Laboratory Studies of Human Respiratory Syncytial Virus and Other Pneumoviruses

我们先前对RSV的各种开放阅读框架（ORF）进行了密码子对去优化（CPD）。这是通过使用计算机算法和从头基因合成重排RSV开放阅读框的密码子来完成的，以增加通常代表性不足的密码子对的含量，而不改变氨基酸编码或总体密码子使用。CPD通常是减毒的，因为认为代表性不足的密码子对的功能不是最佳的。CDP导致ORF表达差和减毒，为疫苗候选物提供了新的途径。由于大量的遗传变化，通常认为CPD的减毒对去减毒是难治的。在过去的几年中，我们生产了RSV突变体，通过CPD的各种组合的ORF减毒，并显示这些病毒出乎意料地是温度敏感的。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，确定了不同的减毒途径。在本财政年度，我们利用这些信息生产了一种额外的候选疫苗，其遗传稳定性进一步提高。改良CPD RSV毒株已被Codageland，Inc.许可用于疫苗开发。 此外，我们使用反向遗传学开发了几种候选的减毒活RSV疫苗病毒。减毒是基于先前开发的辅助蛋白基因和点突变的缺失，并且已经被设计为对减毒难治。通过缺失M2-2 ORF或NS 1或NS 2基因使先导候选物减毒。在目前正在进行的实验中，我们正在进一步表征这些减毒活RSV疫苗候选物的减毒基础。对衰减基础的更深入理解将指导临床评价备用候选药物的开发和优先级排序。虽然主要候选药物正在1期和2期临床研究中进行评估（见我们的报告“呼吸道合胞病毒和相关病毒疫苗的临床试验”），但重要的是要有更多的候选药物可用。如果临床研究结果表明需要不同水平的衰减，则需要能够快速推进临床开发的备用候选物。 在目前正在进行的其他实验中，我们正在提高RSV血清学检测的灵敏度。这些试验将支持RSV候选疫苗的临床前和临床开发。

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.