Abstract
Background: Translational errors can result in bypassing of the main viral protein reading frames and the production of alternate reading frame (ARF) or cryptic peptides. Within HIV, there are many such ARFs in both sense and the antisense directions of transcription. These ARFs have the potential to generate immunogenic peptides called cryptic epitopes (CE). Both antiretroviral drug therapy and the immune system exert a mutational pressure on HIV-1.

Immune pressure exerted by ARF CD8+ T cells on the virus has already been observed in vitro. HAART has also been described to select HIV-1 variants for drug escape mutations. Since the mutational pressure exerted on one location of the HIV-1 genome can potentially affect the 3 reading frames, we hypothesized that ARF responses would be affected by this drug pressure in vivo.

Methodology/Principal findings: In this study we identified new ARFs derived from sense and antisense transcription of HIV-1. Many of these ARFs are detectable in circulating viral proteins. They are predominantly found in the HIV-1 env nucleotide region. We measured T cell responses to 199 HIV-1 CE encoded within 13 sense and 34 antisense HIV-1 ARFs. We were able to observe that these ARF responses are more frequent and of greater magnitude in chronically infected individuals compared to acutely infected patients, and in patients on HAART, the breadth of ARF responses increased.

Conclusions/Significance: These results have implications for vaccine design and unveil the existence of potential new epitopes that could be included as vaccine targets.

Introduction: CD8+ T cell responses are a major component in the immune control of HIV-1 replication [1], [2], [3], [4]. Primate studies suggest that the breadth and magnitude of the vaccine-induced CD8+ T cell response correlates with viral load [5]. In the Step Trial that failed to demonstrate efficacy, the Merck trivalent vaccine induced a low number of CD8+ T cell epitope responses per vaccinee (with a median of three epitopes), suggesting that to be effective, vaccines should induce a greater number of T cell responses [6].

HIV-1 infected cells are recognized by the CD8+ T cell receptor (TCR) through viral peptides presented on MHC-I molecules. Most of the HIV-1 epitopes described have been identified in HIV-1 proteins encoded by primary open reading frames (ORFs) of the viral genome [7], [8], [9], [10], [11], [12]. Aside from these “traditional epitopes”, a new type of epitopes derived from frame-shifted proteins, called alternative reading frame (ARF) epitopes or cryptic epitopes (CE), have been reported for influenza virus [13], [14], malignancies [15], [16], [17], [18], and an autoimmune disease [19]. ARF expression may be explained by several transcriptional and translational mechanisms [20]: ribosomal frame-shifting [21], cryptic promoter activation, internal ribosomal entry sites [22], initiation codon scan-through [23], doublet decoding alternative splicing patterns [24], and initiation from non-AUG codons [25].

These nontraditional Cytotoxic T Lymphocyte (CTL) epitopes are generated during HIV-1 and/or SIV infections [26], [27], [28], [29], [30], [31], [32]. However, another source of cryptic epitopes originates from antisense (3′ to 5′) RNA transcription - such antisense transcripts have been identified in HIV-1 infections [33], [34], [35], and CEs derived from these antisense RNA have been previously reported in SIV and HIV-1 infections [26], [28], [30]. CD8+ specific T cells against HIV-1 CE could potentially contribute to viral control in vivo [36]. In vitro studies show that SIV ARF epitope–specific CTLs are able to select for viral escape variants [30]. These ARF-derived peptides could therefore be an important source of epitopes and could be used in an HIV-1 vaccine to broaden the spectrum of the CD8+ T cell responses.

To date, many studies have shown that immunological pressure exerted by CTL shapes HIV-1 sequences by selecting for CTL escape mutations [32], [37], [38], [39]. HAART has also been described to select HIV-1 variants for drug escape mutations [40], [41], [42], [43], [44], [45], [46], [47], [48]. More recently, there has been some evidence that emergence of drug resistance mutations can potentially abolish HIV-1 CTL responses [49].

In this regard, we anticipated that ARFs would be affected by immunological and drug pressure in vivo. Immune pressure exerted by the ARF CD8+ T cells on the virus has already been reported in vitro [30]. Since the mutational pressure exerted on one location of HIV-1’s genome can potentially affect each of the 3 reading frames, ARF expression could be affected by HAART. HAART mutational pressure on the virus could therefore be responsible for the mutation or conservation of the genome of certain traditional proteins or could disturb the expression of certain locations on HIV-1’s genome, influencing ARF expression and escape from ARF CTL responses.

In this study, we evaluated the influence of HAART on HIV-1 ARF T cell responses over the course of HIV-1 infection. We identified new cryptic epitopes derived from sense and antisense transcription of HIV-1. To evaluate the effect of HAART on the ARF T cell responses, we tested PBMCs from HIV-1 acutely infected patients enrolled in a HAART interruption program and HIV-1 chronically infected patients before and after HAART introduction. We report that ARF responses were more frequent and stronger in magnitude in chronically infected individuals compared to acutely infected patients, and importantly that HAART increased the breadth of the ARF responses. Our results indicate that CE could potentially be used to increase the breadth of an HIV-1 vaccine response.