Innovative tools and approaches to end the transmission of Mycobacterium leprae.

Leprosy control has seen little innovation and only limited progress in the past decade. However, research on the disease has increased and important innovations are underway. Here, we comment on efforts to develop tools and approaches to detect leprosy and to stop the transmission of Mycobacterium leprae, the causative bacillus of the disease. The tracing and screening of contacts of known patients with leprosy promises to strengthen early diagnosis, while preventive chemotherapy will reduce the risk of contacts developing the disease by 50-60% within 2 years of administration. Until now, diagnosis has been mainly based on the presence of signs and symptoms, but efforts are underway to develop inexpensive, reliable, point-of-care tests to diagnose infection. Development of a leprosy-specific vaccine that boosts long-lasting T-cell responses is also a research objective. As for launching a programme to interrupt transmission, two interlinked tools-epidemiological modelling and the concept of an investment case-are being developed to explore the feasibility and costs of such a programme and its overall effect on individuals and society. We believe that sustained innovation is needed and that only a combination of tools and approaches holds promise to end M leprae transmission.

Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region.

CD8(+) cytotoxic T lymphocytes (CTL) are key determinants of immunity to intracellular pathogens and neoplastic cells. Recognition of specific antigens in the form of peptide-MHC class I complexes (pMHCI) presented on the target cell surface is mediated by T cell receptor (TCR) engagement. The CD8 coreceptor binds to invariant domains of pMHCI and facilitates antigen recognition. Here, we investigate the biological effects of a Q115E substitution in the alpha2 domain of human leukocyte antigen (HLA)-A*0201 that enhances CD8 binding by approximately 50% without altering TCR/pMHCI interactions. Soluble and cell surface-expressed forms of Q115E HLA-A*0201 exhibit enhanced recognition by CTL without loss of specificity. These CD8-enhanced antigens induce greater CD3 zeta chain phosphorylation in cognate CTL leading to substantial increases in cytokine production, proliferation and priming of naive T cells. This effect provides a fundamental new mechanism with which to enhance cellular immunity to specific T cell antigens.

Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara.

Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26-35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1(157-165) failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.

Recombinant modified vaccinia Ankara primes functionally activated CTL specific for a melanoma tumor antigen epitope in melanoma patients with a high risk of disease recurrence.

Recombinant plasmid DNA and attenuated poxviruses are under development as cancer and infectious disease vaccines. We present the results of a phase I clinical trial of recombinant plasmid DNA and modified vaccinia Ankara (MVA), both encoding 7 melanoma tumor antigen cytotoxic T lymphocyte (CTL) epitopes. HLA-A*0201-positive patients with surgically treated melanoma received either a "prime-boost" DNA/MVA or a homologous MVA-only regimen. Ex vivo tetramer analysis, performed at multiple time points, provided detailed kinetics of vaccine-driven CTL responses specific for the high-affinity melan-A(26-35) analogue epitope. Melan-A26-35-specific CTL were generated in 2/6 patients who received DNA/MVA (detectable only after the first MVA injection) and 4/7 patients who received MVA only. Ex vivo ELISPOT analysis and in vitro proliferation assays confirmed the effector function of these CTL. Responses were seen in smallpox-vaccinated as well as vaccinia-naive patients, as defined by anti-vaccinia antibody responses demonstrated by ELISA assay. The observations that 1) CTL responses were generated to only 1 of the recombinant epitopes and 2) that the magnitude of these responses (0.029-0.19% CD8(+) T cells) was below the levels usually seen in acute viral infections suggest that to ensure high numbers of CTL specific for multiple recombinant epitopes, a deeper understanding of the interplay between CTL responses specific for the viral vector and recombinant epitopes is required.

Anti-CD8 antibodies can inhibit or enhance peptide-MHC class I (pMHCI) multimer binding: this is paralleled by their effects on CTL activation and occurs in the absence of an interaction between pMHCI and CD8 on the cell surface.

Cytotoxic T lymphocytes recognize short peptides presented in association with MHC class I (MHCI) molecules on the surface of target cells. The Ag specificity of T lymphocytes is conferred by the TCR, but invariable regions of the peptide-MHCI (pMHCI) molecule also interact with the cell surface glycoprotein CD8. The distinct binding sites for CD8 and the TCR allow pMHCI to be bound simultaneously by both molecules. Even before it was established that the TCR recognized pMHCI, it was shown that CTL exhibit clonal heterogeneity in their ability to activate in the presence of anti-CD8 Abs. These Ab-based studies have since been interpreted in the context of the interaction between pMHCI and CD8 and have recently been extended to show that anti-CD8 Ab can affect the cell surface binding of multimerized pMHCI Ags. In this study, we examine the role of CD8 further using point-mutated pMHCI Ag and show that anti-CD8 Abs can either enhance or inhibit the activation of CTL and the stable cell surface binding of multimerized pMHCI, regardless of whether there is a pMHCI/CD8 interaction. We further demonstrate that multimerized pMHCI Ag can recruit CD8 in the absence of a pMHCI/CD8 interaction and that anti-CD8 Abs can generate an intracellular activation signal resulting in CTL effector function. These results question many previous assumptions as to how anti-CD8 Abs must function and indicate that CD8 has multiple roles in CTL activation that are not necessarily dependent on an interaction with pMHCI.

High avidity antigen-specific CTL identified by CD8-independent tetramer staining.

Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.