Characterizing the MHC-II immunopeptidome of HIV using a cell-free antigen processing system and peptide:MHC-specific antibodies

Abstract

Abstract The breadth and specificity of CD4+T cell responses in HIV-1 infection are critical determinants of viral load and an individual’s ability to control infection. Most studies of CD4+T cell responses against HIV have relied on indirect evidence from peptide-pulsing analyses. Direct studies of antigen presentation have been hampered by the technical difficulties inherent in isolating peptide:MHC (p:MHC) complexes from HIV-infected cells. This has made it challenging to understand the HIV immunopeptidome, particularly for MHC-II epitopes. Using a cell-free antigen processing system, we have generated a near complete map of potential DR1-restricted HIV-1 epitopes. To confirm that epitopes identified using this system are actually presented by HIV-infected cells, we generated novel reagents termed single-chain diabodies (scDbs), or bispecific antibodies, which contain one Fab fragment against a p:MHC and another against CD3. These antibodies enable the use of CD8+ or CD4+T cells as readouts for antigen presentation. In proof of concept experiments, scDbs directed against immunodominant HIV MHC-I epitopes enhance cytokine production by CD8+ T cells, confirming presentation of these peptides. Preliminary experiments showed similar results with scDbs targeting MHC-II Gag epitopes on dendritic cells overexpressing the relevant peptides. We are now profiling the use of scDbs to detect antigen presentation in HIV-infected macrophages and CD4+T cells, the two target cells of infection. By establishing an immunopeptide map and tools to detect HIV peptide presentation, we can begin to address the kinetics, pathways, and cell types contributing to HIV antigen presentation on MHC-II, which will inform improved vaccine therapies.