The immunogenicity of adenovirus vectors remains a major obstacle to their safe and efficacious use for gene therapy. In order to identify T-cell epitopes directly from adenoviruses, four viral protein sequences were screened for the well-characterized 9-mer HLA-A2 binding motif. Peripheral blood mononuclear cells (PBMC) from healthy adults were tested for responses to 17 selected viral peptides using a short-term interferon-gamma ELISPOT assay. Memory T-cell responses were identified to a single peptide derived from the major capsid protein hexon in 5 of 6 HLA-A2-positive donors. Unexpectedly, responses to this hexon peptide were also detected in 4 of 6 HLA-A2-negative donors, and responder cells were identified as CD4(+) T cells by immunomagnetic depletion experiments. A longer 15-mer peptide, H910-924, was identified as the optimal CD4(+) T-cell epitope. This hexon epitope induces strong proliferative T-cell responses that can be blocked by a monoclonal antibody against HLA-DR, and molecular HLA typing of donors suggests that the peptide response is restricted by multiple HLA-DR alleles. Additionally, quantitative analysis of responses to H910-924 and whole adenovirus reveals that the frequency of circulating CD4(+) T cells specific for this single hexon epitope (mean = 61 per 10(6) PBMC) represents up to one third of the total adenovirus-specific T-cell response. Finally, comparison of hexon sequences from over 20 different human adenovirus serotypes indicates that H910-924 is highly conserved. In most individuals, therefore, T-cell responses to this hexon epitope will be induced by all adenovirus vectors, including "gutted" vectors packaged with capsid proteins and vectors based on different serotypes.
Adenoviruses (Ads) cause fatal disease in allogeneic stem cell transplant recipients, but there is no established therapy. Ad-specific CD8+ T cells were detected in PBMC from healthy adults at a mean frequency of 77 per 10(5) CD8+ T cells (range 8-260) by interferon-gamma ELISPOT and cytokine flow cytometry assays. CD8+ T cell lines from 7 of 7 donors exhibited MHC-class-I-restricted killing of targets expressing the capsid protein hexon. In contrast, cytotoxicity against the capsid proteins fiber and penton base was weaker or not detected. Two HLA-A2-restricted hexon epitopes and one HLA-B-restricted epitope were identified, all of which are adjacent to or overlap an HLA-DP4-restricted epitope in the highly conserved C-terminus. Thus, hexon is the immunodominant T cell target among capsid proteins and contains multiple C-terminal epitopes conserved among serotypes. These data support evaluation of donor lymphocyte infusions for treatment of Ad disease post-transplant.
The immunogenicity of adenovirus (Ad) vectors is enhanced by virus-specific memory immune responses present in most individuals as a result of past exposure to these ubiquitous pathogens. We previously identified the first human T-cell epitope from the major capsid protein hexon, H910-924, and found that it is highly conserved among different Ad serotypes. Memory/effector T-cell responses to H910-924 were detected in 14 of 18 (78%) healthy adults by an interferon-g ELISPOT assay. Hexon peptide-specific CD4 Tcell lines were generated from three HLA-typed donors and analyzed using a panel of HLA homozygous B-cell lines and monoclonal antibodies to HLA class II loci. These studies reveal that the hexon epitope is restricted by HLA DP4, a class II allele present in 75% of the population. Analysis of overlapping peptides and peptides with single residue mutations identified a HLA DP4-binding motif. Additionally, antibodies to the hexon peptide were detected in all donor sera by dot blot assay and ELISA. Therefore, most individuals exhibit both memory B-and T-cell responses to this highly conserved epitope on hexon, an obligate component of all Ad vectors, including 'gutted' vectors. These data suggest that current strategies for the use of Ad gene therapy vectors will not evade memory immune responses to Ad.
Although nearly all adults are seropositive for adenoviruses, little is known about the cellular immune responses to these ubiquitous pathogens. We have previously identified adenovirus-specific proliferative T-cell responses in peripheral blood mononuclear cells (PBMC) from healthy adults. In this study, memory T-cell responses to adenovirus were further evaluated in healthy adult donors using a short term, quantitative enzyme-linked immunospot assay (ELISPOT) assay. Adenovirus antigen induced specific secretion of interferon-gamma (IFN-gamma) from PBMC within 12 hours of incubation. PBMC from 20 of 22 healthy donors (90.9%) expressed IFN-y in response to adenovirus. Responder cells were identified as CD4+ T cells by immunomagnetic depletion methods. Interleukin-4 (IL-4) secretion was not detected, consistent with a TH1 response. There was a 10-fold variation in the frequencies of adenovirus-specific CD4+ T cells between donors (range, 34 to 294; median, 122 per million PBMC). Adenovirus-specific T cell frequencies remained stable over periods up to 2 years among individual donors, but there was an inverse correlation between frequency and donor age. These quantitative data suggest that most adults retain adenovirus-specific cellular memory after childhood exposure. This assay may be useful for the evaluation of adenovirus-specific CD4+ T-cell responses in patients treated with adenovirus gene therapy vectors and the identification of major T-cell epitopes.
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