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Identification of CD8+ T cell antigens/epitopes expressed by human pathogens with large genomes is especially challenging, yet necessary for vaccine development. Immunity to tuberculosis, a leading cause of mortality worldwide, requires CD8+ T cell immunity, yet the repertoire of CD8 antigens/epitopes remains undefined. We used integrated computational and proteomic approaches to screen 10% of the Mycobacterium tuberculosis (Mtb) proteome for CD8 Mtb antigens. We designed a weighting schema based upon a Multiple Attribute Decision Making:framework to select 10% of the Mtb proteome with a high probability of containing CD8+ T cell epitopes. We created a synthetic peptide library consisting of 15-mers overlapping by 11 aa. Using the interferon-γ ELISPOT assay and Mtb-infected dendritic cells as antigen presenting cells, we screened Mtb-specific CD8+ T cell clones restricted by classical MHC class I molecules (MHC class Ia molecules), that were isolated from Mtb-infected humans, against this library. Three novel CD8 antigens were unambiguously identified: the EsxJ family (Rv1038c, Rv1197, Rv3620c, Rv2347c, Rv1792), PE9 (Rv1088), and PE_PGRS42 (Rv2487c). The epitopes are B5701-restricted EsxJ24–34, B3905-restricted PE953–67, and B3514-restricted PE_PGRS4248–56, respectively. The utility of peptide libraries in identifying unknown epitopes recognized by classically restricted CD8+ T cells was confirmed, which can be applied to other intracellular pathogens with large size genomes. In addition, we identified three novel Mtb epitopes/antigens that may be evaluated for inclusion in vaccines and/or diagnostics for tuberculosis.

Mycobacterium tuberculosis (Mtb) remains a major threat to human health worldwide. Although treatment of infection is an important part of tuberculosis control, an improved vaccine is essential for the elimination of this disease. Control of infection with Mtb is dependent on the cellular immune system, which in turn requires an understanding of those antigens that are capable of stimulating CD4+ and CD8+ T-cell responses. Peptide libraries provide a high-throughput system for identifying novel T-cell epitopes. They can also be used to assess the hierarchy of immunodominance of these novel antigens and epitopes that are associated with infection with Mtb. This T-cell-driven means of antigen discovery is well adapted to vaccine development as well as developing the tools necessary to understand the natural history of this important human pathogen.

The ability of mink cell focus-inducing (MCF) viruses to induce thymomas is determined, in part, by transcriptional enhancers in the U3 region of their long terminal repeats (LTRs). To elucidate sequence motifs important for enhancer function in vivo, we injected newborn mice with MCF 1dr (supF), a weakly pathogenic, molecularly tagged (supF) MCF virus containing only one copy of a sequence that is present as two copies (known as the directly repeated [DR] sequence) in the U3 region of MCF 247 and analyzed LTRs from supF-tagged proviruses in two resulting thymomas. Tagged proviruses integrated upstream and in the reverse transcriptional orientation relative to c-myc provided the focus of our studies. These proviruses are thought to contribute to thymoma induction by enhancer-mediated deregulation of c-myc expression. The U3 region in a tagged LTR in one thymoma was cloned and sequenced. Relative to MCF 1dr (supF), the cloned U3 region contained an insertion of 140 bp derived predominantly from the DR sequence of the injected virus. The inserted sequence contains predicted binding sites for transcription factors known to regulate the U3 regions of various murine leukemia viruses. Similar constellations of binding sites were duplicated in two proviral LTRs integrated upstream from c-myc in a second thymoma. We replaced the U3 sequences in an infectious molecular clone of MCF 247 with the cloned proviral U3 sequences from the first thymoma and generated an infectious chimeric virus, MCF ProEn. When injected into neonatal AKR mice, MCF ProEn was more pathogenic than the parental virus, MCF 1dr (supF), as evidenced by the more rapid onset and higher incidence of thymomas. Molecular analyses of the resultant thymomas indicated that the U3 region of MCF ProEn was genetically stable. These data suggest that the arrangement and/or redundancy of transcription factor binding sites generated by specific U3 sequence duplications are important to the biological events mediated by MCF proviruses integrated near c-myc that contribute to transformation. AKR mice succumb to spontaneous thymomas between 6 and 12 months of age. The virological events leading to leukemia include the inheritance and replication of Akv, a nonleukemogenic, ecotropic murine leukemia virus (MuLV), and the sequential recombination between Akv and two other endogenous retrovirus sequences to produce leukemogenic mink cell focus-inducing (MCF) viruses (6,11,18,26). The first recombination event replaces the long terminal repeat (LTR) sequences in the ecotropic virus with those from the inducible xenotropic virus, Bxv-1, while the second recombination event replaces a portion of the ecotropic envelope (env) gene with polytropic env sequences (6,11,18,26). During the in vivo spread and replication of the recombinant virus containing both the LTR and env substitutions, sequences in the U3 region of the LTR are duplicated, creating the tandemly duplicated sequences commonly referred to as the directly repeated (DR) sequences (18,26). Both recombination events a...

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