Generation of cytotoxic T-cell lines using overlapping pentadecapeptides derived from conserved regions of the adenovirus hexon protein

Abstract

Many of the 51 serotypes of adenovirus have been associated with clinically relevant infection. Adenovirus can disseminate rapidly in patients with a compromised immune system, such as that which occurs secondary to haematopoietic progenitor-cell transplantation. The higher rate of infection in recipients of T cell-depleted grafts and in those undergoing T cell-targeted treatment during graft versus host disease demonstrates the importance of a T-cell response in preventing disseminated infection. Studies have shown that the memory response to adenovirus is directed primarily to the hexon protein and is dominated by CD4 + T cells, probably due to the ability of the virus to block its presentation on HLA class I antigens. We have developed an approach to expand adenovirus-specific T cells using a pool of overlapping pentadecapeptides derived from selected conserved regions of hexon. We characterized responses to identify the peptides that are recognized, the responding T-cell subsets and their HLA restriction. Of eight lines that were characterized extensively, seven included both CD4 + and CD8 + T cells and each recognized between two and eight unique peptide sequences. By focusing the response on the conserved sequences of hexon, the cell lines are likely to recognize most of the serotypes responsible for clinically relevant disease. The 15 aa peptides used to prime the responses are more likely than whole virus or longer peptides to expand the less frequent CD8 + memory subset. Lines prepared by using our method may be more effective in adoptive immunotherapy protocols designed to prevent or treat disseminated adenovirus infections in high-risk patients.

Selection of adenovirus-specific and Epstein-Barr virus–specific T cells with major histocompatibility class I streptamers under Good Manufacturing Practice (GMP)–compliant conditions

“…To date, clinical efficacy of such multimers has been tested in nine patients treated with cytomegalovirus (CMV)-specific tetramers [32]; eight patients with pentamers directed against infections with CMV (n ¼ 6), Epstein-Barr virus (EBV) (n ¼ 1) and HAdV (n ¼ 1) [33]; and four patients with CMV-specific streptamers [34,35]. Although encouraging results were reported for most patients, only removable streptamers are commercially available for GMP-compliant production allowing the transfer of so-called "minimally manipulated" specific T cells and maintaining the original phenotype of differentiated central memory T cells (CD45RAe/ CD62Lþ) [30], the latter being assumed to be important for long-term survival [32,36,37]. In contrast to other conventional multimers [38], streptamers use the high affinity of d-biotin as a competitor for the binding sites of Strept-Tactin.…”

“…Furthermore, even without the infusion of CD4þ helper T cells, the infusion of CMV-and EBVspecific CD8þ T cells showed high protection against viral infections in such patients [32e35]. Nevertheless, the availability of HAdV-specific MHC class II epitopes [36,37] might also facilitate the isolation of CD4þ HAdV-specific T cells for therapeutic applications.…”

HLA-Peptide Multimer Selection of Adenovirus-specific T Cells For Adoptive T-Cell Therapy

“…Peptide-major histocompatibility complex (pMHC) multimers (tetramers or pentamers) are a powerful tool for characterization of antigen-specific T cells 14 and are also valuable reagents for immunotherapy, as the multimerstained cells can be enriched by magnetic bead selection. 13,[18][19][20][21][22] We have developed pMHC tetramers containing 8 of these peptides (Table 1) and have used these to determine the properties of Ad-specific CD8 + T cells directly ex vivo. 17 Twelve Ad-specific class I epitopes have been reported within the hexon and the major capsid protein, and these are conserved across types and species with only minor variations in amino acid sequences.…”

Comparison of Human Memory CD8 T Cell Responses to Adenoviral Early and Late Proteins in Peripheral Blood and Lymphoid Tissue

“…Rooney and colleagues have also identified other CD8+ T cell epitopes from hexon [24]. Most groups are now focused on expanding hexon-specific T cells ex vivo for immunotherapy of Ad disease in SCT recipients [25]–[27].…”