Vaccine Properties of a Novel Marker Gene-Free Recombinant Modified Vaccinia Ankara (MVA) Expressing Immunodominant CMV Antigens.

Abstract

Human CMV continues to be a major risk factor in patients undergoing allogeneic hematopoietic stem cell (HSCT) and solid organ transplant (SOT), despite advancement of antiviral therapy. 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. Similar to healthy adults, CMV tegument protein pp65 and IE1 are frequently recognized by human CD4 and CD8 T cells in HCT and SOT recipients. To better assess their role in host defense, we have constructed a novel attenuated poxvirus (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 during the screening process. Immunogenicity evaluation indicates that pp65-IE1-MVA not only can induce robust primary CMI to both antigens in HLA A2.1, B7 and A11 transgenic mice, but also can stimulate vigorous expansion of memory T lymphocyte responses to pp65 and IE1 in PBMC of CMV-positive donors. The recent discovery that additional early antigens of CMV are also well-recognized by healthy adults prompted us to include the IE2 antigen in the vaccine. Evaluation of in vivo immunogenicity of the vaccine expressing IE2 in both transgenic mice and in vitro antigenicity in human peripheral blood lymphocytes of HSCT and SOT recipients will be presented. A novel overlapping peptide library spanning full-length IE2 has been constructed in our laboratory, and used in conjunction with the vaccine expressing IE2, along with IE1 and pp65. Since MVA has a foreign gene capacity of over 30 kilobases (Kb), we have developed a strategy to maximize coverage of haplotypes through insertion of 6 additional antigens into novel insertion sites of the virus, for a total of just 12.0 Kb of the CMV genome. The selected antigens cover 83% of the population, while representing over 22% of the combined CD4 and CD8 T cell repertoire in seropositives. Our studies of the recognition of these antigens expressed in MVA and monitored with our own synthesized over-lapping peptide libraries in both HSCT and SOT recipients will be presented. 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 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.