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

• 批准号: 8660624
• 负责人: Peter A Barry
• 金额: $ 75.03万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8660624
• 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; 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; dosage; experience; immunogenicity; improved; in utero; in vitro testing; in vivo; meetings; member; neutralizing antibody; novel; prevent; public health relevance; reconstitution; research clinical testing; response; safety study; success; vaccine development; vector; viral DNA

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）：我们对人巨细胞病毒（HCMV）感染的理解已经通过发现由五聚体病毒体糖蛋白复合物介导的病毒进入上皮-内皮细胞（Epi/EC）的第二种途径得到增强。对HCMV进入原始成纤维细胞（Fibro）途径的多年研究的高潮是一项临床试验，在该试验中，将配制的gB疫苗重复给予HCMV阴性女性，发现对原发感染的保护率为50%。我们使用恒河猴CMV（RhCMV）阴性恒河猴（RM）复制了这一成功，证明对强毒RhCMV攻击的保护率为50%，使用由RhgB和被膜蛋白Rhpp 65组成的修饰的安卡拉牛痘（MVA）疫苗。我们假设，为了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的操作的革命性的方法，我们连续克隆的5个亚基蛋白构成的RhCMV UL 128 C（RhUL 128 C）在单独的MVA插入位点的BAC质粒。我们最近发表了用RhUL 128 C-MVA接种RhCMV阴性RM产生高滴度中和抗体（NAb），其抑制强毒RhCMV天然分离株感染Epi/EC和Fibro，证实了RhUL 128 C的功能。基于这些新的结果，在RhCMV/RM模型中，我们构建了一个HCMV对应物的RhUL 128 C在BAC-MVA，和重组MVA病毒用于接种BALB/c小鼠产生NAb，以防止在体外HCMV感染Epi/EC。以下3个特定目标（SA）将测试这一概念，形成临床评价的路径。在 SA 1中，我们将MVA（1974-MVA）克隆到可自切除的BAC中，随后插入构成HCMV UL 128 C的5个亚基基因（H-UL 128 C）以构建功能性疫苗。将通过测量对Epi细胞（ARPE-19）的HCMV感染的直接体外抑制以及通过在BALB/c小鼠中体内产生将抑制ARPE-19细胞的HCMV感染的NAb来间接地对从MVA表达的H-UL 128 C进行功能性评价。在SA 2中，H-UL 128 C- MVA将用于接种RM，并且将评估来自接种组和对照组的血清和唾液中针对五聚体复合物产生的NAb的性质，以预防ARPE-19细胞的天然HCMV分离株感染。接种后血清的亲合力测定将在与含有裂解物的五聚体结合后使用尿素变性进行评估。在SA 3中，我们将选择通过包括表达H-UL 128 C和HCMV pp 65/gB亚基的疫苗来抑制2种HCMV进入途径的方案，所述疫苗作为2种单独的疫苗或单一的多插入物形式。在BALB/c小鼠中的初步研究将允许向下选择性能更好的构建体。将基于干扰ARPE-19细胞和Fibro的体外HCMV感染的上级NAb亲合力和滴度的产生来选择制剂和方案。将首先在接种疫苗的小鼠中评估rAdv感染的原始细胞的溶细胞识别和T细胞活化， 然后在SA 2和3中进行RM。这些研究的预期结果将是准备用于临床开发的基于HCMV的亚单位疫苗。