Publications

BACKGROUND

Antigen-specific CD8 T cells following infection or immunization are typically assessed by measuring interferon-gamma production after stimulation with overlapping peptides spanning the region of interest. The effect of epitope location within such peptides is not known but may influence recognition.

OBJECTIVE

To examine if peptides containing the appropriate C-terminal anchor amino acid residue would provide more sensitive detection of T cell responses. The impact was examined of epitope location within overlapping peptides on recognition of epitope-specific CD8 T cell responses.

METHODS

C-terminal amino acid residues were analyzed in well-defined optimal epitopes for HIV, Epstein-Barr virus, cytomegalovirus and influenza and in peptide-binding motifs. Recognition of known epitopes within longer synthesized peptides by peripheral blood mononuclear cells or CD8 T cell lines was tested using interferon-gamma Elispot at various peptide concentrations.

RESULTS

Only 9 of 20 amino acids served as the C-terminal anchor position in 96% of described optimal epitopes and in 95% of peptide-binding motifs. A CD8 T cell response to an epitope within a longer peptide is best detected when the epitope is situated at the C-terminal end of the longer peptide, both when using peptides designed to include the optimal epitope at every possible position and when comparing responses towards optimal epitopes and corresponding overlapping peptides in a larger group of subjects.

CONCLUSION

When using overlapping peptides to screen for CD8 T cell responses, more sensitive detection will be achieved using known C-terminal anchor amino acid residues at the C-terminus.

Mutations within cytotoxic T lymphocyte (CTL) epitopes impair T cell recognition, but escape mutations arising in flanking regions that alter antigen processing have not been defined in natural human infections. In human histocompatibility leukocyte antigen (HLA)-B57+ HIV-infected persons, immune selection pressure leads to a mutation from alanine to proline at Gag residue 146 immediately preceding the NH2 terminus of a dominant HLA-B57-restricted epitope, ISPRTLNAW. Although N-extended wild-type or mutant peptides remained well-recognized, mutant virus-infected CD4 T cells failed to be recognized by the same CTL clones. The A146P mutation prevented NH2-terminal trimming of the optimal epitope by the endoplasmic reticulum aminopeptidase I. These results demonstrate that allele-associated sequence variation within the flanking region of CTL epitopes can alter antigen processing. Identifying such mutations is of major relevance in the construction of vaccine sequences.

Antiretroviral (ARV)-treated patients often maintain low to moderate levels of viremia, despite the emergence of drug-resistant human immunodeficiency virus (HIV). We studied host and viral factors that may contribute to the control of viral replication in a cohort of 189 adults. Among ARV-treated patients with detectable viremia, there was a bell-shaped relationship between Gag-specific CD4+ T cell responses and viremia, with the highest cellular immune responses observed in patients with plasma HIV RNA levels of 1000-10,000 copies/mL. In contrast, there was a negative association between Gag-specific CD4+ T cell responses and viremia among ARV-untreated individuals with wild-type HIV. Strong cellular immune responses among individuals with drug-resistant HIV predicted subsequent lack of virological progression. Finally, there was a positive correlation between replicative capacity and viremia. Collectively, these data suggest that the selection of drug-resistance mutations may reduce the pathogenic potential of HIV, which leads to a balanced state of enhanced cellular immunity and low-level viremia.

Gag-specific CD4 proliferative responses correlate inversely with HIV-1 RNA levels in infected adults, and robust responses are characteristic of long-term nonprogressive infection. However, strong responses are seldom detected in adult subjects with progressive infection and are not generally reconstituted on highly active antiretroviral therapy (HAART). To date, the role of HIV-1-specific Th responses in children has not been thoroughly examined. We characterized Gag-specific CD4 responses among 35 perinatally infected subjects, including 2 children who spontaneously control viremia without antiretroviral therapy, 21 children with viral loads (VL) of <400 on HAART, and 12 viremic children. Gag-specific Th activity was assessed by lymphoproliferative assay, and responses were mapped using overlapping Gag peptides in an IFN-gamma ELISPOT. Robust proliferative responses were detected in the children exhibiting spontaneous control of viremia, and mapping of targeted Gag regions in one such subject identified multiple epitopes. Among children >or=5 years old, 14 of 17 subjects with VL of <400 on HAART demonstrated a significant p24 proliferative response (median p24 stimulation index, 20), in contrast with only 1 of 9 viremic children (median p24 stimulation index, 2.0; p = 0.0008). However, no subject younger than 5 years of age possessed a significant response, even when viremia was fully suppressed. When compared with adults with VL of <400 on HAART, Th responses among children with VL of <400 were both more frequent (p = 0.009) and of greater magnitude (p = 0.002). These data suggest that children may have a greater intrinsic capacity to reconstitute HIV-1-specific immunity than adults, and may be excellent candidates for immune-based therapies.