Refinement in the production and purification of recombinant HCMV IE1–pp65 protein for the generation of epitope-specific T cell immunity

Human cytomegalovirus (HCMV) remains one of the most common opportunistic infections causing disease following stem cell transplantation, despite the availability of anti-viral therapies. Adoptive immunotherapy has the potential to further aid in counteracting chronic viral reactivation and subsequent disease by restoring viral immunity through the transfer of virus-specific T cells from transplant donors to their recipients. Our study refines the production and purification of a recombinant HCMV protein containing two of the most immunodominant antigens (IE1 and pp65) for the generation of polyclonal HCMV-specific T cells. In doing so, a 6× His-tagged IE1-pp65 protein was generated using a serum-free baculovirus/insect cell expression system and soluble IE1–pp65 protein was subsequently purified using Ni–NTA affinity chromatography under stringent conditions to obtain a highly pure product. The ability of the recombinant IE1–pp65 protein to elicit a functional T cell mediated immune response was demonstrated by the vigorous reactivation and expansion of HLA-A2-restricted pp65 495–503-specific CD8 + T cells. This recombinant IE1–pp65 protein can potentially generate a multitude of HLA-restricted HCMV-specific T cells, providing a better alternative to using costly overlapping peptides or HCMV lysates for expansion of T cells for use in adoptive immunotherapy strategies.