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

描述（由申请人提供）：我们对人类巨细胞病毒（HCMV）感染的理解已经通过五聚体病毒粒子糖蛋白复合物介导的病毒进入上皮内皮细胞（Epi/EC）的第二途径的发现得到加强。对HCMV进入原成纤维细胞（Fibro）途径的多年研究成果是一项临床试验，在该试验中，配制的gB疫苗反复接种于HCMV阴性妇女，发现对原发性感染的保护率为50%。我们在恒河巨细胞病毒（RhCMV）阴性的恒河猴（RM）身上重现了这一成功，使用由RhgB和被膜蛋白Rhpp65组成的改良安卡拉牛苗（MVA）疫苗，展示了对强毒性RhCMV攻击的50%保护。我们假设，为了进一步提高疫苗的成功率；需要有效地抑制CMV进入Epi/EC。利用一种革命性的方法来操作细菌人工染色体（BAC）衍生的MVA，我们在BAC质粒的不同MVA插入位点连续克隆了构成RhCMV UL128C （RhUL128C）的5个亚基蛋白。我们最近发表了RhUL128C- mva疫苗接种RhCMV阴性RM产生高滴度中和抗体（NAb），抑制强毒RhCMV天然分离株感染Epi/EC和Fibro，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RhUL128C的HCMV对应物，并将重组的MVA病毒用于接种BALB/c小鼠，产生NAb以阻止体外HCMV感染Epi/EC。以下3个具体目标（SA）将测试这一概念，形成临床评估的路径。在

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.