Identification and biological validation of HPV16 E6/E7-derived T cell target epitopes and their use for performance assessment of MHC class I binding predictors

Persistent infection with high-risk types of human papillomavirus (HPV) can cause several malignancies, in particular oropharyngeal and anogenital cancers. HPV16 has been identified as the most prevalent high-risk type, being related to 60% of cervical cancers, 75% of oropharyngeal cancers, 71% of anal cancers and the majority of precancerous lesions. As standard of care treatment is invasive, and harbors risks and side effects, there is a need for new approaches. For rationally designing a therapeutic vaccine against HPV-induced malignancies, it is essential to identify suitable target epitopes, which are presented on the surface of an HPV-transformed cell and induce immune responses that eventually mediate target cell death. The HPV16 oncoproteins E6 and E7 represent ideal targets for immunotherapy as they mediate the transforming potential of the virus and are constitutively expressed in all malignant cells. In order to define HPV16 target epitopes, in this thesis several algorithms were used to predict potential HPV16 E6- and E7-derived binders of human leukocyte antigen (HLA) class I in silico. Predicted peptides were synthesized and HLA binding capacity was validated in competition-based cellular binding assays. To ensure broad population coverage, predictions and validations were performed for seven frequent HLA alleles: A*01:01, A*02:01, A*03:01, A*11:01, A*24:02, B*07:02 and B*15:01. Including peptides derived from HPV16 E6/E7 variants containing amino acid changes, 271 peptides were experimentally assessed and 69 binders were identified. Combined with previous results, the total HPV16 E6/E7 dataset comprised 779 peptide-HLA measurements. The HPV16 E6/E7 dataset was used to evaluate the performance of employed predictors. No single algorithm was outperforming other methods, but different predictors were found to be best for different settings, depending on investigated HLA type and peptide length. As applying commonly used decision threshold yielded only low sensitivity, criteria for optimal decision thresholds were defined and optimal thresholds were calculated for individual predictors, HLA-types and peptide lengths. Comparing threshold-dependent performance of predictors showed that using criteria-based thresholds allowed more sensitive prediction of HLA-binding peptides without a strong negative influence on prediction accuracy. To identify T cell epitopes among the HPV16 E6- and E7-derived HLA ligands, their capacity to induce immune responses was investigated. To this end, peripheral blood mononuclear cells of healthy donors were HLA-typed and stimulated with respective peptides to generate epitope-specific T cell lines. By assessing interferon-γ-secretion of these T cells, 31 immunogenic peptides were identified. Further characterizing the functionality of epitopes in cytotoxicity assays, five of ten immunogenic HLA-A*02:01-peptides mediated specific killing of HPV16+ target cells by CD8+ T cells. In conclusion, several immunogenic HPV16 E6-and E7-derived epitopes were identified, which are the basis for rational design of a therapeutic HPV vaccine. Additionally, this thesis provides an evaluation of peptide–HLA class-I binding prediction method and recommendations to increase prediction sensitivity to extend the number of potential epitopes as targets for immunotherapy

The shared frameshift mutation landscape of microsatellite-unstable cancers suggests immunoediting during tumor evolution

The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers. DNA mismatch repair (MMR)-deficient cancers with microsatellite-instability are characterized by a high load of frameshift mutation-derived neoantigens. Here, by mapping the frameshift mutation landscape and predicting the immunogenicity of the resulting peptides, the authors show evidence of immunoediting in MMR-deficient colorectal and endometrial cancers.Peer reviewe

Consultation pluridisciplinaire "Souffrance au travail": une expérience romande [Multidisciplinary consultation "Suffering at work": an experience in western Switzerland].

Les problèmes de santé mentale au travail constituent un défi à la fois clinique, professionnel, économique et de santé publique. Les coûts totaux qu'ils génèrent en Suisse équivalent à 3,2 % du produit intérieur brut (PIB) suisse et ils aboutissent très souvent à un licenciement. La grande majorité des personnes sont soignées par un médecin de premier recours. L'Institut de Santé au Travail propose une consultation spécialisée dans les questions de souffrance au travail, offrant aux soignants de première ligne un avis ou un soutien pluridisciplinaire, dans une perspective collaborative des soins. Son action, adaptée aux besoins de chaque situation, va d'un avis à une orientation vers des spécialistes pouvant étoffer durablement le réseau (suivi psychiatrique, programme de soutien à l'emploi, avis juridique ou social). Mental health problems at work constitute a challenge in the clinical feld, as well in the professional, the economic and the public health perspective. The total costs they generate in Switzerland are equivalent to 3.2% of the Swiss gross domestic product and they very often lead to dismissal. The vast majority of people are treated by their primary care physician. The Institute for Work and Health features a specialized consultation on the topic of suffering at work, offering the primary care physicians a pluridisciplinary advice or support, in a collaborative care prospect. Its action, adapted to each situation's needs, goes from an advice to a referral to specialists that can strengthen the network on a long-term basis (mental health follow-up, supported employment program, legal or social advice)

Definition and Characterization of SOX11-Derived T Cell Epitopes towards Immunotherapy of Glioma

The transcription factor SOX11 is a tumor-associated antigen with low expression in normal cells, but overexpression in glioblastoma (GBM). So far, conventional surgery, chemotherapy, and radiotherapy have not substantially improved the dismal prognosis of relapsed/refractory GBM patients. Immunotherapy is considered a promising strategy against GBM, but there is a fervent need for better immunotargets in GBM. To this end, we performed an in silico prediction study on SOX11, which primarily yielded ten promising HLA-A*0201-restricted peptides derived from SOX11. We defined a novel peptide FMACSPVAL, which had the highest score according to in silico prediction (6.02 nM by NetMHC-4.0) and showed an exquisite binding affinity to the HLA-A*0201 molecule in the peptide-binding assays. In the IFN-γ ELISPOT assays, FMACSPVAL demonstrated a high efficiency for generating SOX11-specific CD8+ T cells. Nine out of thirty-two healthy donors showed a positive response to SOX11, as assessed by the ELISPOT assays. Therefore, this novel antigen peptide epitope seems to be promising as a target for T cell-based immunotherapy in GBM. The adoptive transfer of in vitro elicited SOX11-specific CD8+ T cells constitutes a potential approach for the treatment of GBM patients

Definition and Characterization of SOX11-Derived T Cell Epitopes towards Immunotherapy of Glioma

The transcription factor SOX11 is a tumor-associated antigen with low expression in normal cells, but overexpression in glioblastoma (GBM). So far, conventional surgery, chemotherapy, and radiotherapy have not substantially improved the dismal prognosis of relapsed/refractory GBM patients. Immunotherapy is considered a promising strategy against GBM, but there is a fervent need for better immunotargets in GBM. To this end, we performed an in silico prediction study on SOX11, which primarily yielded ten promising HLA-A*0201-restricted peptides derived from SOX11. We defined a novel peptide FMACSPVAL, which had the highest score according to in silico prediction (6.02 nM by NetMHC-4.0) and showed an exquisite binding affinity to the HLA-A*0201 molecule in the peptide-binding assays. In the IFN-γ ELISPOT assays, FMACSPVAL demonstrated a high efficiency for generating SOX11-specific CD8+ T cells. Nine out of thirty-two healthy donors showed a positive response to SOX11, as assessed by the ELISPOT assays. Therefore, this novel antigen peptide epitope seems to be promising as a target for T cell-based immunotherapy in GBM. The adoptive transfer of in vitro elicited SOX11-specific CD8+ T cells constitutes a potential approach for the treatment of GBM patients