R. Laird

High-avidity, high-IFNγ-producing CD8 T-cell responses following immune selection during HIV-1 infection

HIV‐1 mutations, which reduce or abolish CTL responses against virus‐infected cells, are frequently selected in acute and chronic HIV infection. Among population HIV‐1 sequences, immune selection is evident as human leukocyte antigen (HLA) allele‐associated substitutions of amino acids within or near CD8 T‐cell epitopes. In these cases, the non‐adapted epitope is susceptible to immune recognition until an escape mutation renders the epitope less immunogenic. However, several population‐based studies have independently identified HLA‐associated viral changes, which lead to the formation of a new T‐cell epitope, suggesting that the immune responses that these variants or ‘neo‐epitopes’ elicit provide an evolutionary advantage to the virus rather than the host. Here, we examined the functional characteristics of eight CD8 T‐cell responses that result from viral adaptation in 125 HLA‐genotyped individuals with chronic HIV‐1 infection. Neo‐epitopes included well‐characterized immunodominant epitopes restricted by common HLA alleles, and in most cases the T‐cell responses against the neo‐epitope showed significantly greater functional avidity and higher IFNγ production than T cells for non‐adapted epitopes, but were not more cytotoxic. Neo‐epitope formation and emergence of cognate T‐cell response coincident with a rise in viral load was then observed in vivo in an acutely infected individual. These findings show that HIV‐1 adaptation not only abrogates the immune recognition of early targeted epitopes, but may also increase immune recognition to other epitopes, which elicit immunodominant but non‐protective T‐cell responses. These data have implications for immunodominance associated with polyvalent vaccines based on the diversity of chronic HIV‐1 sequences.

Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, p<0.05). The precision results from the automated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.