Clinical human vaccine studies have so far failed to demonstrate that induction of either antibody or T cells in isolation confer protection against HIV-1 infection or delay disease progression (Flynn et al., 2005
; Buchbinder et al., 2008
). A clinical trial recently completed in Thailand (RV-144) demonstrated partial efficacy (Rerks-Ngarm et al., 2009
). In this study, the use of an HIV-1 recombinant canarypox vector (ALVAC-HIV) boosted with a bivalent glycoprotein subunit vaccine (AIDSVAX B/E) was able to induce envelope-specific antibody and T cell responses. Because the correlates of protection using this latter vaccine regimen are not understood, it would be prudent to develop strategies to increase both the humoral and cell-mediated immune responses induced by HIV-1 vaccines.
Recent nonhuman primate studies suggest a correlation of viral load with the breadth and magnitude of vaccine-induced CD8 T cell responses (Liu et al., 2009
; Wilson et al., 2009
). Perhaps importantly, the breadth of the CD8 T cell responses induced by these SIV-based vaccines far exceeded those elicited by any HIV-1 vaccine tested to date (McElrath et al., 2008
). Foremost among these, the recent Merck trivalent vaccine (Step Trial) induced a median of only three CD8 T cell epitope (a median of one Gag, one Pol, and one Nef response) responses per vaccinee, and only 31% of vaccinees demonstrated both CD8 and CD4 T cell responses (McElrath et al., 2008
). These studies would suggest that to be effective, at minimum, vaccines will need to induce a considerably greater number of T cell responses.
The three letter codon alphabet recognized by unique tRNA during protein synthesis allows for three potential overlapping reading frames in each direction of DNA transcription. A reading frame that encodes a potential peptide sequence that is not part of a functional protein is called an alternative reading frame (ARF), and within HIV-1 there are many such ARFs in both the sense and the antisense directions of transcription (Fig. S1
). Although all functional HIV-1 proteins are thought to be transcribed from the positive sense DNA strand, several studies have shown that antisense RNA is transcribed during HIV-1 infection and that translation of several ARFs occurs, with the potential to generate immunogenic and antigenic peptides called cryptic epitopes (CEs), a potential source of HLA class I (HLA-I)–presented peptides (He et al., 2008
; Seila et al., 2008
). After transcription, there are multiple mechanisms through which the RNA strand can be manipulated or otherwise used to generate CEs, including alternative splicing patterns, ribosomal frameshifting (Weiss et al., 1987
), internal ribosomal entry sites (McBratney et al., 1993
), initiation codon scanthrough (Bullock et al., 1997
), doublet decoding (Bruce et al., 1986
), and initiation from non-AUG codons (Malarkannan et al., 1999
CD8 T cell responses targeting CEs derived from the positive-strand RNA have been described previously for both SIV and HIV-1 (Cardinaud et al., 2004
; Maness et al., 2007
); however, the frequency and biological significance of these responses are unknown. Furthermore, CD8 T cell targeting of CEs derived from antisense transcription has not been described despite evidence that HIV-1 proteins are produced from this process (Michael et al., 1994
; Ludwig et al., 2006
; Landry et al., 2007
). This latter production of peptides could be an important source of antigenic epitopes considering the relatively large amount of CEs that can be generated from antisense open reading frames (Fig. S1).
In this report, we predict frequent targeting of CEs derived from ARFs in chronic infection by identifying HIV-1 polymorphisms associated with specific HLA-I alleles. The majority of these CE responses are predicted to occur in the pol region and most CEs are derived from antisense transcription, possibly because of the large number of potential peptides synthesized from this region. Using PBMCs from early and chronically HIV-1–infected patients, we show that CD8 T cell targeting of CEs is a frequent occurrence. Finally, we show evidence of evolution at predicted CEs during the first year of HIV-1 infection. Immune targeting of protein products derived from antisense transcription is a previously unrecognized process that, in addition to providing insight into the natural immune response to HIV-1, could potentially be used to significantly increase the breadth of a vaccine response.