Characterization of cancer/testis antigen MAGE-A11 for immunotherapy of prostate cancer

Les antigènes testiculaires du cancer sont des cibles idéales pour l’immunothérapie du cancer car ce sont des protéines immunogéniques dont l’expression est restreinte aux cellules germinales et au cancer. Le but de cette étude est d’évaluer le potentiel de MAGE-A11, un antigène testiculaire du cancer, comme cible pour développer un vaccin contre le cancer de la prostate. Pour ce faire, l’anticorps monoclonal 5C4 qui a la capacité de reconnaître la présence de MAGE-A11 dans les tissus fixés et inclus en paraffine a été produit. De plus, l’expression de MAGE-A11 a été analysée sur plusieurs lignées de cellules cancéreuses. Il a été démontré que MAGE-A11 est exprimé dans plusieurs types de cancers notamment dans le cancer du côlon et du cerveau. Finalement, nous avons identifié trois épitopes du CMH classe II HLA-DR1 dans la protéine MAGE-A11 confirmant ainsi l’immunogénicité de cet antigène et son potentiel comme cible pour l’immunothérapie du cancer.

Rôle de la conformation des glycoprotéines de l’enveloppe du VIH-1 dans la réponse cytotoxique cellulaire dépendante des anticorps et impact des protéines virales Nef et Vpu

Alors que d’énormes efforts sont mis de l’avant pour mettre en place des stratégies thérapeutiques contre l’infection au VIH-1, il est nécessaire de mieux cerner les déterminants viraux qui aideront à l’efficacité de celles-ci. En ce sens, une volumineuse littérature scientifique suggère que les anticorps contre le VIH-1 possédant une capacité à induire une réponse effectrice dépendante de leur portion Fc puissent jouer un rôle important dans la prévention de l’infection et dans la progression de la maladie. Cependant, peu d’information est disponible concernant les déterminants reconnus par ces anticorps et comment le virus s’en protège. Le but des travaux présentés dans cette thèse est donc d’élucider les mécanismes viraux contrôlant la reconnaissance des cellules infectées par ces anticorps capables d’induire une réponse effectrice. De par les corrélats de protection identifiés au cours de l’essai vaccinal RV144, les travaux présentés ici se concentrent sur la réponse cytotoxique dépendante des anticorps (ADCC), puisqu’il s’agit d’une réponse effectrice suggérée pour avoir joué un rôle dans la protection observée dans le RV144, seul essai vaccinal anti-VIH à avoir démontré un certain degré de protection. De plus, plusieurs anticorps capables d’induire cette réponse contre le VIH sont connus pour reconnaître les glycoprotéines de surface du virus (Env) dans une conformation dite ouverte, c’est-à-dire la conformation adoptée lors de la liaison d’Env avec son récepteur CD4 (épitopes CD4i). Nous avons mis au point deux techniques in vitro permettant d’étudier ces changements de conformation ainsi que leur impact sur la réponse ADCC. Les techniques mises au point, un ÉLISA sur base cellulaire pour mesurer les changements de conformation d’Env ainsi que la mesure de la réponse ADCC par cytométrie en flux, nous ont permis de démontrer comment le virus empêche l’exposition des épitopes d’Env CD4i. L’activité simultanée des protéines accessoires virales Nef et Vpu sur le retrait du récepteur CD4 de la surface des cellules infectées et l’inhibition du facteur de restriction Tétherine / BST-2 par Vpu contrôlent à la fois les niveaux d’Env et de CD4 à la surface cellulaire et donc modulent l’interaction Env-CD4 et ultimement la susceptibilité à la réponse ADCC contre les épitopes CD4i reconnus par des anticorps hautement prévalents lors de l’infection au VIH. Également, nous démontrons comment de petits composés mimant la liaison de CD4 sur Env sont capables de forcer l’exposition des épitopes CD4i, même en présence des protéines Nef et Vpu, et donc d’augmenter la susceptibilité des cellules infectées à la réponse ADCC. Une autre découverte présentée ici est la démonstration que la portion soluble d’Env produite par les cellules infectées peut interagir avec le récepteur CD4 des cellules non-infectées avoisinantes et induire leur reconnaissance et élimination par la réponse ADCC contre Env. Somme toute, la modulation de la réponse ADCC par l’interaction Env–CD4 représente un important pilier de la relation hôte – pathogène du VIH-1 de la perspective des réponses Fc-dépendantes. Les travaux présentés dans cette thèse ont le potentiel d’être utilisés dans l’élaboration de nouvelles stratégies antivirales tout en élargissant les connaissances fondamentales de cette interaction hôte – pathogène.

Towards the Knowledge-based Design of Universal Influenza Epitope Ensemble Vaccines

Motivation: Influenza A viral heterogeneity remains a significant threat due to unpredictable antigenic drift in seasonal influenza and antigenic shifts caused by the emergence of novel subtypes. Annual review of multivalent influenza vaccines targets strains of influenza A and B likely to be predominant in future influenza seasons. This does not induce broad, cross protective immunity against emergent subtypes. Better strategies are needed to prevent future pandemics. Cross-protection can be achieved by activating CD8+ and CD4+ T cells against highly-conserved regions of the influenza genome. We combine available experimental data with informatics-based immunological predictions to help design vaccines potentially able to induce cross-protective T-cells against multiple influenza subtypes. Results: To exemplify our approach we designed two epitope ensemble vaccines comprising highlyconserved and experimentally-verified immunogenic influenza A epitopes as putative non-seasonal influenza vaccines; one specifically targets the US population and the other is a universal vaccine. The USA-specific vaccine comprised 6 CD8+ T cell epitopes (GILGFVFTL, FMYSDFHFI, GMDPRMCSL, SVKEKDMTK, FYIQMCTEL, DTVNRTHQY) and 3 CD4+ epitopes (KGILGFVFTLTVPSE, EYIMKGVYINTALLN, ILGFVFTLTVPSERG). The universal vaccine comprised 8 CD8+ epitopes: (FMYSDFHFI, GILGFVFTL, ILRGSVAHK, FYIQMCTEL, ILKGKFQTA, YYLEKANKI, VSDGGPNLY, YSHGTGTGY) and the same 3 CD4+ epitopes. Our USA-specific vaccine has a population protection coverage (portion of the population potentially responsive to one or more component epitopes of the vaccine, PPC) of over 96% and 95% coverage of observed influenza subtypes. The universal vaccine has a PPC value of over 97% and 88% coverage of observed subtypes.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+ T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Fruit cell culture as a model system to study cell wall changes during strawberry fruit ripening

Strawberry (Fragaria x ananassa, Duch.) fruit is characterized by its fast ripening and soft texture at the ripen stage, resulting in a short postharvest shelf life and high economic losses. It is generally believed that the disassembly of cell walls, the dissolution of the middle lamella and the reduction of cell turgor are the main factors determining the softening of fleshy fruits. In strawberry, several studies indicate that the solubilisation and depolymerisation of pectins, as well as the depolymerisation of xyloglucans, are the main processes occurring during ripening. Functional analyses of genes encoding pectinases such as polygalacturonase and pectate lyase also point out to the pectin fraction as a key factor involved in textural changes. All these studies have been performed with whole fruits, a complex organ containing different tissues that differ in their cell wall composition and undergo ripening at different rates. Cell cultures derived from fruits have been proposed as model systems for the study of several processes occurring during fruit ripening, such as the production of anthocyanin and its regulation by plant hormones. The main objective of this research was to obtain and characterize strawberry cell cultures to evaluate their potential use as a model for the study of the cell wall disassembly process associate with fruit ripening. Cell cultures were obtained from cortical tissue of strawberry fruits, cv. Chandler, at the stages of unripe-green, white and mature-red. Additionally, a cell culture line derived from strawberry leaves was obtained. All cultures were maintained in solid medium supplemented with 2.5 mg.l-1 2,4-D and incubated in the dark. Cell walls from the different callus lines were extracted and fractionated to obtain CDTA and sodium carbonate soluble pectin fractions, which represent polyuronides located in the middle lamella or the primary cell wall, respectively. The amounts of homogalacturonan in both fractions were estimated by ELISA using LM19 and LM20 antibodies, specific against demethylated and methyl-esterified homogalacturonan, respectively. In the CDTA fraction, the cell line from ripe fruit showed a significant lower amount of demethylated pectins than the rest of lines. By contrast, the content of methylated pectins was similar in green- and red-fruit lines, and lower than in white-fruit and leaf lines. In the sodium carbonate pectin fraction, the line from red fruit also showed the lowest amount of pectins. These preliminary results indicate that cell cultures obtained from fruits at different developmental stages differ in their cell wall composition and these differences resemble to some extent the changes that occur during strawberry softening. Experiments are in progress to further characterize cell wall extracts with monoclonal antibodies against other cell wall epitopes.

Development of synthetic light-chain antibodies as novel and potent HIV fusion inhibitors

This is a non-final version of an article published in final form in AIDS. 2016 Jul 17;30(11):1691-701.

Comparative Genomics of Field Isolates of Mycobacterium bovis and M. caprae Provides Evidence for Possible Correlates with Bacterial Viability and Virulence

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to develop tools to evaluate risks for TB disease caused by M. bovis/M.caprae and for TB control in humans and animals.

Development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to T cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/-recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts. This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

Development of metabotropic glutamate receptors from trigeminal nuclei to barrel cortex in postnatal mouse

Expression patterns of group I (mGluR1α and mGluR5)and group II (mGluR2/3) metabotropic glutamate receptor subtypes were examined immunocytochemically in the trigeminal system of mice during the first 3 weeks of postnatal development, when somatotopic whisker representations are sequentially established from brainstem through thalamus to cerebral cortex. Immunostaining for all three epitopes formed whisker-related patterns in the trigeminal nuclei from postnatal day (P) 0, in the ventral posterior thalamic nucleus from P2, and in the posteromedial barrel subfield of somatosensory cortex (SI) from P4. The appearance of whisker-related patterns was preceded by increased levels of immunostaining of the neuropil, which subsequently declined from the trigeminal nuclei upward. In SI, mGluR1α-positive neurons were observed in all cortical layers from P2. mGluR5 was localized in neurons, glial cells, and neuropil from P2. mGluR2/3 immunostaining was distributed only in the neuropil at all ages. The three receptor subtypes showed moderate to high expression in deep layer V throughout development. Transient expression peaked in the hollows of layer IV barrels from P4 to P9, and then fell off as expression increased in supragranular layers from P14 to P21. The deep aspect of the cortical subplate (layer VIb) showed dense mGluR5 and less dense mGluR1α immunostaining throughout development. Up-regulation of expression of group I and II mGluRs is correlated with the growth and refinement of connectivity and the establishment of somatotopic patterns in the three main relay stations of the trigeminal system. This finding suggests roles for mGluRs in the early processing of sensory information and in developmental plasticity.