Laboratory And Pre-clinical Studies Of Parainfluenza Viruses

副流感病毒的实验室和临床前研究

• 批准号: 10272021
• 负责人: Ursula Buchholz
• 金额: $ 163.32万
• 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/10272021
• 关键词: Age; Amino Acid Sequence; Animals; Antigens; Antiviral Agents; Attenuated; Attenuated Live Virus Vaccine; Attenuated Vaccines; Blood group antigen f; Cattle; Cell Culture Techniques; Characteristics; Child; Childhood; Chimeric Proteins; Clinical Trials; Codon Nucleotides; Complement; Cooperative Research and Development Agreement; Country; Cytoplasmic Tail; Development; Disease; Engineering; Evaluation; Exhibits; Gene Order; Genes; Glycoproteins; Hamsters; Human; Human Parainfluenza Virus 2; Immunization; In Vitro; Infant; Intranasal Administration; Laboratory Study; Lead; Life; Lower respiratory tract structure; Missense Mutation; Modification; Molecular Biology; Molecular Conformation; Molecular Virology; Mutation; NIH Vaccine Research Center; Nasal turbinate bone structure; Nose; Open Reading Frames; Para-Influenza Virus Type 1; Para-Influenza Virus Type 3; Phase; Phase I Clinical Trials; Phenotype; Pneumovirus; Positioning Attribute; Protein Conformation; Proteins; Reporting; Resources; Respiratory Syncytial Virus Vaccines; Respiratory Tract Diseases; Respiratory syncytial virus; Respiratory syncytial virus RSV F proteins; Serotyping; Serum; Site; System; Ursidae Family; Vaccine Clinical Trial; Vaccines; Vertebral column; Viral; Viral Antigens; Virion; Virus; Work; attachment protein G; attenuation; base; disulfide bond; immunogenic; immunogenicity; insight; neutralizing antibody; nonhuman primate; parainfluenza virus; particle; pathogen; preclinical study; programs; research clinical testing; respiratory; reverse genetics; vaccine candidate; vector; vector genome

In recent years, we have focused on using a PIV3-based vector to express RSV antigen, providing a bivalent vaccine against the two most important pediatric viral respiratory pathogens. The vector is one that we previously developed, called rB/HPIV3, which consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3. This results in a chimeric virus that is attenuated in non-human primates and humans due to the BPIV3 backbone, and which bears the neutralization and major protective F and HN antigens of HPIV3. Both the empty B/HPIV3 vector and B/HPIV3 expressing the unmodified RSV F protein were previously shown to be well-tolerated in infants and young children. Therefore, this vector appears to be in the desired range of attenuation. In work continuing from previous years, we continued to focus on expressing the RSV fusion F glycoprotein because it generally is considered to be the most important RSV neutralization and protective antigen. RSV F also is much more highly conserved among RSV strains than the attachment G protein, which is the other neutralization antigen and the second most important protective antigen. We continued to evaluate a number of strategies to optimize the immunogenicity of rB/HPIV3 expressing RSV F protein. This involved increasing both the quantity and the quality of the expressed RSV F antigen. Evaluation of several different positions for the RSV F gene in the vector genome identified the second gene position as generally being optimal. Evaluation of several versions of codon-optimization of the RSV F ORF identified the most efficient one, provided by GenScript (GS). The RSV F protein was modified with two missense mutations (called HEK) to be identical to an early-passage isolate of this RSV strain, which reduced fusion and stabilized the trimer. Two additional modifications in particular substantially increased the immunogenicity of vector-expressed F protein: (i) one modification was to increase the stability of the pre-fusion conformation of the F protein - the conformation that is the most effective in inducing RSV-neutralizing antibodies - by introducing mutations that have been reported by colleagues in the NIH Vaccine Research Center and elsewhere. The most successful mutations involved addition of a disulfide bond (called the DS mutation) in combination with two cavity-filling missense mutations (called Cav1). (ii) The other modification was to engineer RSV F to be efficiently packaged in the B/HPIV3 vector particle. This was done by replacing the transmembrane and cytoplasmic tail (TMCT) domains of RSV F with those of BPIV3 F. Each of these two modifications, DS-Cav1 and TMCT, resulted in a substantial increase in the induction of serum RSV-neutralizing antibodies, and in particular antibodies that neutralized RSV efficiently in vitro without added complement and thus are highly effective in neutralization. In work continuing from previous years, we constructed and evaluated more than 35 versions of rB/HPIV3-RSV-F in pre-clinical studies. These constructs included a variety of missense mutations identified by workers in the field as well as with deletion of the F cleavage site, resulting in a single chain protein. These studies resulted in the identification of two lead versions. One is called rB/HPIV3-F2/HEK/GS-opt/DS-Cav1 and has the following characteristics: insertion of RSV F at the second gene position (F2), an early-passage amino acid sequence (HEK), GenScript optimization (GS-opt), and the DS-Cav1 pre-F stabilization. The second lead version, called rB/HPIV3-F2/HEK/GS-opt/DS-Cav1/B3TMCT, is identical except that it also contains the TMCT modification. These candidates presently are being manufactured into clinical trial material for pediatric clinical evaluation. We also used HPIV3 as a vector to express the RSV fusion protein. One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of a modified RSV F gene, which may occur with rB/HPIV3. This project built on scientific insights obtained with B/HPVI3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.

近年来，我们专注于使用基于PIV3的载体表达RSV抗原，为两种最重要的小儿病毒呼吸道病原体提供了二价疫苗。该向量是我们先前开发的称为RB/HPIV3的载体，该向量由牛PIV3组成，其中F和HN基因已被HPIV3所取代。这导致由于BPIV3骨架而导致非人类灵长类动物和人类减弱的嵌合病毒，并具有HPIV3的中和和主要的保护性F和HN抗原。以前，在婴儿和幼儿中，表达未修饰的RSV F蛋白的空的B/HPIV3载体和B/HPIV3均被证明具有良好的耐受性。因此，该矢量似乎处于所需的衰减范围。 从往年开始的工作中，我们继续专注于表达RSV融合蛋白蛋白，因为它通常被认为是最重要的RSV中和和保护性抗原。在RSV菌株中，RSV F也比附着G蛋白要高得多，后者是另一种中和抗原和第二重要的保护性抗原。我们继续评估许多策略，以优化表达RSV F蛋白的RB/HPIV3的免疫原性。这涉及增加表达的RSV F抗原的数量和质量。对载体基因组中RSV F基因的几个不同位置的评估确定第二个基因位置通常是最佳的。评估Genscript（GS）提供的最有效效率的RSV F ORF的几种版本的密码子优化。 RSV F蛋白通过两个错义突变（称为HEK）进行了修饰，与该RSV菌株的早期分离株相同，从而降低了融合并稳定了三聚体。特别是另外两种修饰显着提高了向量表达的F蛋白的免疫原性：（i）一种修饰是提高F蛋白的融合前构象的稳定性 - F蛋白的融合构象的稳定性 - 该构象在诱导RSV中性化抗体中最有效的构象 - 通过引入了核核的突变，该突变是在NIH研究中引入了NIH IH IH IH IH IH IH IH IH IH IH IH IH IH IH的构型。最成功的突变涉及添加二硫键（称为DS突变）以及两个腔填充的错义突变（称为CAV1）。 （ii）另一个修改是要在B/HPIV3矢量粒子中有效打包RSV F。这是通过更换RSV F的跨膜和细胞质尾部（TMCT）结构域与BPIV3 F的结构域来完成的。这两种修饰（DS-CAV1和TMCT）中的每一个都可以实质上增加了血清中性抗体的抗体，并在不具有中性的抗体中诱导了中性抗体，并具有高效的有效性无效，并有效地有效地构成了有效的有效性无效，并且有效地构成了有效的有效性，并具有有效的有效性的抗体能力。中和。 在往年开始的工作中，我们在临床前研究中构建和评估了35个以上版本的RB/HPIV3-RSV-F。这些结构包括该场中工人确定的各种错义突变以及F裂解位点的缺失，从而产生单链蛋白。这些研究导致了两个铅版本的鉴定。一种称为RB/HPIV3-F2/HEK/GS-OPT/DS-CAV1，具有以下特征：在第二基因位置（F2）处插入RSV F，早期通用氨基酸序列（HEK），Genscript优化（GS-OPT）和DS-CAV1 Pre-F Pre-F Pre-F Pre-F Pre-F Pre-F Pre-F稳定性。第二个名为RB/HPIV3-F2/HEK/GS-OPT/DS-CAV1/B3TMCT的铅版本相同，除了它还包含TMCT修改。这些候选人目前正在制造成临床试验材料，以进行小儿临床评估。 我们还使用HPIV3作为载体来表达RSV融合蛋白。一个优点是RHPIV3表达所有HPIV3抗原，而RB/HPIV3仅表示两种。此外，在添加了修饰的RSV F基因后，将RHPIV3用作载体应避免过度衰减，而RB/HPIV3可能会发生。这个项目建立在B/HPVI3获得的科学见解上。为了增强其免疫原性，对RSV F进行了修改（i），以提高预融合（PER-F）构象的稳定性和（ii）通过更换其跨膜（TM）和细胞质尾巴（CT）结构域的稳定性，并用HPIV3 F（H3TMCT）增加了载体中的稳定性。 RSV F（+/- H3TMCT）从RHPIV3的第一个（F/PREN）或第二（F/N-P）基因位置表示。 H3TMCT修饰将RSV F的包装大大增加到载体病毒体中，并且在仓鼠中，除了由前稳定稳定性所赋予的增加外，还导致高质量血清RSV中和抗体的滴度显着增加。 RSV F插入物在鼻涡轮上仅显着衰减F-H3TMCT/PREN复制。 F-H3TMCT/PREN，F/N-P和F-H3TMCT/N-P提供了针对WT RSV挑战的完全保护。 F-H3TMCT/N-P表现出RSV F的最稳定和最高表达，为其进一步发展提供了动力。