Laboratory Studies of Human Respiratory Syncytial Virus and Other Pneumoviruses

人类呼吸道合胞病毒和其他肺病毒的实验室研究

• 批准号: 10692018
• 负责人: Ursula Buchholz
• 金额: $ 135.61万
• 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/10692018
• 关键词: Age; Amino Acids; Animal Model; Attenuated; Attenuated Vaccines; Back; Basic Science; Biological Assay; Cardiopulmonary; Characteristics; Childhood; Clinical Research; Code; Codon Nucleotides; Computational algorithm; Cooperative Research and Development Agreement; Development; Elderly; Evaluation; Family member; Gene Mutation; Gene Proteins; Genes; Genetic; Genome; Glycoproteins; Goals; Human; Human Metapneumovirus; Hydrophobicity; Immune; Immunobiology; Individual; Laboratory Study; Messenger RNA; Molecular Biology; Molecular Genetics; Molecular Virology; Morbidity - disease rate; Murine pneumonia virus; Mutation; Nonstructural Protein; Nucleoproteins; Open Reading Frames; Phase; Phosphoproteins; Play; Pneumovirus; Point Mutation; Polymerase; Population; Proteins; RNA; RNA Viruses; Reagent; Refractory; Reporting; Respiratory Syncytial Virus Vaccines; Respiratory Tract Diseases; Respiratory syncytial virus; Rodent; Role; Route; Serology test; System; Temperature; Vaccine Clinical Trial; Vaccines; Variant; Viral; Viral Proteins; Virus; attenuation; base; clinical candidate; clinical development; design; experimental study; gene synthesis; genome-wide; glycoprotein G; improved; lead candidate; member; mortality; multiple myeloma M Protein; mutant; pathogen; preclinical development; preclinical study; programs; research clinical testing; reverse genetics; vaccine candidate; vaccine development; viral fitness

We previously performed codon-pair deoptimization (CPD) of various open reading frames (ORFs) of RSV. This is done by rearranging codons of RSV open reading frames using computer algorithms and de novo gene synthesis to increase the content of normally underrepresented codon-pairs without changing amino acid coding or overall codon usage. CPD typically is attenuating, because under-represented codon pairs are thought to function suboptimally. CDP leads to poor ORF expression and attenuation, providing a new path to vaccine candidates. Due to the large number of genetic changes, it is generally thought that attenuation by CPD is refractory to de-attenuation. In previous years, we produced RSV mutants, attenuated by CPD of various combinations of ORFs, and showed that these viruses unexpectedly were temperature-sensitive. We used this characteristic to evaluate the genetic stability of these vaccine candidates when cultured at restrictive temperatures. These studies revealed a surprising level of adaptability of these attenuated viruses, identifying different routes to de-attenuation. During this fiscal year, we used this information to generate an additional vaccine candidate with further improved genetic stability. Improved CPD RSV strains have been licensed for vaccine development by Codagenix, Inc. In addition, we developed several candidate live-attenuated RSV vaccine viruses using reverse genetics. Attenuation is based on deletion of accessory protein genes and point mutations that were previously developed and had been designed to be refractory to de-attenuation. Lead candidates are attenuated by deletion of the M2-2 ORF, or the NS1 or NS2 genes. In experiments that are currently in progress, we are further characterizing the basis of attenuation of these live-attenuated RSV vaccine candidates. A deeper understanding of the basis of attenuation will guide the development and prioritization of backup candidates for clinical evaluation. While lead candidates are being evaluated in Phase 1 and 2 clinical studies (see our report "Clinical Trials of Vaccines for Respiratory Syncytial Virus and Related Viruses"), it is important that additional candidates are available. If clinical study results suggest that a different level of attenuation is needed, backup candidates are needed that can be advanced rapidly to clinical development. In other experiments that are currently in progress, we are improving the sensitivity of RSV serology assays. These assays will support preclinical and clinical development of RSV vaccine candidates.

我们之前对RSV的各种开放阅读框（orf）进行了密码子对反优化（CPD）。这是通过使用计算机算法和从头基因合成重新排列RSV开放阅读框的密码子来完成的，以增加通常未被代表的密码子对的含量，而不改变氨基酸编码或整体密码子使用。CPD通常是衰减的，因为代表性不足的密码子对被认为是次优功能。CDP导致ORF表达和衰减不良，为候选疫苗提供了新的途径。由于大量的遗传变化，一般认为CPD的衰减难以去衰减。在前几年，我们生产了RSV突变体，通过各种orf组合的CPD减毒，并表明这些病毒出乎意料地对温度敏感。我们利用这一特性来评估这些候选疫苗在限制性温度下培养时的遗传稳定性。这些研究揭示了这些减毒病毒令人惊讶的适应性水平，确定了不同的去衰减途径。在本财政年度，我们利用这些信息产生了进一步提高遗传稳定性的额外候选疫苗。Codagenix公司已批准改良CPD RSV菌株用于疫苗开发。