，所有病毒均具有免疫原性，可有效保护AGM免受HPIV1 WT挑战。 RHPIV1-C（DEL84-85）+P将作为HPIV1的潜在实时疫苗候选者进行研究。 我们进一步试图了解C蛋白在凋亡中的作用。 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，C，Y1和Y2）被沉默而不影响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-）在感染过程中与HPIV1 WT一样有效地复制了体外，但随后其效率与Rhpiv1 WT观察到的广泛的细胞性效应（CPE）的发作降低了，这是Rhpiv1-P（C-Piv1-wt）的结果。 凋亡是通过固有（caspase 9）和外部（caspase 8）途径激活而引发的，但发现并不是病毒复制减少的原因。 HPIV2疫苗的开发：在野生型人parainfluenza病毒2型（WT HPIV2）中，一个基因（P/V基因）编码了聚合酶相关的磷酸蛋白（P）和辅助性半胱氨酸V蛋白。 P和V蛋白具有相同的N末端序列，但由于RNA编辑而具有不同的C末端结构域。 V是一种辅助蛋白，在模型的帕马伏病毒中，除了执行其他较不确定的功能外，还可以抑制细胞干扰素（IFN）反应并调节病毒RNA合成。 我们生成了HPIV2病毒（RHPIV2-VKO），其中P/V基因仅编码P蛋白来检查V在体内复制中的作用并用作潜在的活衰减病毒疫苗。 防止V蛋白表达的表达严重受损的病毒从cDNA和体外生长，尤其是在IFN能力的细胞中。 与WT HPIV2不同，RHPIV2-VKO病毒强烈诱导了I型IFN并通过IFN受体允许的信号传导，从而建立了强大的抗病毒态。 RHPIV2-VKO感染诱导广泛的合胞菌，并引起巨大的细胞疗法，这既是凋亡和坏死。 RHPIV2-VKO的复制在非洲绿猴的上和下呼吸道中受到了高度限制，在分化的原发性人类气道上皮（HAE）培养物中未检测到，这表明V蛋白对于在体内和HAE培养物中有效地复制V蛋白对于有效地复制了HPIV2。 非人类灵长类动物和原发性HAE培养物中RHPIV2-VKO的高度限制表明，该突变体被过度稳定，并且不适合作为活的减毒病毒疫苗，但在HPIV2感染过程中研究V功能是有用的。接下来，我们在人类气道上皮（HAE）的体外模型中表征了野生型HPIV2（RHPIV2-WT）感染，并透露该病毒仅以顶端脱落，靶向纤毛细胞，并诱导细胞病理学最小的细胞。 由于目前正在开发HPIV2突变体作为候选疫苗的活疫苗，因此我们接下来试图确定HAE是否与HPIV2疫苗候选者感染，该疫苗候选者在去年的年度报告中进行了描述，目前正在临床试验中，反映了非人类灵长类动物的复制。 实验性HPIV2疫苗菌株RHPIV2-V94（15C）/948L/DEL1724均包含温度敏感（TS）和非TS衰减突变，在上部（URT）和下呼吸道（LRT）和非洲绿色怪兽（LRT）的复制中受到限制。与HAE培养物在32C的HAE培养物相比，RHPIV2-V94（15C）/948L/DEL1724的复制减少了30倍以上，并且在37C的培养物中的生产培养很少，反映出在冷却器和温暖的LRT agms中相似的复制限制。 这些数据表明，HAE模型提供了一个方便的实验系统，用于检查HPIV2疫苗候选物的细胞向量，细胞毒性和衰减表型，以及表征人类气道上皮组织对感染的先天宿主反应。 由于临床试验是疫苗安全性和功效的唯一真正检验，因此本研究的结果鼓励在临床试验中继续评估RHPIV2-VAC。