Laboratory Studies of Human Respiratory Syncytial Virus and Other Pneumoviruses

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的使用。CPD仍然是一种新技术，尚未完全理解。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。在过去的几年里，我们用CPD方法生产了四种不同orf组合的RSV突变体，并表明这些病毒确实是减毒的，出乎意料的是，它们对温度敏感。我们还评估了其中两种病毒在限制性温度下的遗传稳定性，确定了潜在的去衰减突变，并利用这些信息制作了一种有希望的候选疫苗，该疫苗带有CPD L聚合酶ORF，具有理想的衰减、免疫原性和遗传稳定性。CPD RSV菌株已被授权给科达吉尼公司进行开发。

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.