Background:
Cytolytic T lymphocytes (CTLs) help control viremia in people with HIV. HIV evades this response through escape mutations in CTL epitopes and downregulation of HLA molecules. The dimorphic non-canonical HLA-E molecules, however, are upregulated during infection, making them promising targets for CTL-engaging reagents such as single-chain diabodies (scDbs) provided that they present HIV epitopes. Here, we show that HIV-infected cells can be targeted for CTL-mediated lysis using novel scDbs specific for HLA-E-bound epitopes.
Methods:
To validate HLA-E as a suitable scDb target, we used the previously characterized [i]M. tuberculosis[/i]-derived peptide Mtb44 that binds with high affinity to HLA-E. Through phage biopanning, we isolated a novel scDb (RLP13) that recognizes this epitope bound to HLA-E. This scDB also recognizes CD3 and is able to induce CTL activation. The specificity of RLP13 was tested through killing assays with CD8+ effector T cells and target cells presenting cognate Mtb44 peptide or the irrelevant canonical HLA-E epitope VL9. To test whether HIV-encoded peptides can be targeted, an Mtb44-encoding DNA segment was inserted into an HIV reporter virus (NL4.3 ΔEnv-GFP). Target cells were infected with this virus and co-cultured with RLP13 scDb and autologous CTLs to assess killing of infected cells.
Results:
RLP13 scDb induced antigen-specific lysis of Mtb44-presenting target cells, regardless of the donor HLA type, in an scDb- and antigen-dose-dependent manner. Furthermore, addition of the RLP13 scDb to co-cultures of primary CD8+ T cells and autologous CD4+ T cells infected with viral reporter constructs carrying the Mtb44 epitope led to a 50% reduction of the HIV+ population, indicating that targeting of HIV-1 peptides presented on HLA-E can potentially lead to the elimination of infected cells.
Conclusions:
Our findings with scDb RLP13 demonstrate that virally encoded peptides presented in the context of HLA-E can be targeted by scDbs to induce CTL-mediated lysis of infected cells, regardless of HLA-genotype. The development of novel scDbs recognizing HIV-derived epitopes that bind HLA-E would provide a universal reagent that could be used to promote the killing of HIV-1 infected cells in shock and kill strategies for HIV cure.