Laboratory/Pre-clinical Studies Of Parainfluenza Viruses

副流感病毒的实验室/临床前研究

• 批准号: 7192830
• 负责人: Brian R. Murphy
• 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/7192830
• 关键词: Cercopithecidae; Macaca mulatta; Pan; Paramyxoviridae; Paramyxovirus; Rodentias; active immunization; attenuated microorganism; biotechnology; communicable disease control; enzyme linked immunosorbent assay; guinea pigs; live vaccine; nonhuman therapy evaluation; paramyxovirus vaccine; recombinant proteins; recombinant virus; respiratory infections; site directed mutagenesis; vaccine development; vaccine evaluation; virus antigen; virus genetics; virus protein; virus replication

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. The development of a reverse genetics system for HPIV1 provides the capability to generate live attenuated recombinant HPIV1 (rHPIV1) vaccine candidates by the introduction of one or more ts and non-ts att mutations into wild type HPIV1. Since respiratory viruses with mutations in proteins with anti-interferon activities are attenuated in vivo, the C accessory proteins are prime targets for inactivation by mutation. In addition, mutations in L have been identified that attenuate respiratory viruses for rodents or primates. Attenuating mutations identified in the L genes of RSV and HPIV3 and the C genes of MPIV1 and HPIV3 were previously transferred to the homologous loci of HPIV1 identified by sequence alignments to generate live attenuated HPIV1 vaccine candidates. Specifically, amino acid substitutions introduced individually at position 170 in the C protein of HPIV1 and at positions 456, 942, 992 and 1558 in L attenuated HPIV1 for replication in the respiratory tract of hamsters. The mutation at position 170 in C specified a non-ts att phenotype whereas those in L specified either a ts or non-ts att phenotype. The combination of L gene mutations rendered viruses more ts and more attenuated in hamsters than either mutation alone. The codons at positions 942 and 992 were systematically mutated to achieve enhanced phenotypic stability and increased attenuation. At position 942, the original rL-Y942H virus was mutated to generate rL-Y942A, a virus that possessed a similar level of temperature sensitivity and attenuation as rL-Y942H but that would require three nucleotide substitutions in the Y942A codon to generate a codon that specified a wild type phenotype. The rL-Y942A mutant was confirmed to exhibit increased genetic and phenotypic stability over that of rL-Y942H. Similarly, a 2-nucleotide substitution at position 992 (Leu to Cys) was found to specify the highest level of temperature sensitivity and attenuation among recombinants with a change at codon 992. These previous observations were extended in several respects. First, rHPIV1 vaccine candidates were generated to contain new combinations of mutations that included the stabilized codon at 942 and the partially-stabilized codon at 992 in L. Second, a pair of novel spontaneous mutations in C (R84G) and HN (T553A) was identified, characterized, and used to generate novel vaccine candidates. Third, new rHPIV1 combination vaccine candidates were evaluated in hamsters. Fourth, the rHPIV1 vaccine candidates were evaluated in African green monkeys (AGMs), whose anatomical and phylogenic relatedness to humans makes them suitable as the penultimate step prior to clinical trials. rHPIV1 vaccine candidates were identified that exhibited a spectrum in their level of attenuation, immunogenicity, and efficacy in hamsters or AGMs. Several of the rHPIV1 vaccine candidates appeared to have achieved an acceptable balance between attenuation and immunogenicity for AGMs and thus represent promising vaccine candidates for use in humans. In addition, progress has been made on the development of a live-attenuated HPIV2 vaccine. Recombinant human parainfluenza virus type 2 (rHPIV2) vaccine candidates were created using reverse genetics by importing known attenuating mutations in the L polymerase protein from heterologous paramyxoviruses into the homologous sites of the HPIV2 L protein. Four recombinants (rF460L, rY948H, rL1566I, and rS1724I) were recovered and three were attenuated for replication in hamsters. The genetic stability of the imported mutations at three of the four sites was enhanced by use of alternative codons or by deletion of a pair of amino acids. rHPIV2s bearing these modified mutations exhibited enhanced attenuation. The genetically stabilized mutations conferring a high level of attenuation will be useful in generating a live-attenuated virus vaccine for HPIV2.

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。 用于HPIV1的反向遗传系统的开发提供了通过将一个或多个TS和非TS ATT突变引入野生型HPIV1的候选物质候选的活减弱重组HPIV1（RHPIV1）疫苗的能力。由于在体内会减弱具有抗互干霉活性的蛋白质突变的呼吸道病毒，因此C辅助蛋白是通过突变失活的主要靶标。此外，已经确定了L中的突变，可减弱啮齿动物或灵长类动物的呼吸道病毒。先前先前将RSV和HPIV3和MPIV1和HPIV3的C基因鉴定出的衰减突变转移到通过序列比对确定的HPIV1的同源基因座，以产生活减弱的HPIV1疫苗候选者。具体而言，在HPIV1的C蛋白和位置456、942、992和1558的位置在L衰减的HPIV1中单独引入的氨基酸取代，以在仓鼠的呼吸道中复制。 C中位置170的突变指定了非TS ATT表型，而L中的突变指定为TS或非TS ATT表型。 L基因突变的组合使病毒在仓鼠中比单独的任何一个突变更多。将942和992位置的密码子系统突变，以达到增强的表型稳定性和增加的衰减。在942位置，将原始的RL-Y942H病毒突变为生成RL-Y942A，该病毒具有与RL-Y942H相似的温度敏感性和衰减水平的病毒，但它需要在Y942A密码子中产生Y942A密码子中的三个核苷酸取代，以生成指定野生类型概况的密码子。 RL-Y942A突变体被证实在RL-Y942H的遗传和表型稳定性上表现出增加。类似地，发现位置992（LEU至CYS）处的2-核苷酸取代指定重组的最高温度敏感性和衰减水平，而密码子992的变化也会变化。 这些先前的观察结果在几个方面进行了扩展。首先，生成了RHPIV1疫苗候选物，以包含包括942时稳定的密码子的新组合，以及L.第二的992的部分稳定密码子，C（R84G）和HN（R84G）和HN（T553A）中的一对新型自发突变被确定，并用来鉴定出用于生成新颖的无性液。第三，在仓鼠中评估了新的RHPIV1组合疫苗。第四，在非洲绿猴（AGMS）中评估了RHPIV1疫苗的候选物，其解剖学和系统性相关性使其与人类合适，作为临床试验前的倒数第二步。鉴定出RHPIV1疫苗的候选物在仓鼠或AGMS中表现出其衰减，免疫原性和疗效水平的频谱。 RHPIV1疫苗的几种候选物似乎在AGM的衰减和免疫原性之间取得了可接受的平衡，因此代表了有希望的候选疫苗候选者。 此外，在开发实时衰减的HPIV2疫苗方面取得了进展。 通过反向遗传学，通过将已知的衰减突变从异源性帕托粘病毒中导入到HPIV2 L蛋白的同源性位点，使用反向遗传学创建了重组人副帕弗鲁氏菌病毒2型（RHPIV2）候选疫苗候选。回收了四种重组（RF460L，RY948H，RL1566I和RS1724I），并在仓鼠中衰减了三只。通过使用替代密码子或删除一对氨基酸，进口突变在四个位点中的三个位置的遗传稳定性得到了增强。带有这些修饰突变的RHPIV2表现出增强的衰减。赋予高水平衰减的遗传稳定突变将有助于生成用于HPIV2的活衰减病毒疫苗。