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is one that we previously developed, called rB/HPIV3, which consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3.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PIV 3的载体表达RSV抗原，提供针对两种最重要的儿科病毒性呼吸道病原体的二价疫苗。该载体是我们以前开发的一种载体，称为rB/HPIV 3，它由牛PIV 3组成，其中F和HN基因已被HPIV 3的基因取代。这导致嵌合病毒由于BPIV 3骨架而在非人灵长类动物和人中减毒，并且其携带HPIV 3的中和和主要保护性F和HN抗原。空B/HPIV 3载体和表达未修饰的RSV F蛋白的B/HPIV 3之前均显示在婴儿和幼儿中耐受良好。因此，该矢量似乎在期望的衰减范围内。 在前几年继续进行的工作中，我们继续专注于表达RSV融合F糖蛋白，因为它通常被认为是最重要的RSV中和和保护性抗原。RSV F在RSV毒株中也比附着G蛋白高度保守，附着G蛋白是另一种中和抗原和第二重要的保护性抗原。我们继续评估多种策略来优化表达RSV F蛋白的rB/HPIV 3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价鉴定出第二个基因位置通常是最佳的。对RSV F ORF的密码子优化的几种版本的评估鉴定了由GenScript（GS）提供的最有效的一种。用两个错义突变（称为HEK）修饰RSV F蛋白，使其与该RSV毒株的早期传代分离株相同，这减少了融合并稳定了三聚体。特别是两个额外的修饰显著增加了载体表达的F蛋白的免疫原性：（i）一个修饰是通过引入NIH疫苗研究中心和其他地方的同事已经报道的突变来增加F蛋白的融合前构象的稳定性，所述融合前构象是诱导RSV中和抗体最有效的构象。最成功的突变涉及添加二硫键（称为DS突变）与两个空腔填充错义突变（称为Cav 1）的组合。(ii)另一种修饰是将RSV F工程化以有效包装在B/HPIV 3载体颗粒中。这是通过用BPIV 3 F的跨膜和胞质尾区（TMCT）结构域替换RSV F的跨膜和胞质尾区（TMCT）结构域完成的。这两种修饰DS-Cav 1和TMCT中的每一种导致血清RSV中和抗体的诱导显著增加，特别是在体外有效中和RSV而不添加补体的抗体，因此在中和中非常有效。 在前几年继续的工作中，我们在临床前研究中构建并评估了超过35个版本的rB/HPIV 3-RSV-F。这些构建体包括由本领域工作者鉴定的各种错义突变以及F切割位点的缺失，从而产生单链蛋白。这些研究识别出两种电极导线版本。一种称为rB/HPIV 3-F2/HEK/GS-opt/DS-Cav 1，具有以下特征：在第二个基因位置（F2）插入RSV F，早期传代氨基酸序列（HEK），GenScript优化（GS-opt）和DS-Cav 1 pre-F稳定化。第二个电极导线版本称为rB/HPIV 3-F2/HEK/GS-opt/DS-Cav 1/B3 TMCT，除了还包含TMCT修饰外，其余均相同。这些候选药物目前正被制成临床试验材料，用于儿科临床评价。 我们还使用HPIV 3作为载体来表达RSV融合蛋白。一个优点是rHPIV 3表达所有HPIV 3抗原，而rB/HPIV 3仅表达两种抗原。此外，使用rHPIV 3作为载体应避免在添加修饰的RSV F基因后过度减毒，这可能发生在rB/HPIV 3中。该项目建立在使用B/HPVI 3获得的科学见解的基础上。为了增强其免疫原性，对RSV F进行修饰（i）以增加融合前（pre-F）构象的稳定性，以及（ii）通过用HPIV 3 F（H3 TMCT）的跨膜（TM）和胞质尾区（CT）结构域替换其跨膜（TM）和胞质尾区（CT）结构域，以增加在载体病毒体中的掺入。RSV F（+/-H3 TMCT）从rHPIV 3的第一个（F/preN）或第二个（F/N-P）基因位置表达。H3 TMCT修饰显著增加了RSV F包装到载体病毒体中，并且在仓鼠中，除了前F稳定化所赋予的增加之外，还导致高质量血清RSV中和抗体滴度的显著增加。只有F-H3 TMCT/preN复制在鼻甲中被RSV F插入物显著减弱。F-H3 TMCT/preN、F/N-P和F-H3 TMCT/N-P提供针对wt RSV攻击的完全保护。F-H3 TMCT/N-P是RSV F基因表达最稳定、表达量最高的菌株，为RSV F基因的进一步发展提供了动力。