Comparison of overlapping peptide sets for detection of antiviral CD8 and CD4 T cell responses
Introduction
In recent years, methods for detecting both CD4 and CD8 T cell responses have been improved using interferon γ based Elispot assays and intracellular cytokine staining (ICS) assays Czerkinsky et al., 1983, Schmittel et al., 1997, Goulder et al., 2000, Maecker et al., 2001. This has led to more sensitive detection and quantitation of cellular immune responses to pools of peptides (Betts et al., 2001), defined optimal epitopes (Dalod et al., 1999) and, more recently, comprehensive mapping of responses to all expressed viral proteins (Addo, M.M., et al., in press. Comprehensive epitope analysis of HIV-1-specific T cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J. Virol.).
Despite greater ease and rapidity of assessment of immune responses, these approaches have left a number of issues unresolved. Important among these are the extent to which the sequence of the reference strain as well as the length and degree of peptide overlap influence the detection of T cell responses. These are important issues not only for understanding viral immunopathogenesis but also because such comprehensive screening approaches will be important in evaluating immunotherapy and vaccine trials in both the cancer and virology fields. The length and degree of overlap of peptides also has a major impact on cost and labor intensiveness of such studies, and therefore identifying optimal techniques is a great priority.
In the present study, we compared the breadth and magnitude of CD8 and CD4 T cell responses by assaying induction of interferon γ production in ICS and Elispot assays using six sets of overlapping peptides differing in length, overlap and reference sequence. We selected HIV-1 Nef for analysis because of its demonstrated immunogenicity for cellular immune responses Betts et al., 2001, Couillin et al., 2001 and because it is reported to have among the highest density of epitopes per peptide length. Although the protein is only 206 amino acids (aa) in length, a total of 27 optimally defined CTL epitopes within Nef are listed in the Los Alamos Database. Our results using Elispot and intracellular cytokine staining for interferon γ indicate that peptides ranging in length from 15 to 20 amino acids and in overlap from 10 to 11 amino acids have similar ability to detect CD8 T cell responses. The shorter peptides showed a trend to detect more CD8 T cell responses but the longer peptides are slightly better for detection of CD4 T cell responses. Overall no peptide set was able to detect all responses, indicating that present approaches are likely to underestimate the magnitude and breadth of responses to reference strains of virus.
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Subjects
Twenty chronically HIV-1-infected individuals were studied at the Massachusetts General Hospital (MGH) and the Lemuel Shattuck Hospital, Boston, USA. Eighteen were untreated; two were treated with highly active antiretroviral therapy (HAART). Viral loads were determined by Roche Amplicor Assay Version 1.0 and ranged from <50 to 750,000 cp/ml. HLA class I typing was performed at the MGH Tissue Typing Laboratory using sequence-specific primer-PCR. The HLA class I genotypes of the 20 randomly
Immunogenicity of Nef peptides for CD8 and CD4 T cell responses by intracellular cytokine staining
The HIV-1 Nef protein has been shown to be a dominant target for CD8 T cell responses Evans et al., 2000, Couillin et al., 2001 (Addo, M.M., et al., in press. Comprehensive epitope analysis of HIV-1-specific T cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J. Virol.). However, the optimal peptide length for detection of such responses has not been determined, nor has the impact of use of different
Discussion
In this study, we compared six sets of overlapping peptides varying in length, overlap and sequence for their ability to detect CD8 and CD4 T cell responses to HIV-1 Nef. We demonstrate that none of these sets can be considered the clear winner in terms of sensitivity for magnitude of CD4 or CD8 responses by ICS or for detecting magnitude and breadth of CD8 responses by Elispot. Adapting the C terminal position of the set of 18 mers resulted in the detection of more responses compared to the
Acknowledgements
We thank all study participants, the dedicated clinical research staff at the collaborating sites and Margaret E. Feeney and Hang Lee for statistical advice.
This project was supported by the Deutsche Forschungsgemeinschaft, Germany (DFG, DR 424/1-1), the National Institute of Allergy and Infectious Disease, National Institute of Health, US, under Contract No. N01-A1-15442, and the EU Program EVA/MRC Centralized Facility for AIDS Reagents, NIBSC, UK (Grant No. QLK2-CT-1999-00609 and GP828102).
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