Wang Zhongde, La Rosa Corinna, Lacey Simon F, Maas Rebecca, Mekhoubad Shahram, Britt William J, Diamond Don J
Laboratory of Vaccine Research, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
J Clin Virol. 2006 Mar;35(3):324-31. doi: 10.1016/j.jcv.2005.09.018. Epub 2006 Jan 4.
Human cytomegalovirus (CMV) infection is an important risk factor in the post-transplant (Tx) recovery phase for both hematopoietic stem cell Tx (HSCT) and solid organ Tx (SOT) recipients. CMV infection may be prevented or controlled by simultaneously inducing both CMV-specific neutralizing antibody (nAb) and cellular immunity. Soluble (s) UL55 (surface glycoprotein), UL83 (tegument protein) and UL123/e4 (nuclear protein) are immunodominant in eliciting both CMV nAb and cellular immunity. An attenuated poxvirus, modified vaccinia Ankara (MVA) was selected to develop this vaccine strategy in Tx recipients, because of its clinical safety record, large foreign gene capacity, and capability to activate strong humoral and cellular immune responses against recombinant antigens.
A subunit vaccine that targets multiple CMV antigens will be used to gain maximal coverage and protective function against CMV infection. rMVA simultaneously expressing sUL55, UL83 and UL123/e4 will be generated, and humoral and cellular immunity it elicits will be characterized, after murine immunization and in vitro to amplify clinical recall responses.
rMVA will be constructed in two steps using UL123/e4-pLW22 followed by sUL55-UL83-pLW51 transfer plasmids. Western blots will be used to characterize expression levels of each antigen. Primary immunity will be evaluated in mouse models, while recall responses to the virally expressed CMV antigens will be assessed in human peripheral blood.
We generated CMV-MVA via homologous recombination, and demonstrated high expression levels of sUL55, UL83 and UL123/e4 by Western blot. CMV-MVA immunization potently induced both humoral and cellular immunity to sUL55, UL83 and UL123 after murine immunization, and cellular immunity to UL83 and UL123 by in vitro amplification of T cell recall responses in human PBMC.
rMVA promotes high level expression of three immunodominant CMV antigens, which is reflected in results of immunization studies in which high titers of UL55-specific antibodies and CD4+ T-help are detected, as well as high levels of UL83-specific and moderate levels of UL123-specific CD8+ CTL.
人巨细胞病毒（CMV）感染是造血干细胞移植（HSCT）和实体器官移植（SOT）受者移植后恢复阶段的一个重要风险因素。通过同时诱导CMV特异性中和抗体（nAb）和细胞免疫，可预防或控制CMV感染。可溶性（s）UL55（表面糖蛋白）、UL83（被膜蛋白）和UL123/e4（核蛋白）在引发CMV nAb和细胞免疫方面具有免疫优势。由于其临床安全记录、较大的外源基因承载能力以及激活针对重组抗原的强大体液和细胞免疫反应的能力，减毒痘病毒安卡拉痘苗病毒（MVA）被选用于在移植受者中开发这种疫苗策略。
使用一种靶向多种CMV抗原的亚单位疫苗，以获得对CMV感染的最大覆盖范围和保护功能。将构建同时表达sUL55、UL83和UL123/e4的重组MVA（rMVA），并在小鼠免疫后以及体外扩增临床回忆反应，以表征其引发的体液和细胞免疫。
rMVA将分两步构建，首先使用UL123/e4-pLW22，然后使用sUL55-UL83-pLW51转移质粒。蛋白质免疫印迹法将用于表征每种抗原的表达水平。将在小鼠模型中评估初次免疫，同时在人外周血中评估对病毒表达的CMV抗原的回忆反应。
我们通过同源重组产生了CMV-MVA，并通过蛋白质免疫印迹法证明了sUL55、UL83和UL123/e4的高表达水平。在小鼠免疫后，CMV-MVA免疫有力地诱导了对sUL55、UL83和UL123的体液和细胞免疫，并通过体外扩增人外周血单个核细胞（PBMC）中的T细胞回忆反应诱导了对UL83和UL123的细胞免疫。
rMVA促进了三种免疫优势CMV抗原的高水平表达，这在免疫研究结果中得到体现，其中检测到高滴度的UL55特异性抗体和CD4+辅助性T细胞，以及高水平的UL83特异性和中等水平的UL123特异性CD8+细胞毒性T淋巴细胞（CTL）。
