CMV tegument protein pp65 and CMV immediate early gene product IE1 are both considered immunodominant targets of cell-mediated immunity (CMI) and potentially capable of controlling CMV infection. To better assess their role in host defense, we have constructed a novel MVA transfer vector named pZWIIA and generated a recombinant MVA (rMVA) expressing both full-length pp65 and exon4 of IE1 (pp65-IE1-MVA) at high levels, followed by the genetic removal of the bacterial marker gene used to distinguish recombinant forms. Immunogenicity evaluation indicates that pp65-IE1-MVA not only can induce robust primary CMI to both antigens in HLA A2.1 Tg mice, but also can stimulate vigorous expansion of memory T lymphocyte responses to pp65 and IE1 in PBMC of CMV-positive donors. These properties make the MVA-based vaccine ideal for the dual role of priming and boosting CMV-specific T cell immunity as a means to control CMV disease in recipients of hematopoietic cell or solid organ transplantation (HCT or SOT). pZWIIA alone or in combination with other MVA transfer vectors can be used to generate MVA based multiple-antigen vaccine which have application in vaccine development for a wide spectrum of infectious diseases and cancer.