Identification and characterization of L. major T cell epitopes based on quantitative proteomics
Final Report Abstract
Leishmaniasis is a protozoan infectious disease transmitted by the bite of a sand fly. It is endemic in 88 countries, and 12 million people are currently affected worldwide. 350 Mio. people live at daily risk of infection. Infections with Leishmania spp. represent a serious health problem. Overall, despite of a strong need for a suitable vaccination against leishmaniasis, the optimal antigens for vaccine development have not yet been identified. The project initially aimed to identify and characterize of stage-specific CD4+ and CD8+ T cell epitopes from L. major, based on the hypothesis that L. major-specific T cells are primed by antigens derived from amastigote-specific proteins or proteins expressed in both life forms as DC preferentially internalize amastigotes. The project was planned to address the following specific aims: a) Identification of stage-specific Leishmania CD8+ T cell epitopes by a combination of quantitative proteome analysis and in silico epitope prediction b) Identification of CD4+ T cell epitopes from soluble Leishmania lysate (SLA) by proteome subfractionation and peptide library screening c) Verification of natural processing of identified epitopes my mass spectrometric analysis of MHC ligands from L. major-infected DC by nanoUPLC-ESI-TOF mass spectrometry d) Validation and characterization of identified epitopes: analysis of relevant mechanisms for immunodominance, antigen processing and presentation of Leishmania antigens. In Aim a) we successfully established a high-coverage proteomic workflow and successfully applied the methodology towards the characterization of the L. major promastigote and amastigote life stages. The Top300 most abundant proteins were selected for epitope predictions and 300 H2-Db and H2-Kb peptides derived from both Leishmania life-forms were selected for further analysis. In cooperation with Soren Buus (Kopenhagen, Denmark) all selected peptides were analysed for their binding stability towards MHC class I molecules. Surprisingly, the correlation between binding affinity, SYFPEITHI predictions, ex vivo screening and vaccination results were not as strong as expected. Nevertheless, we successfully identified a MHC class I restricted epitope, peptide p54, in the framework of this proposal. p54 protected mice against challenge compared to control mice, as shown by decreased lesion volumes, reduced numbers of local and systemic parasites as well as reduced levels of secreted Th2-related cytokines, whereas all other tested peptides failed to do so. In Aim b), we developed and applied proteomic subfractionation strategies for soluble Leishmania lysate (SLA). Based on quantitative proteomic analyses of biologically active subfractions, we successfully identified several novel CD4 antigens, which provided protective immunity in a prime-boost-boost setting. In line, we have shown that these do not only hold potential as vaccination tools in C57BL/6 mice, but also in BALB/c. Finally, human reactivity as observed in patients with prior L. major, but also with L. infantum infection suggested a strong cross-reactivity among species highlighting the potential of the identified proteins as future vaccine targets. The identity and potential application as immunoprotective vaccines has been filed as an international patent. In addition, the application of the proteins in the context of therapeutic vaccines (starting at a time when lesion development has occurred mimicking the clinical situation with patients) have been set up and will be followed upon in the context of a follow-up proposal. Other adjuvants may also need to be tested. Finally, using overlapping peptide libraries, relevant protein-specific epitopes are currently screened. Further characterization and commercial exploitation of theses vaccine candidates will be addressed in follow-up projects. Most likely, using a similar approach, more antigens can be identified, also those that are preferentially recognized by other Th subsets, such as Th2, Th17, or Treg cells. Within Aim c) we successfully established a mass spectrometric workflow for the characterization of MHC presented ligandomes. However, despite successful identification of >3000 epitopes from human cancer cell lines, we were not successful in identifying L. major derived MHC presented epitopes from infected dendritic cells, likely due to insufficient sensitivity of the mass spectrometric instrumentation using currently available protocols for ex vivo DC isolation and/or generation from bone marrow. To address this problem, we have approached cooperation partners and have recently obtained SP37A3 cells (among others) that represent a DC cell line derived from spleens of C57BL/6 mice. Similar cell lines derived from BALB/c and one additional from C57BL/6 bone marrow were also obtained and are available for experiments aiming towards the validation and characterization of identified epitopes as well as for the analysis of relevant mechanisms for immunodominance, antigen processing and presentation of Leishmania antigens in the framework of a follow-up proposal.
Publications
- Dendritic cells in Leishmania major infections: mechanisms of parasite uptake, cell activation and evidence for physiological relevance. Med Microbiol Immunol. 2012; 201(4): 581-92
Kautz-Neu K, Schwonberg K, Fischer MR, Schermann AI, von Stebut E
(See online at https://doi.org/10.1007/s00430-012-0261-2) - Isolation of T cells from the skin. Methods Mol Biol. 2014; 1193: 3-13
Lorenz B, von Stebut E
(See online at https://doi.org/10.1007/978-1-4939-1212-4_1) - IL-10 signaling in dendritic cells attenuates anti-Leishmania major immunity without affecting protective memory responses. J Invest Dermatol. 2015; 135(11): 2890-2894
Girard-Madoux MJH, Kautz-Neu K, Lorenz B, Ober-Blöbaum JL, von Stebut E, Clausen BE
(See online at https://doi.org/10.1038/jid.2015.236) - A multicenter study benchmarks software tools for label-free proteome quantification. Nat Biotechnol. 2016; 34(11): 1130-1136
Navarro P, Kuharev J, Gillet LC, Bernhardt OM, MacLean B, Röst HL, Tate SA, Tsou CC, Reiter L, Distler U, Rosenberger G, Perez-Riverol Y, Nesvizhskii AI, Aebersold R, Tenzer S
(See online at https://doi.org/10.1038/nbt.3685) - Label-free quantification in ion mobility-enhanced dataindependent acquisition proteomics. Nat Protoc. 2016; 11(4): 795-812
Distler U, Kuharev J, Navarro P, Tenzer S
(See online at https://doi.org/10.1038/nprot.2016.042) - Myeloid cells do not contribute to gender-dependent differences in disease outcome in murine cutaneous leishmaniasis. Cell Immunol. 2016; 308: 13-18
Fischer MR, Kunz C, Dietze-Schwonberg K, Lorenz B, von Stebut E
(See online at https://doi.org/10.1016/j.cellimm.2016.07.009) - NFATc1 supports imiquimod-induced skin inflammation by suppressing IL-10 synthesis in B cells. Nat Commun. 2016; 7: 11724
Alrefai H, Muhammad K, Rudolf R, Pham DA, Klein-Hessling S, Patra AK, Avots A, Bukur V, Sahin U, Tenzer S, Goebeler M, Kerstan A, Serfling E
(See online at https://doi.org/10.1038/ncomms11724) - Parasite Clearance in Leishmaniasis in Resistant Animals Is Independent of the IL-23/IL-17A Axis. J Invest Dermatol. 2016; 136(9): 1906-1908
Dietze-Schwonberg K, Lorenz B, Lopez Kostka S, Waisman A, von Stebut E
(See online at https://doi.org/10.1016/j.jid.2016.05.111) - In silico prediction of Leishmania major-specific CD8+ epitopes. Exp Dermatol. 2017; 26(9): 838-840
Dietze-Schwonberg K, Grewe B, Brosch S, Kuharev J, van Zandbergen G, Rammensee HG, Tenzer S, von Stebut E
(See online at https://doi.org/10.1111/exd.13295)
