Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I–associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity.

It is widely assumed that CD4+ T cells recognize antigenic peptides (epitopes) derived solely from incoming, exogenous, viral particles or proteins. However, alternative sources of MHC class II (MHC-II)–restricted Ags have been described, in particular epitopes derived from newly synthesized proteins (so-called endogenous). In this study, we show that HIV-infected dendritic cells (DC) present MHC-II–restricted endogenous viral Ags to HIV-specific (HS) CD4+ T cells. This endogenous pathway functions independently of the exogenous route for HIV Ag presentation and offers a distinct possibility for the immune system to activate HS CD4+ T cells. We examined the implication of autophagy, which plays a crucial role in endogenous viral Ag presentation and thymic selection of CD4+ T cells, in HIV endogenous presentation. We show that infected DC do not use autophagy to process MHC-II–restricted HIV Ags. This is unlikely to correspond to a viral escape from autophagic degradation, as infecting DC with Nef- or Env-deficient HIV strains did not impact HS T cell activation. However, we demonstrate that, in DC, specific targeting of HIV Ags to autophagosomes using a microtubule-associated protein L chain 3 (LC3) fusion protein effectively enhances and broadens HS CD4+ T cell responses, thus favoring an endogenous MHC-II–restricted presentation. In summary, in DC, multiple endogenous presentation pathways lead to the activation of HS CD4+ T cell responses. These findings will help in designing novel strategies to activate HS CD4+ T cells that are required for CTL activation/maintenance and B cell maturation.