Laboratory And Pre-clinical Studies Of Parainfluenza Vir

副流感病毒的实验室和临床前研究

• 批准号: 7299914
• 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/7299914
• 关键词: Laboratory; Pre; clinical; Studies; Parainfluenza

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 temperature (ts) and non-ts attenuating (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. The P/C gene of human parainfluenza virus type 1 (HPIV1) encodes a nested set of related accessory C proteins, C??/C/Y1/Y2, which have been shown in other paramyxoviruses to have a role in evasion of the type I interferon (IFN) response following virus infection. We previously demonstrated that a set of two amino acid substitutions, CR84G/HNT553A, and a separate amino acid substitution, CF170S, are independently attenuating for HPIV1 in African green monkeys (AGMs). However, in each case the attenuation (att) phenotype is vulnerable to reversion by a single nucleotide change back to wild type. Using reverse genetics, recombinant HPIV1 (rHPIV1) vaccine candidates were generated that were designed for increased genetic and phenotypic stability by: i) creating a two-amino acid deletion and substitution at the site of the CF170S mutation, yielding C??170; ii) introducing a 6 amino acid deletion in the N-terminal region of C, C??10-15; and iii) combining these stable deletion mutations with the att CR84G/HNT553A mutation. The resulting rHPIV1 vaccine candidates were evaluated for attenuation in hamsters and AGMs and for immunogenicity and protective efficacy in AGMs. The C??10-15 mutation was attenuating in hamsters but not in AGMs, and likely will be of limited value for an HPIV1 vaccine. Conversely, the CR84G/HNT553A mutation set was attenuating in AGMs but not in hamsters. Thus, these two mutations demonstrated reciprocal host range phenotypes involving different regions of C. The C??170 mutation conferred a significant level of attenuation in hamsters and AGMs that closely resembled that of CF170S and will be of particular utility for vaccine development because it involves a deletion of six nucleotides rendering it highly refractory to reversion. The combination of the CR84G/HNT553A mutation set and the C??170 deletion mutation yielded a virus, rCR84G/??170HNT553A, that exhibited a satisfactory level of attenuation in hamsters and AGMs and was immunogenic and highly protective against HPIV1 wt challenge. This virus was modified to contain the stabilized codon at 942, and this virus will be manufactured by Charles River Laboratories and evaluated clinically as a live intranasal HPIV1 vaccine. Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion (??) mutations in the P/C gene (r-C??10-15HNT553A, r-CR84G, r-CF170S and r-C??170), 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-C??170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-C??170 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 P/C 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 P/C mutants, r-CF170S and r-C??170, was necessary for significant attenuation in African green monkeys (AGMs). 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. A live attenuated HPIV2 vaccine candidate has been identified.

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引入一个或多个温度（TS）和非TS减弱（ATT）突变（ATT）突变的野生型HPIV1，从而产生活减弱的重组HPIV1（RHPIV1）疫苗。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。 人副帕氟氏菌病毒1型（HPIV1）的P/C基因编码一组相关的辅助C蛋白，C ??/C/C/Y1/Y2，在其他Paramyxovires中已显示出在其他Paramyxovirues中显示出在病毒感染后逃避I型I Interferon（IFN）反应中发挥作用的作用。我们先前证明，在非洲绿猴（AGMS）中，一组两种氨基酸取代CR84G/HNT553A和单独的氨基酸取代CF170S，正在独立衰减HPIV1。但是，在每种情况下，衰减（ATT）表型很容易通过单个核苷酸转换为野生型的衰减。使用反向遗传学，生成了重组HPIV1（RHPIV1）候选疫苗，该疫苗的设计是为了提高遗传和表型稳定性的设计，这是通过：）在CF170S突变部位创建两氨基酸的缺失和取代，产生C ?? 170； ii）在C，C ?? 10-15的N末端区域引入6个氨基酸缺失； iii）将这些稳定的缺失突变与ATT CR84G/HNT553A突变相结合。评估了最终的RHPIV1疫苗候选物在仓鼠和AGM中的衰减以及AGM的免疫原性和保护性疗效。 C ?? 10-15突变在仓鼠中衰减，但在AGM中没有衰减，HPIV1疫苗的价值可能有限。相反，CR84G/HNT553A突变集在AGM中衰减，但在仓鼠中没有衰减。因此，这两个突变表现出涉及C的不同区域的相互宿主范围表型。C?? 170突变在仓鼠和AGM中赋予了显着水平的衰减水平，与CF170S的衰减非常相似，并且会特别是疫苗开发的实用性，因为它涉及六核位核定剂对造成高度反应的六核局限性，以使其反复反复反应。 CR84G/HNT553A突变集和C ?? 170缺失突变的组合产生了一种病毒RCR84G/?? 170HNT553A，在仓鼠和AGM中表现出令人满意的衰减水平，并且是免疫原性的，并且具有高度保护性，并且具有很高的保护性，并且对HPIV1 WT挑战具有很高的保护性。该病毒经过修饰，以在942处包含稳定的密码子，该病毒将由Charles River Laboratories生产，并在临床上作为实时鼻内HPIV1疫苗进行评估。 重组人副帕氟二氮病毒1型（HPIV1）和突变体包含P/C基因中的点和缺失（??）突变（R-C ?? 10-15HNT553A，R-CR84G，R-CR84G，R-CF170S和R-C ?? 170）先前已评估为HPIV1 visten vistern vistern vistern vistern vistern visten in tyne in the the the ways in the ways in at ways in at ways in at ways Inters in at ways Inters in at ways Inters in at ways in at ways in vers in ways in vers inters in ty的响应（体外。 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-C ?? 170因抑制IRF-3激活和IFN产生以及R-CF170S和R-C ?? 170的有缺陷，并未抑制IFN信号传导。因此，HPIV1在诱导水平和信号传导水平上拮抗IFN响应，并且在P/C基因中的突变破坏了两种水平的拮抗作用。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P/C突变体，R-CF170S和R-C？170上观察到IFN反应的拮抗作用丧失是非洲绿色猴子（AGMS）的大量衰减所必需的。 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。已经确定了活的HPIV2疫苗候选者。