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: 10272025
• 负责人: Ursula Buchholz
• 金额: $ 125.84万
• 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/10272025
• 关键词: Adult; Age; Amino Acid Sequence; Amino Acids; Animal Model; Antigen Presentation; Attenuated; Attenuated Live Virus Vaccine; Back; Basic Science; Blood; CCR5 gene; CD86 gene; Cardiopulmonary; Cells; Childhood; Clinical Research; Code; Codon Nucleotides; Complex; Computational algorithm; Cooperative Research and Development Agreement; Deletion Mutation; Dendritic Cells; Development; Dinucleoside Phosphates; Disease; Down-Regulation; Elderly; Epitopes; Evaluation; Exhibits; Family member; Genetic; Genome; Glycoproteins; Goals; Hamsters; Human; Human Genome; Human Metapneumovirus; Hydrophobicity; Immune; Immune response; Immunobiology; Immunologics; In Vitro; Individual; Infant; Influenza A virus; Laboratory Study; Life; Lymphoid Tissue; Messenger RNA; Missense Mutation; Molecular Biology; Molecular Genetics; Morbidity - disease rate; Murine pneumonia virus; Mus; Mutation; Myelogenous; Nonstructural Protein; Nucleoproteins; Open Reading Frames; Peripheral; Phosphoproteins; Play; Pneumovirus; Polymerase; Population; Production; Protein Biosynthesis; Proteins; RNA; RNA Viruses; Reagent; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus Vaccines; Respiratory Tract Diseases; Respiratory syncytial virus; Rodent; Role; Secondary to; Serum; Surface; System; Techniques; Temperature; Tissues; Translations; Umbilical Cord Blood; Vaccine Clinical Trial; Vaccines; Variant; Viral; Viral Genome; Viral Proteins; Virus; Virus Replication; Work; adaptive immune response; attenuation; base; cell motility; cytokine; design; gene synthesis; genome-wide; glycoprotein G; immunogenicity; member; mortality; multiple myeloma M Protein; mutant; neutralizing antibody; pathogen; preclinical study; programs; reverse genetics; vaccine candidate; viral fitness

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.

我们以前对RSV的各种开放式阅读框（ORF）进行了密码子对Deoptimization（CPD）。这是通过使用计算机算法和从头基因合成来重新安装密码子来完成的，以增加正常代表的密码子对的含量，而无需更改氨基酸编码或整体密码子使用。 CPD仍然是一项新技术，尚不完全理解。它通常具有衰减的效果。认为这主要是由于翻译效率降低，尽管这是有争议的，并且其他机制也可能有助于。在过去的几年中，我们通过各种ORF组合的CPD生产了四个RSV突变体，并表明这些病毒确实被衰减，并且出乎意料的是温度敏感。我们还评估了在限制性温度下这些病毒中的两种病毒的遗传稳定性，确定了潜在的去衰减突变，并使用此信息来制造具有CPD L聚合酶ORF的有希望的疫苗候选者，具有衰减，免疫原性和遗传稳定性具有可取的特征。 CPD RSV菌株已获得开发的许可。 虽然CPD主要用于生成活体内疫苗的候选物，但产生密码子对优化病毒的相反方法的影响仍然很大程度上是未知的。为了评估密码子对优化的效果（CPO），我们通过增加人类基因组中代表过多的密码子对的含量而没有改变整体密码子使用情况和氨基酸序列，从而使RSV基因组中的各种开放式读取框（ORF）通过CPO进行了CPO。这有可能增加编码蛋白（S）和抗原决定因素的表达，并且对于产生具有增加免疫原性的疫苗候选物可能是有用的。制作了四种病毒：Max A（具有NS1，NS2，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（用除M2-1和M2-2的所有ORF的CPO）。由于可能增加了病毒复制的可能性，因此通过纳入密码子缺失突变（DEL1313）和LONSINSION突变（I1314L）在L聚合酶中抑制了每种CPO病毒。我们发现CPO对体外，温度敏感性或特定感染性的多环流病毒复制没有影响。 Max A和L具有共同的CPO，其中一个或多种聚合酶复合物的ORF蛋白质表现出病毒蛋白合成的全球增加。 Max B（带有RSV的主要抗原决定因素的CPO，G和F糖蛋白）表现出降低的蛋白质合成，并且仅在体外降低了单周期病毒复制。所有CPO RSV均显示出小鼠和仓鼠复制的边缘减少。令人惊讶的是，CPO RSV诱导仓鼠中血清RSV中和抗体的较低水平。这种降低的免疫原性可能反映了病毒复制的降低，也可能反映出CpG和UPA二核苷酸作为免疫刺激剂的降低。总体而言，我们的研究描述了RNA病毒CPO对病毒复制和适应性体液免疫反应的矛盾作用。 呼吸综合病毒（RSV）感染并引起婴儿的疾病，并在整个生命中降低疾病而没有显着抗原性变化。相反，通过流感病毒（IAV）重新感染了很大程度上需要抗原性变化。自适应免疫反应取决于树突状细胞（DC）的抗原表现，在年轻婴儿中可能过于不成熟，无法诱导针对RSV恢复的完全保护性免疫反应。因此，我们比较了RSV和IAV激活原代人索血液（CB）和成人血（AB）髓样DC（MDC）的能力。尽管RSV和IAV感染了相似的效率，但RSV在CB和AB MDC中诱导的成熟和细胞因子产生较差。在CB MDC中，RSV和IAV之间的这种差异更为深刻。虽然IAV在一定程度上激活了CB MDC，但RSV并未诱导CB MDC增加了将直流迁移到淋巴组织的成熟标记CD38和CD86或CCR7。低CCR7表面表达与CCR5的高表达有关，CCR5保持在发炎的周围组织中。为了评估RSV可能的抑制作用，我们对RSV接种的AB MDC进行了继发IAV接种。尽管RSV接种的AB MDC通过有效上调激活标记和细胞因子的产生来对次级IAV接种做出反应，但IAV诱导的CCR5下调在表现出强大的RSV感染的细胞中略有抑制。因此，次优刺激和弱且大部分可逆的抑制似乎是RSV效率低下的MDC激活的原因。年轻婴儿的MDC刺激和免疫不成熟的效率低下可能导致免疫反应降低和针对RSV恢复的不完全保护。