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：
8839199
负责人：
Peter A Barry
金额：
$ 73.77万
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 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8839199
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 Drug Formulations Embryo Endothelial Cells Engineering Ensure Epithelial Epithelial Cells Evaluation Extracellular Domain Fetus Fibroblasts Flow Cytometry Formalin Frequencies Future Generations Genes Glycoproteins Goals Harvest Health Human Human Cytomegalovirus Infection Pathway 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 Primates Property Protein Subunits Proteins Publishing Qualifying 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）：通过发现病毒进入于由五型病毒素糖蛋白复合蛋白复合物介导的上皮细胞（EPI/EC）的第二个途径，我们对人类巨细胞病毒（HCMV）感染的理解得到了增强。 HCMV进入的原始成纤维细胞（Fibro）途径多年的研究的高潮是一项临床试验，在该试验中，发现了对HCMV阴性妇女的配方GB疫苗，发现了50％的针对原发性感染的保护。我们使用修饰的Rhgb和Tegument Protein RHPPP65的修饰疫苗ANKARA（MVA）疫苗，使用修饰的Vaccinia Ankara（MVA）疫苗，使用修饰的Vaccinia Ankara（MVA）疫苗，通过证明50％的保护RHCMV挑战，通过证明50％的保护对强烈的RHCMV挑战，通过证明50％的保护对强烈的RHCMV挑战，通过证明50％的保护措施，通过证明50％的保护措施，证明了50％的保护，我们通过Rhcmv（RHCMV）阴性猕猴（RM）重现了这一成功。我们假设这进一步改善了疫苗成功；将需要有效抑制CMV进入EPI/EC。利用一种革命性的方法来操纵细菌人造染色体（BAC）衍生的MVA，我们在BAC质粒中的单独的MVA插入位点中串联克隆了5种亚基蛋白中的每一种。我们最近发表了，用RHUL128C-MVA接种RHCMV阴性RM产生了高滴度中和抗体（NAB），从而抑制了从感染EPI/EC和纤维的毒性RHCMV天然分离物，从而抑制RHUL128C的功能。基于这些新颖的结果，在RHCMV/RM模型中，我们在BAC-MVA中构建了与RHUL128C的HCMV对应物，并用于疫苗接种BALB/C小鼠的重构MVA病毒，以防止EPI/EC的体外HCMV感染。以下3个特定目标（SA）将测试这一概念，构成临床评估的途径。在 SA1我们将克隆MVA（1974-MVA）进入一个可自我验证的BAC，然后插入构成HCMV UL128C（H-UL128C）的5个亚基基因以构建功能疫苗。通过MVA表达的H-UL128C将通过测量EPI细胞HCMV感染的直接体外抑制（ARPE-19）（ARPE-19），并通过体内在BALB/CILE中间接生成NAB，从而在功能上进行评估，而BALB/c小鼠将抑制ARPE-19细胞的HCMV感染。在SA2中，H-UL128C-MVA将用于接种RM和针对pentamer络合物在血清和唾液中产生的NAB的特性，并从接种疫苗和对照组中进行评估，以防止天然HCMV分离ARPE-19细胞的天然HCMV分离型感染。疫苗接种后血清的亲和力测定将在结合含有裂解物的裂解物后使用尿素变性评估。在SA3中，我们将通过包括表达H-UL128C和HCMV PP65/GB亚基的疫苗作为2种单独的疫苗或单个多重插入形式来选择一种抑制2 HCMV进入途径的方案。在BALB/C小鼠中的初步研究将使表现更好的构建体降低。将基于产生高级NAB亲和力和干扰ARPE-19细胞和纤维的体外HCMV感染的滴度来选择该制剂和方案。评估RADV感染的爆炸和T细胞激活的细胞溶解识别将在接种的小鼠中进行第一进行。然后在SA2和3中进行RM。这些研究的预期结果将是一种基于HCMV的亚基疫苗，准备进行临床开发。