Rapid Ex Vivo Expansion of CMV Specific CTL Suitable for Clinical Immunotherapy Using CpG-DNA Matured PBMC Infected with Recombinant MVA.

Abstract

Adoptive transfer of ex vivo expanded CMV-specific T cells is an effective approach, and an attractive alternative to using anti-virals to manage CMV infection for HSCT recipients. We recently published a robust approach to expanding CMV-specific CTL based on infection of autologous EBV-LCL with the attenuated poxvirus, Modified Vaccinia Ankara (MVA), expressing CMV pp65, pp150, and IE1 proteins. This approach causes vigorous, up to 500fold expansions in as little as 12–14 days of memory CD8+ T cells specific for these immunodominant antigens. In order to improve the specificity of the expanded T cells, a method was sought to derive effective antigen presenting cells (APC) that avoided the use of EBV-LCL. Of equal importance is to develop an expansion approach that avoids the need to involve virally infected APC in developing a clinical product. Our preliminary observation is that rMVA can infect PBMC in vitro, causing high levels of expression of recombinant CMV antigens. To be permissible for high level expression from rMVA, fresh PBMC were treated with different combinations of single-stranded CpG-containing phosphorothioate backbone oligonucleotides (ODN). A three-day incubation with a combination of two ODN (ODN # 2006 and 2216) which are known to stimulate both plasmacytoid dendritic and B-cells were found to reproducibly generate a highly rMVA infectable population of PBMC. In all five healthy CMV-positive donors tested, CpG ODN treated autologous PBMC, infected with recombinant rMVA, elicited a 20-fold average expansion of CMV-specific CD8+ T cells, in 10 days. Several different rMVA expressing CMV genes were evaluated, including a novel vector expressing the UL44 gene product, an immunodominant target of the host cellular immune response. The expanded T cell populations showed minimal alloreactivity, and exhibited high levels of CMV-specific MHC Class I tetramer binding, epitope-specific cytokine production, and cytotoxic activity. The availability of a source of autologous professional APC that can be used after only 3 days of priming, enhances the attractiveness of using rMVA for adoptive immunotherapy for HSCT recipients or donor vaccination.