The Adenovirus Capsid Protein Hexon Contains a Highly Conserved Human CD4⁺T-Cell Epitope

Abstract

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.

Adenovirus infection in children after allogeneic stem cell transplantation: diagnosis, treatment and immunity

“…In contrast to EBV and CMV, HAdV-derived epitopes recognized by T cells are only recently coming to be identified. 16,17 Furthermore, HadVinfected cells may escape from recognition by the immune system through several mechanisms. 18 Evidence has been obtained that recovery of immunity after SCT is essential for the elimination of HAdV infection.…”

Adenovirus hexon T-cell epitope is recognized by most adults and is restricted by HLA DP4, the most common class II allele

“…These peptides, which contain HLA A2-binding motifs, were previously used to screen PBMC for virus-specific T-cell responses in the IFN-g ELISPOT assay. 8 First, sera were screened for responses to Ad5 virion and Ad peptides in a dot blot assay. Antibodies to both H910-924 and H913-921 were detected from all 12 donors.…”

“…8 For blocking experiments, targets were preincubated with 40 mg/ml class II locus-specific mAbs against monomorphic determinants on HLA-DR (clone L243), HLA-DP (clone B7/21), and HLA-DQ (clone Ia3) (Leinco Technologies, St Louis, MO, USA) for 30 0 at RT and then added to an equal volume of effectors.…”

“…8 All mAbs were purchased as fluorescein or phycoerythrin conjugates (Becton Dickinson, San Jose, CA, USA). MAb cocktails used were CD45/CD14, CD3/CD8, and CD3/ CD4.…”

“…8 Responses to the hexon peptide were detected in PBMC from nine of 12 healthy adults by an interferon-g (IFN-g) ELISPOT assay and determined to be mediated by CD4 T cells. Moreover, this epitope was found to be highly conserved among different human Ad serotypes, and analysis of the X-ray crystallography structure revealed that it is located in a crucial b-pleated sheet structure that helps form the base of the hexon molecule.…”