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68 Matrix-Based ELISPOT Assay for Identification of Cross-Clade HIV-1-gag T Lymphocyte Reactivity
S. M. Keating*, J. B. Jackson, R. C. Bollinger, and L. M. Carruth
Johns Hopkins Univ. Sch. of Med., Baltimore, MD, USA
Background: The success of global vaccine strategies may be dependent upon a better understanding of the cross-clade T-lymphocyte immune response to HIV-1. Identifying regions of HIV-1 that are recognized by CD8+ T lymphocytes of infected individuals may be important for the design and evaluation of candidate HIV vaccines, particularly for developing countries. The goal of this study was to evaluate an efficient ELISPOT assay algorithm to identify peptide targets of T-cell responsiveness in the gag region of HIV-1 subtypes A, B, and C.
Methods: Cryopreserved peripheral blood mononuclear cells (PBMC) from 22 chronically HIV-1-infected US volunteers currently off of anti-retroviral therapy were screened for IFN-gamma production in an ELISPOT assay using a matrix format of overlapping HIV-1 subtype A, B, and C gag peptide pools.
Results: ELISPOT responses to HIV-1 subtype B gag were detected in 13 of 22 (59%) of seropositive individuals and in none of 4 HIV-1 seronegative controls. Seven of the 13 responders recognized multiple gag peptides. The regions of T-cell reactivity corresponded with previously identified immunologic “hot spots” in the p17 and p24 regions of the HIV-1 subtype B gag protein. Nine of 9 subtype B responders screened thus far were also found to recognize the corresponding subtype C gag peptide pools. Responses to A clade peptides were present, but less frequent.
Conclusions: A matrix-based IFN-gamma ELISPOT assay can be utilized to identify T-cell cross-clade reactivity to 3 different strains of HIV-1. This screening algorithm may be an efficient way of characterizing CD8+ T-cell responses in either HIV-1-infected individuals or HIV-1 vaccine recipients. However, clade/sequence of antigen used may impact the sensitivity of the assay.
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