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: 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
• 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

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)：通过发现病毒进入上皮-内皮细胞的第二条途径(EPI/EC)，我们对人巨细胞病毒(HCMV)感染的了解得到了加强。多年来对人巨细胞病毒进入的原始成纤维细胞(FIBO)途径的研究达到了顶峰，这是一项临床试验，在该试验中，对HCMV阴性的妇女反复接种配方GB疫苗，发现对初次感染有50%的保护作用。我们用Rheus CMV(RhCMV)阴性恒河猴(RM)复制了这一成功，通过使用由RhgB和被蛋白Rhpp65组成的改良牛痘Ankara(MVA)疫苗展示了对强毒RhCMV攻击的50%保护。我们推测，为了进一步提高疫苗的成功率，将需要有效地抑制CMV进入Epi/EC。利用一种革命性的操作细菌人工染色体(BAC)来源的MVA的方法，我们将组成RhCMV UL128C(RhUL128C)的5个亚基蛋白分别序列克隆到BAC质粒中不同的MVA插入位点。我们最近发表了用RhUL128C-MVA免疫RhCMV阴性的RM产生高滴度的中和抗体(NAB)，以抑制强毒株感染Epi/EC和Fibo，证实了RhUL128C的功能。基于这些在RhCMV/RM模型中的新结果，我们在BAC-MVA中构建了与RhUL128C类似的HCMV，并且用于免疫BALB/c小鼠的重组MVA病毒产生了NAB以防止体外HCMV感染Epi/EC。以下3个具体目标(SA)将检验这一概念，形成临床评估的途径。在……里面 SA1我们将MVA(1974-MVA)克隆到一个自切除的BAC中，然后插入组成HCMV UL128C(H-UL128C)的5个亚单位基因，以构建功能疫苗。由MVA表达的H-UL128C将通过在体外直接抑制EPI细胞(ARPE-19)的HCMV感染，以及间接通过在BALB/c小鼠体内产生抑制ARPE-19细胞的HCMV感染的NAB来间接进行功能评估。在SA2中，H-UL128C-MVA将用于接种RM，并将评估接种组和对照组产生的针对血清和唾液中五聚体复合体的NAB的性质，以防止HCMV分离株自然感染ARPE-19细胞。疫苗接种后血清的亲和力分析将在与含有裂解物的五聚体结合后用尿素变性来评估。在SA3中，我们将选择一种抑制2条HCMV进入途径的方案，将表达H-UL128C和HCMV pp65/GB亚单位的疫苗作为2个单独的疫苗或单个多重插入形式。对BALB/c小鼠的初步研究将允许向下选择性能更好的结构。处方和方案的选择将基于产生的优越的NAB亲和力和滴度，干扰体外感染的ARPE-19细胞和纤维。首先将在接种疫苗的小鼠中进行rAdv感染的母细胞的溶细胞识别和T细胞激活的评估， 这些研究的预期结果将是一种基于HCMV的亚单位疫苗，为临床开发做好准备。