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  • Title: Refinement in the production and purification of recombinant HCMV IE1-pp65 protein for the generation of epitope-specific T cell immunity.
    Author: Nguyen TH, Mifsud NA, Stewart LA, Rose MJ, Etto TL, Williamson NA, Purcell AW, Kotsimbos T, Schwarer AP.
    Journal: Protein Expr Purif; 2008 Sep; 61(1):22-30. PubMed ID: 18539483.
    Abstract:
    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 6x 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.
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