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: 9161440
• 负责人: PETER LEON COLLINS
• 金额: $ 152.01万
• 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/9161440
• 关键词: 1 year old; Affect; Amino Acid Sequence; Animals; Antibodies; Antibody Response; Antigens; Attenuated; Attenuated Live Virus Vaccine; Biological Assay; Biology; Cattle; Cell Culture Techniques; Cells; Child; Childhood; Clinical; Clinical Research; Codon Nucleotides; Country; Cultured Cells; Deletion Mutation; Development; Disease; Exhibits; Family; Filament; Filtration; Gene Expression Regulation; Gene Order; Gene Proteins; Genes; Genetic; Genetic Transcription; Genome; Goals; Hamsters; Hemagglutinin; Human; Human Parainfluenza Virus 1; Human respiratory syncytial virus; Immune response; Immunity; Immunization; Immunofluorescence Immunologic; In Vitro; Infant; Intranasal Administration; Laboratories; Laboratory Study; Life; Location; Lower respiratory tract structure; Lung; Membrane Glycoproteins; Messenger RNA; Missense Mutation; Modeling; Modification; Molecular Biology; Molecular Conformation; Molecular Genetics; Molecular Virology; Mutate; Mutation; Nasal turbinate bone structure; National Institute of Allergy and Infectious Disease; Neuraminidase; Nose; Nucleoproteins; Nucleotides; Open Reading Frames; Paramyxoviridae; Phenotype; Phosphoproteins; Polymerase; Positioning Attribute; Primates; Production; Protein Biosynthesis; Proteins; RNA Viruses; Reading; Recombinants; Reporting; Resources; Respiratory Syncytial Virus Vaccines; Respiratory Tract Diseases; Respiratory syncytial virus; Respiratory syncytial virus RSV F proteins; Respiratory syncytial virus RSV proteins; Role; Serotyping; Serum; Signal Transduction; Staining method; Stains; System; Temperature; Testing; Time; Transcript; Translations; Ursidae Family; Vaccines; Vertebral column; Viral; Viral Antigens; Virion; Virus; Virus Diseases; Virus Replication; adaptive immunity; attenuation; design; human disease; immunogenic; immunogenicity; improved; in vivo; infancy; multiple myeloma M Protein; mutant; novel; parainfluenza virus; particle; phase 1 study; preclinical study; repaired; response; reverse genetics; vaccine candidate; vector; vector vaccine

Human parainfluenza viruses 1, 2, and 3 are significant causes of severe pediatric respiratory tract disease worldwide. The HPIVs are enveloped, non-segmented, negative strand RNA viruses of the family Paramyxoviridae. The broad outlines of their biology and molecular genetics have been defined in previous studies by this laboratory and others. The HPIV genome encodes a nucleoprotein N, phosphoprotein P, large polymerase protein L, internal matrix protein M, and fusion F and hemagglutinin-neuraminidase HN transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and the major protective antigens. In addition, the P gene encodes various accessory protein(s)from one or more additional ORFs: C (HPIV1), V (HPIV2), and C, D, and possibly V (HPIV3). These accessory proteins have a number of functions that antagonize the host response to viral infection, as described in previous years. We are developing attenuated versions of HPIV1, 2, and 3 that also express the fusion F protein of human respiratory syncytial virus (RSV). RSV is the most important viral agent of severe pediatric respiratory tract disease, with a contribution to human disease comparable to that of the HPIVs combined, and the F protein is the major RSV neutralization and protective antigen. HPIV1, 2, and 3 expressing the RSV F protein would provide bivalent vaccines against each respective HPIV and RSV. Compared to RSV strains, the HPIVs replicate more efficiently in cell culture and have much greater physical stability. They also form spherical particles compared to the large filaments of RSV, making them more amenable to filtration and other steps in manufacture. These attributes make HPIV vectors much easier to manufacture, distribute, store, and use compared to attenuated RSV strains. The greater physical stability in particular may be essential for extending RSV vaccines to resource-challenged countries. Furthermore, in experimental animals, boosting RSV responses was more efficient using HPIV/RSV vectors as opposed to attenuated RSV strains, since the latter are subject to greater restriction by prior RSV-specific immunity. We have been evaluating a number of parameters of vaccine vector design using, as proof of principle, an attenuated HPIV3 virus called B/HPIV3 This consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3, yielding a chimeric virus that is attenuated in primates due to the bovine backbone and bears the neutralization and major protective F and HN antigens of HPIV3. Previously, B/HPIV3 has been evaluated in clinical phase 1 studies, both as an empty vector (LID/NIAID study) and as a vector for RSV-F (MedImmune study), and was shown to be well-tolerated in either role in infants and young children. In the initial clinical study of B/HPIV3-RSV-F, the RSV F insert exhibited substantial instability and was not as immunogenic as hoped. Our goal therefore has been to increase the immunogenicity and stability of the RSV F insert. We previously evaluated the effects of the position of insertion of the RSV F gene into the B/HPIV3 backbone, and found that the first (pre-N) and second (N-P) gene positions readily accommodated the RSV F insert. This resulted in greatly increased expression of RSV F protein compared to downstream locations (up to 69-fold increase) and greatly increased fusion. Surprisingly, this did not appear to interfere with vector replication in vitro or in hamsters. In the past year, we found that expression of the RSV F protein was further enhanced 5-fold by codon-optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypo-fusogenic phenotype that presumably reflects the original clinical isolate, and suggests that this strain of RSV may have mutated to acquire a hyper-fusogenic phenotype during passage in vitro. We then compared vectors expressing stabilized pre- and post-fusion versions of RSV F protein. In a hamster model, pre-fusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wt RSV challenge, compared to native F. In contrast, vector expressing the post-fusion F was more immunogenic and protective than native RSV F, but less than pre-fusion F. Use of a double-staining immunofluorescence assay showed that the stability of expression of the RSV F protein was high and was not affected by enhanced expression or the pre- or post-fusion conformations of RSV F. These studies provide an improved version of the rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. HPIV1 also was developed as a vector for RSV F during the past year. The RSV F gene was inserted individually into three different genome locations (pre-N (F1), N-P (F2), or P-M (F3)) in each of two attenuated rHPIV1 backbones. Each backbone contained a single previously-described attenuating mutation that was stabilized against de-attenuation: (1) a non-temperature-sensitivity deletion mutation involving six nucleotides in the overlapping P/C ORFs (Cdel170), or (2) a temperature-sensitivity missense mutation in the L ORF (LY942A). In vitro, the presence of the F insert reduced the rate of virus replication, but the final titers were the same as wt HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-Cdel170-F1, -F2, and -F3, and rHPIV1-LY942A-F1. In hamsters, the rHPIV1-Cdel170-F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates and highly restricted in lungs, and were genetically stable in vivo. Among the Cdel170 vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-LY942A vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of over-attenuation. The Cdel170-F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine. We also investigated regulation of gene expression in HPIV3. The gene end (GE) transcription signals of the HPIV3 genes are highly conserved except that the M GE signal contains an apparent 8-nucleotide insert. This is associated with increased synthesis of a read-through transcript of the M gene plus the downstream F protein gene. We hypothesized that this insert may function to down-regulate expression of F protein by interfering with termination/re-initiation at the M-F gene junction, thus promoting the production of M-F read-through mRNA at the expense of monocistronic F mRNA. To test this hypothesis, two similar recombinant HPIV3 viruses were generated from which this insert in the M-GE signal was removed. The M-GE mutants exhibited a reduction in M-F read-through mRNA and an increase in monocistronic F mRNA. This resulted in a substantial increase in F protein synthesis in the infected cells as well as enhanced incorporation of F protein into virions. The efficiency of mutant virus replication was similar to that of wt HPIV3 both in vitro and in vivo. However, the F protein-specific serum antibody response in hamsters was increased for the mutants as compared to wt HPIV3. This study identifies a novel viral mechanism for reducing stimulation of the host adaptive immune response. Repairing the M-GE signal should provide a means to increase the antibody response to a live attenuated HPIV3 vaccine without affecting viral replication and attenuation.

人类副流感病毒 1、2 和 3 型是全世界严重儿科呼吸道疾病的重要原因。 HPIV 是副粘病毒科的有包膜、不分段、负链 RNA 病毒。该实验室和其他实验室之前的研究已经确定了它们的生物学和分子遗传学的大致轮廓。 HPIV基因组编码核蛋白N、磷蛋白P、大聚合酶蛋白L、内部基质蛋白M、融合F和血凝素-神经氨酸酶HN跨膜表面糖蛋白。 F和HN是两种病毒中和抗原，也是主要的保护性抗原。此外，P基因编码来自一个或多个附加ORF的各种辅助蛋白：C (HPIV1)、V (HPIV2)和C、D，并且可能还包括V (HPIV3)。正如前几年所述，这些辅助蛋白具有许多拮抗宿主对病毒感染反应的功能。 我们正在开发 HPIV1、2 和 3 的减毒版本，它们也表达人呼吸道合胞病毒 (RSV) 的融合 F 蛋白。 RSV 是严重儿科呼吸道疾病最重要的病毒因子，其对人类疾病的贡献可与 HPIV 的总和相媲美，而 F 蛋白是主要的 RSV 中和和保护性抗原。表达RSV F蛋白的HPIV1、2和3将提供针对各自的HPIV和RSV的二价疫苗。 与 RSV 毒株相比，HPIV 在细胞培养中复制更有效，并且具有更高的物理稳定性。与 RSV 的大丝相比，它们还形成球形颗粒，使它们更适合过滤和其他制造步骤。与减毒 RSV 毒株相比，这些特性使得 HPIV 载体更容易制造、分发、储存和使用。尤其是，更高的物理稳定性对于将 RSV 疫苗推广到资源匮乏的国家可能至关重要。此外，在实验动物中，与减毒RSV毒株相比，使用HPIV/RSV载体增强RSV反应更有效，因为后者受到先前RSV特异性免疫的更大限制。 我们一直在评估疫苗载体设计的许多参数，作为原理证明，使用一种称为 B/HPIV3 的减毒 HPIV3 病毒。该病毒由牛 PIV3 组成，其中 F 和 HN 基因已被 HPIV3 的基因取代，产生一种嵌合病毒，由于牛骨架，在灵长类动物中减毒，并具有中和和主要保护性 F 和 HN 抗原 HPIV3。此前，B/HPIV3 已在临床 1 期研究中进行了评估，既作为空载体（LID/NIAID 研究），又作为 RSV-F 载体（MedImmune 研究），并显示在婴儿和幼儿中这两种作用均具有良好的耐受性。在 B/HPIV3-RSV-F 的初步临床研究中，RSV F 插入片段表现出很大的不稳定性，并且免疫原性不如希望的那样。因此，我们的目标是提高 RSV F 插入片段的免疫原性和稳定性。 我们之前评估了 RSV F 基因插入 B/HPIV3 主链的位置的影响，发现第一个（前 N）和第二个（N-P）基因位置很容易容纳 RSV F 插入。与下游位置相比，这导致 RSV F 蛋白的表达大大增加（高达 69 倍）并大大增加了融合。令人惊讶的是，这似乎并没有干扰体外或仓鼠中的载体复制。 去年，我们发现通过密码子优化以及将氨基酸序列修改为与原始临床分离株早期传代相同，RSV F 蛋白的表达进一步增强了 5 倍。这赋予了低融合表型，可能反映了最初的临床分离株，并表明该RSV菌株可能在体外传代期间发生突变以获得高融合表型。然后我们比较了表达稳定的 RSV F 蛋白融合前和融合后版本的载体。在仓鼠模型中，与天然 F 相比，融合前 F 诱导 RSV 中和血清抗体的数量和质量增加，并增强对野生型 RSV 攻击的保护。相反，表达融合后 F 的载体比天然 RSV F 更具免疫原性和保护性，但低于融合前 F。 使用双重染色免疫荧光测定表明，RSV F 蛋白表达的稳定性很高，并且不受 RSV F 表达增强或融合前或融合后构象的影响。这些研究提供了 rB/HPIV3-RSV F 候选疫苗的改进版本，可诱导优异的 RSV 中和血清抗体反应。 去年，HPIV1 还被开发为 RSV F 的载体。 RSV F 基因分别插入两个减毒 rHPIV1 主链的三个不同基因组位置（前 N (F1)、N-P (F2) 或 P-M (F3)）。每个主链包含一个先前描述的减毒突变，该突变可稳定抵抗去减毒：(1) 涉及重叠 P/C ORF (Cdel170) 中六个核苷酸的非温度敏感性缺失突变，或 (2) L ORF (LY942A) 中的温度敏感性错义突变。在体外，F插入片段的存在降低了病毒复制速率，但最终滴度与wt HPIV1相同。使用 rHPIV1-Cdel170-F1、-F2 和 -F3 以及 rHPIV1-LY942A-F1 观察到培养细胞中高水平的 RSV F 表达。在仓鼠中，rHPIV1-Cdel170-F1、-F2 和 -F3 载体在鼻甲中中度限制，在肺中高度限制，并且在体内遗传稳定。在 Cdel170 载体中，F1 病毒是最具免疫原性和针对 wt RSV 攻击的保护性的。 rHPIV1-LY942A 载体在体内受到高度限制，未检测到免疫原性或保护性，表明过度减毒。 Cdel170-F1 构建体似乎具有适当的减毒和免疫原性，可进一步开发为二价鼻内儿科疫苗。 我们还研究了 HPIV3 基因表达的调控。 HPIV3 基因的基因末端 (GE) 转录信号高度保守，但 M GE 信号包含明显的 8 核苷酸插入。这与 M 基因通读转录物和下游 F 蛋白基因的合成增加有关。我们假设该插入片段可能通过干扰 M-F 基因连接处的终止/重新启动来下调 F 蛋白的表达，从而以单顺反子 F mRNA 为代价促进 M-F 通读 mRNA 的产生。为了检验这一假设，生成了两种相似的重组 HPIV3 病毒，并从中删除了 M-GE 信号中的插入片段。 M-GE突变体表现出M-F通读mRNA的减少和单顺反子F mRNA的增加。这导致受感染细胞中 F 蛋白合成的大幅增加以及 F 蛋白与病毒粒子的掺入增强。突变病毒在体外和体内的复制效率与wt HPIV3相似。然而，与wt HPIV3相比，突变体在仓鼠中的F蛋白特异性血清抗体反应增加。这项研究确定了一种新的病毒机制，可以减少对宿主适应性免疫反应的刺激。修复 M-GE 信号应该提供一种方法来增强对 HPIV3 减毒活疫苗的抗体反应，而不影响病毒复制和减毒。