HCMV Vaccine produced from BAC-MVA that Blocks Epithelial and Fibroblast Entry

由 BAC-MVA 生产的 HCMV 疫苗可阻止上皮细胞和成纤维细胞进入

基本信息

批准号：
9054798
负责人：
Peter A Barry
金额：
$ 72.42万
依托单位：
BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-15 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9054798
关键词：
Activities of Daily Living Antibody Avidity Antigens Avidity Bacterial Artificial Chromosomes Binding Biological Assay Blast Cell Cells Chickens Clinical Clinical Trials Combined Vaccines Complex Congenital Abnormality Control Groups Cytomegalovirus Cytomegalovirus Infections Cytomegalovirus Vaccines Development Dose Embryo Endothelial Cells Engineering Ensure Epithelial Epithelial Cells Evaluation Extracellular Domain Fetus Fibroblasts Flow Cytometry Formalin Formulation Frequencies Future Generations Genes Glycoproteins Goals Harvest Health Human Immunization In Vitro Inbred BALB C Mice Individual Infant Infection Licensure Macaca mulatta Measures Mediating Methods Modeling Modified Vaccinia Virus Ankara Mus Natural History Pathway interactions Plasmids Primary Infection Primates Property Protein Subunits Proteins Publishing Qualifying Recombinant modified vaccinia virus Ankara Regimen Residual state Route Saliva Serial Passage Serum Site Staging Structure of retinal pigment epithelium Subunit Vaccines T-Cell Activation T-Lymphocyte Techniques Technology Testing Time Toxic effect Translations Urea Vaccinated Vaccination Vaccine Design Vaccines Viral Viremia Virion Virulent Virus Woman bacterial vector base cell type clinical investigation dosage experience immunogenicity improved in utero in vitro testing in vivo meetings member neutralizing antibody novel prevent reconstitution research clinical testing response safety study success vaccine development vector viral DNA

项目摘要

DESCRIPTION (provided by applicant): Our understanding of human cytomegalovirus (HCMV) infection has been enhanced by discovery of a 2nd pathway of virus entry into epithelial-endothelial cells (Epi/EC) mediated by a pentameric virion glycoprotein complex. The culmination of many years of study on the original Fibroblast (Fibro) pathway of HCMV entry was a clinical trial in which a formulated gB vaccine was repeatedly administered to HCMV-negative women and 50% protection against primary infection was found. We have reproduced this success using Rhesus CMV (RhCMV)-negative rhesus macaques (RM) by demonstrating 50% protection against a virulent RhCMV challenge, using a modified vaccinia Ankara (MVA) vaccine composed of RhgB and the tegument protein Rhpp65. We hypothesize that to further improve vaccine success; efficient inhibition of CMV entry into Epi/EC will be required. Utilizing a revolutionary approach of manipulation of a bacterial artificial chromosome (BAC) derived MVA, we serially cloned each of 5 subunit proteins constituting the RhCMV UL128C (RhUL128C) in separate MVA insertion sites in a BAC plasmid. We recently published that vaccination of RhCMV-negative RM with RhUL128C-MVA produced high titer neutralizing antibodies (NAb) that inhibit virulent RhCMV natural isolates from infecting Epi/EC and Fibro, confirming RhUL128C function. Based on these novel results in the RhCMV/RM model, we constructed an HCMV counterpart to RhUL128C in BAC-MVA, and the reconstituted MVA virus used to vaccinate BALB/c mice generated NAb to prevent in vitro HCMV infection of Epi/EC. The following 3 Specific Aims (SA) will test this concept forming a path to clinical evaluation. In SA1 we will clone MVA (1974-MVA) into a self-excisable BAC, followed by insertion of 5 subunit genes constituting HCMV UL128C (H-UL128C) to construct a functional vaccine. H-UL128C expressed from MVA will be functionally evaluated by measuring direct in vitro inhibition of HCMV infection of Epi cells (ARPE-19), and indirectly by in vivo generation of NAb in BALB/c mice that will inhibit HCMV infection of ARPE-19 cells. In SA2, H-UL128C- MVA will be used to vaccinate RM and properties of NAb generated against the pentamer complex in sera and saliva from vaccinated and control groups will be assessed in preventing natural HCMV isolate infection of ARPE-19 cells. Avidity assays of post-vaccination sera will be assessed using urea denaturation after binding to pentamer containing lysates. In SA3 we will choose a regimen for inhibition of 2 HCMV entry pathways by including vaccines expressing H-UL128C and HCMV pp65/gB subunits either as 2 individual vaccines or a single multiple insert form. Preliminary studies in BALB/c mice will allow down-selection of better performing constructs. The formulation and regimen will be selected based on generation of superior NAb avidity and titers that interfere with in vitro HCMV infection of ARPE-19 cells and Fibro. Assessment of cytolytic recognition of rAdv-infected blasts and T cell activation will be 1st conducted in vaccinated mice, then RM in SA2 & 3. The anticipated result of these studies will be an HCMV-based subunit vaccine ready for clinical development.

描述（由申请人提供）：通过发现由五聚体病毒颗粒糖蛋白复合物介导的病毒进入上皮内皮细胞（Epi/EC）的第二条途径，我们对人巨细胞病毒（HCMV）感染的理解得到了增强。对 HCMV 进入的原始成纤维细胞 (Fibro) 途径进行多年研究的成果是一项临床试验，其中对 HCMV 阴性女性重复施用配制的 gB 疫苗，发现对原发感染有 50% 的保护作用。我们利用恒河猴 CMV (RhCMV) 阴性恒河猴 (RM) 重现了这一成功，通过使用由 RhgB 和皮膜蛋白 Rhpp65 组成的改良痘苗安卡拉 (MVA) 疫苗，证明对强毒力 RhCMV 攻击具有 50% 的保护作用。我们假设要进一步提高疫苗的成功率；需要有效抑制 CMV 进入 Epi/EC。利用细菌人工染色体 (BAC) 衍生的 MVA 的革命性操作方法，我们在 BAC 质粒的单独 MVA 插入位点中连续克隆了构成 RhCMV UL128C (RhUL128C) 的 5 个亚基蛋白。我们最近发表了用 RhUL128C-MVA 接种 RhCMV 阴性 RM 产生的高滴度中和抗体 (NAb)，该抗体可抑制强毒力 RhCMV 天然分离株感染 Epi/EC 和 Fibro，从而证实了 RhUL128C 的功能。基于RhCMV/RM模型中的这些新结果，我们在BAC-MVA中构建了RhUL128C的HCMV对应物，并且用于接种BALB/c小鼠的重组MVA病毒产生NAb以预防Epi/EC的体外HCMV感染。以下 3 个具体目标 (SA) 将测试这一概念，形成临床评估的途径。在 SA1我们将MVA（1974-MVA）克隆到可自我切除的BAC中，然后插入构成HCMV UL128C（H-UL128C）的5个亚基基因以构建功能性疫苗。 MVA 表达的 H-UL128C 将通过测量 Epi 细胞 (ARPE-19) HCMV 感染的直接体外抑制进行功能评估，并通过 BALB/c 小鼠体内产生 NAb 间接进行功能评估，NAb 将抑制 ARPE-19 的 HCMV 感染细胞。在 SA2 中，H-UL128C-MVA 将用于接种 RM，并且将评估针对来自接种组和对照组的血清和唾液中的五聚体复合物产生的 NAb 的特性，以防止 ARPE-19 细胞的自然 HCMV 分离株感染。与含有五聚体的裂解物结合后，将使用尿素变性来评估疫苗接种后血清的亲合力测定。在 SA3 中，我们将选择一种抑制 2 条 HCMV 进入途径的方案，包括表达 H-UL128C 和 HCMV pp65/gB 亚基的疫苗，作为 2 种单独的疫苗或单一的多重插入形式。 BALB/c 小鼠的初步研究将允许向下选择性能更好的构建体。将根据干扰 ARPE-19 细胞和 Fibro 的体外 HCMV 感染的优异 NAb 亲和力和滴度的产生来选择制剂和方案。首先将在接种疫苗的小鼠中进行对 rAdv 感染的母细胞的溶细胞识别和 T 细胞活化的评估，然后是 SA2 和 3 中的 RM。这些研究的预期结果将是一种基于 HCMV 的亚单位疫苗，可供临床开发。