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.

人类副磷素病毒1、2和3是全球严重的小儿呼吸道疾病的重要原因。 HPIV是家族paramyxoviridae的包裹，非细分的，负的RNA病毒。该实验室和其他人在先前的研究中定义了其生物学和分子遗传学的广泛概述。 HPIV基因组编码A核蛋白N，磷酸蛋白P，大型聚合酶蛋白L，内部基质蛋白M，融合F和hemagglutin-noraminidase hn跨膜表面糖蛋白。 F和HN是两个病毒中和抗原和主要的保护抗原。另外，P基因从一个或多个附加的ORF中编码各种辅助蛋白：C（HPIV1），V（HPIV2）和C，D和可能的V（HPIV3）。这些附件蛋白具有许多功能，可以拮抗宿主对病毒感染的反应，如前几年所述。 我们正在开发HPIV1、2和3的衰减版本，这些版本也表达了人类呼吸道合胞病毒（RSV）的融合F蛋白。 RSV是严重的小儿呼吸道疾病的最重要的病毒剂，对与HPIV合并的人类疾病的贡献有效，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将对每个相应的HPIV和RSV提供二价疫苗。 与RSV菌株相比，HPIV在细胞培养中更有效地复制，并且具有更大的身体稳定性。与RSV的大丝相比，它们还形成了球形颗粒，使它们更适合过滤和其他制造步骤。这些属性使HPIV矢量与衰减的RSV菌株相比更容易制造，分发，存储和使用。尤其是较高的身体稳定性对于将RSV疫苗扩展到资源挑战的国家可能至关重要。此外，在实验动物中，使用HPIV/RSV载体而不是减弱的RSV菌株，提高RSV反应的效率更高，因为后者受到先前的RSV特异性免疫力受到更大的限制。 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 HPIV3。以前，B/HPIV3已在临床1期研究中进行了评估，无论是作为空载体（盖子/NIAID研究）还是作为RSV-F研究的载体（Medimmune研究），并且被证明在婴儿和幼儿中都具有良好的作用。在B/HPIV3-RSV-F的最初临床研究中，RSV F插入物表现出很大的不稳定性，并且不如所希望的那样免疫原性。因此，我们的目标是增加RSV F插入物的免疫原性和稳定性。 我们先前评估了RSV F基因插入到B/HPIV3骨架中的位置的影响，并发现第一个（PRE-N）和第二个（N-P）基因位置很容易容纳RSV F插入物。与下游位置相比，RSV F蛋白的表达大大增加（增加了69倍），并且融合量大大增加。令人惊讶的是，这似乎并没有干扰体外或仓鼠的矢量复制。 在过去的一年中，我们发现RSV F蛋白的表达通过密码子优化进一步增强了5倍，并通过修改氨基酸序列与原始临床分离株的早期通过相同。这赋予了一种低舒张的表型，大概反映了原始的临床分离株，并表明这种RSV菌株可能已突变以在体外通过过程中获得高效原表型。然后，我们比较了表达RSV F蛋白的稳定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诱导的RSV中和血清抗体的数量和质量增加和对WT RSV挑战的保护量增加。相比之下，表达融合后F的矢量比天然RSV F比本地RSV F更具免疫原性和保护性。 使用双染色免疫荧光测定法表明，RSV F蛋白表达的稳定性很高，并且不受RSV F的表达或融合前或融合后的影响。这些研究提供了RB/HPIV3-RSV F疫苗候选者的改进版本，可诱导RSV-RSV-RSV-NEDANTRAD ANTIBIMANE ANTIMARMAIMARAM MINIDERAMPODY。 HPIV1在过去一年中也被开发为RSV F的向量。将RSV F基因单独插入三个不同的基因组位置（PRE-N（F1），N-P（F2）或P-M（F3））中的三个减弱的RHPIV1骨架中的每个主体。每个骨干都包含一个先前描述的衰减突变，该突变稳定在脱位：（1）非温度敏感性缺失突变突变，​​涉及六个核苷酸的P/C ORF（CDEL170）中的六个核苷酸，或（2）在L ORF（ly942a）中的温度敏感性遗传突变。在体外，F插入物的存在降低了病毒复制的速率，但最终滴度与WT HPIV1相同。用RHPIV1-CDEL170-F1，-F2和-F3和RHPIV1-LY942A-F1观察到培养细胞中的RSV F表达高水平的表达。在仓鼠中，RHPIV1 -CDEL170 -F1，-F2和-F3载体在鼻涡轮上受到适度限制，并且在肺中受到了高度限制，并且在体内遗传稳定。在CDEL170载体中，F1病毒是针对WT RSV挑战的最免疫原性和保护性。 RHPIV1-LY942A载体在体内受到高度限制，并且未检测到免疫原性或保护性，表明过度衰减过度。 CDEL170-F1构建体似乎是适当地减弱和免疫原性的，以进一步发育，作为一种双重鼻内小儿疫苗。 我们还研究了HPIV3中基因表达的调节。 HPIV3基因的基因端（GE）转录信号是高度保守的，除了M GE信号包含明显的8-核苷酸插入物。这与M基因和下游F蛋白基因的读入转录物的合成增加有关。我们假设该插入物可以通过干扰M-F基因连接处的终止/重新定位来下调F蛋白的表达，从而促进M-F读取mRNA的产生，而以单科发电率F mRNA为代价。为了检验该假设，产生了两个类似的重组HPIV3病毒，从中去除了M-GE信号中的该插入物。 M-GE突变体表现出M-F读取mRNA的降低，并且单科传播FRNA的增加。这导致了感染细胞中F蛋白合成的大幅增加，并增强了F蛋白在病毒体中的掺入。突变病毒复制的效率与体内和体内的WT HPIV3相似。然而，与WT HPIV3相比，突变体中F蛋白特异性血清抗体反应增加。这项研究确定了一种新型的病毒机制，用于减少宿主适应性免疫反应的刺激。修复M-GE信号应提供一种增加对活衰减HPIV3疫苗的抗体反应的方法，而不会影响病毒复制和衰减。