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新排列密码子来完成的，以增加正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仍然是一项新技术，人们对它的了解还不完全。它通常具有衰减的效果。这被认为主要是由于翻译效率降低，尽管这是有争议的，其他机制也可能起作用。在过去的几年中，我们通过CPD的ORF的各种组合产生了四个RSV突变体，并表明这些病毒确实是减毒的，出乎意料的是，是温度敏感的。我们还评估了这些病毒中的两种在限制性温度下的遗传稳定性，鉴定了潜在的去减毒突变，并使用这些信息来制备具有减毒、免疫原性和遗传稳定性的期望特征的携带CPD L聚合酶ORF的有希望的疫苗候选物。CPD RSV毒株已被许可开发给Codageland，Inc.。 虽然CPD主要用于产生减毒活疫苗候选物，但产生密码子对优化病毒的匡威方法的效果在很大程度上仍然未知。为了评估密码子对优化（CPO）的效果，我们通过增加在人类基因组中过度表达的密码子对的含量，而不改变总体密码子使用和氨基酸序列，将RSV基因组中的各种开放阅读框（ORF）进行CPO。这有可能增加编码的蛋白质和抗原决定簇的表达，并且可用于产生具有增加的免疫原性的疫苗候选物。制作了四种病毒：Max A（具有NS 1、NS 2、N、P、M和SH ORF的CPO）、Max B（具有G和F的CPO）、Max L（具有L的CPO）和Max FLC（具有除M2-1和M2-2之外的所有ORF的CPO）。由于增加病毒复制的可能性，通过在L聚合酶中包含密码子缺失突变（del 1313）和错义突变（I1314 L）来减毒每种CPO病毒。我们发现，CPO在体外，温度敏感性，或特定的感染性对多重冠状病毒复制没有影响。Max A和L，其共同具有聚合酶复合物的蛋白质的一个或多个ORF的CPO，表现出病毒蛋白质合成的整体增加。Max B（含RSV主要抗原决定簇的CPO，即G和F糖蛋白）表现出蛋白质合成减少，并且其单独在体外具有减少的单循环病毒复制。所有CPO RSV在小鼠和仓鼠中的复制均表现出轻微减少。令人惊讶的是，CPO RSV在仓鼠中诱导较低水平的血清RSV中和抗体。这种降低的免疫原性可能反映了病毒复制的减少，也可能反映了作为免疫刺激剂的CpG和UpA二核苷酸的减少。总的来说，我们的研究描述了RNA病毒的CPO对病毒复制和适应性体液免疫应答的矛盾作用。 呼吸道合胞病毒（RSV）感染并导致婴儿疾病，并在一生中再次感染，疾病减少，而没有显著的抗原变化。相比之下，甲型流感病毒（IAV）的再次感染在很大程度上需要抗原变化。适应性免疫应答依赖于树突状细胞（DC）的抗原呈递，树突状细胞在年幼婴儿中可能太不成熟而不能诱导针对RSV再感染的完全保护性免疫应答。因此，我们比较了RSV和IAV激活原代人脐带血（CB）和成人血（AB）髓样DC（mDC）的能力。虽然RSV和IAV感染具有相似的效率，但RSV在CB和AB mDC中诱导成熟和细胞因子产生较差。RSV和IAV之间的这种差异在CB mDC中更深刻。虽然IAV在一定程度上激活CB mDC，但RSV并不诱导CB mDC增加成熟标志物CD 38和CD 86或CCR 7，其指导DC向淋巴组织迁移。低CCR 7表面表达与CCR 5的高表达相关，CCR 5将DC保持在发炎的外周组织中。为了评估RSV的可能抑制，我们将RSV接种的AB mDC进行第二次IAV接种。虽然RSV接种的AB mDC通过有效上调活化标志物和细胞因子产生来响应二次IAV接种，但IAV诱导的CCR 5下调在表现出强RSV感染的细胞中被轻微抑制。因此，次优刺激和弱且大部分可逆的抑制似乎是RSV无效激活mDC的原因。低效率的mDC刺激和年幼婴儿的免疫不成熟可能导致免疫应答降低和对RSV再感染的不完全保护。

We previously performed codon-pair deoptimization (CPD) of various open reading frames (ORFs) of RSV. This is done by rearranging codons 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 is still a new technique and is incompletely understood. It typically has the effect of attenuation. This is thought to be due primarily to reduced efficiency of translation, although this is controversial and other mechanisms may also contribute. In previous years, we produced four RSV mutants by CPD of various combinations of ORFs, and showed that these viruses indeed were attenuated and, unexpectedly, were temperature-sensitive. We also evaluated the genetic stability of two of these viruses under restrictive temperatures, identified potential de-attenuating mutations, and used this information to make a promising vaccine candidate bearing a CPD L polymerase ORF that had desirable feature of attenuation, immunogenicity, and genetic stability. CPD RSV strains have been licensed for development to Codagenix, Inc. While CPD has been used mostly to generate live-attenuated vaccine candidates, the effects of the converse approach of generating codon pair optimized viruses are still largely unknown. To evaluate effects of codon pair optimization (CPO), we subjected various open reading frames (ORFs) in the RSV genome to CPO by increasing the content of codon pairs that are overrepresented in the human genome without changing overall codon usage and amino acid sequences. This has the potential to increase the expression of the encoded protein(s) and antigenic determinants, and could be useful to generate vaccine candidates with increased immunogenicity. Four viruses were made: Max A (with CPO of NS1, NS2, N, P, M, and SH ORFs), Max B (with CPO of G and F), Max L (with CPO of L), and Max FLC (with CPO of all ORFs except M2-1 and M2-2). Because of the possibility of increased viral replication, each CPO virus was attenuated by the inclusion of a codon deletion mutation (del1313) and a missense mutation (I1314L) in the L polymerase. We found that CPO had no effect on multicycle virus replication in vitro, temperature sensitivity, or specific infectivity. Max A and L, which in common had CPO of one or more ORFs of proteins of the polymerase complex, exhibited global increases in viral protein synthesis. Max B (with CPO of the major antigenic determinants of RSV, the G and F glycoproteins) exhibited decreased protein synthesis, and it alone had reduced single-cycle virus replication in vitro. All CPO RSVs exhibited marginal reductions in replication in mice and hamsters. Surprisingly, the CPO RSVs induced lower levels of serum RSV-neutralizing antibodies in hamsters. This reduced immunogenicity might reflect reduced viral replication and possibly also the decrease in CpG and UpA dinucleotides as immune stimulators. Overall, our study describes paradoxical effects of CPO of an RNA virus on viral replication and the adaptive humoral immune response. Respiratory syncytial virus (RSV) infects and causes disease in infants and reinfects with reduced disease throughout life without significant antigenic change. In contrast, reinfection by influenza A virus (IAV) largely requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC), which may be too immature in young infants to induce a fully protective immune response against RSV reinfections. We therefore compared the ability of RSV and IAV to activate primary human cord blood (CB) and adult blood (AB) myeloid DC (mDC). While RSV and IAV infected with similar efficiencies, RSV poorly induced maturation and cytokine production in CB and AB mDC. This difference between RSV and IAV was more profound in CB mDC. While IAV activated CB mDC to some extent, RSV did not induce CB mDC to increase the maturation markers CD38 and CD86 or CCR7, which directs DC migration to lymphatic tissue. Low CCR7 surface expression was associated with high expression of CCR5, which keeps DC in inflamed peripheral tissues. To evaluate a possible inhibition by RSV, we subjected RSV-inoculated AB mDC to secondary IAV inoculation. While RSV-inoculated AB mDC responded to secondary IAV inoculation by efficiently upregulating activation markers and cytokine production, IAV-induced CCR5 downregulation was slightly inhibited in cells exhibiting robust RSV infection. Thus, suboptimal stimulation and weak and mostly reversible inhibition seem to be responsible for inefficient mDC activation by RSV. The inefficient mDC stimulation and immunological immaturity in young infants may contribute to reduced immune responses and incomplete protection against RSV reinfection.