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 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.
Nguyen
Thi H O
TH
Bone Marrow Transplant Research Laboratory, 1st Floor William Buckland Building, Alfred Hospital, Melbourne, Vic. 3004, Australia. o.nguyen@alfred.org.au
Mifsud
Nicole A
NA
Stewart
Lisbeth A
LA
Rose
Mingus J
MJ
Etto
Tamara L
TL
Williamson
Nicholas A
NA
Purcell
Anthony W
AW
Kotsimbos
Tom
T
Schwarer
Anthony P
AP
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D016056:Immunodominant Epitopes / Q000276:immunology*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018411:Spodoptera; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*
D017931:DNA Primers; C464970:IE1-pp65 recombinant protein, Human cytomegalovirus; D017874:Immediate-Early Proteins; D016056:Immunodominant Epitopes; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins