abstract: CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self/nonself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2017

No description available.

Da Filarieninfektionen noch immer chemotherapeutisch schwer bekämpfbar sind, ist die Aufklärung von potentiell protektiven Molekülen, die zur Impfstoffentwicklung von Nutzen sein könnten, von Bedeutung. Ein vielversprechendes Antigen stellt in diesem Zusammenhang die Chitinase von infektiösen Drittlarven von Onchocerca volvulus dar. In dieser Arbeit sollte deshalb dieses Protein immunologisch charakterisiert und immundominante Bereiche identifiziert werden. Dazu wurde die cDNA des gesamten Proteins (OvL3-Chitinase) und die cDNA der Domäne, die für die Chitin-Bindung verantwortlich ist (OvL3-CBD), in einen Expressionsvektor kloniert, in E. coli exprimiert und aufgereinigt. Die aufgereinigte OvL3-Chitinase zeigte enzymatische Aktivität. Die OvL3-Chitinase und die OvL3-CBD wurden in Immunisierungsstudien im Tiermodel der Nagetierfilarie A. viteae und M. unguiculatus eingesetzt. Während die Immunisierung mit OvL3-CBD mit dem Adjuvans Alum zu keiner Reduzierung der Adultwurmlast führte, war nach Gabe der OvL3-Chitinase die Anzahl der Adultwürmer um 40 % bzw. um 17,7% reduziert. Zusätzlich wurde die OvL3-Chitinase allein und in Kombination mit zwei verschiedenen Adjuvantien (STP und Alum) in Immunisierungsstudien von BALB/c-Mäusen eingesetzt, deren Immunantworten anschließend charakterisiert wurden. Durch diese Versuche konnte gezeigt werden, daß die Chitinase ohne zusätzliche Gabe eines Adjuvans unter den getesteten Bedingungen eine TH2-Immunantwort induziert. Dies wurde durch die Anwesenheit von Antikörpern der Subklasse IgG1 deutlich. Außerdem waren Milzzellen dieser Mäuse nicht in der Lage, nach Restimulation mit Chitinase mit Proliferation zu reagieren. Durch den Einsatz der Adjuvantien STP und Alum konnte eine Polarisation der Immunantworten erfolgen. Während die Immunisierung mit Chitinase und Alum zur deutlichen Bildung von IgG1-Antikörpern und zu einer leichten Erhöhung der Antikörper IgG2a und IgG2b führte, konnte nach Immunisierung mit Chitinase und STP neben Antikörpern der Subklasse IgG1 deutliche erhöhte OD-Werte von IgG2a und IgG2b gemessen werden. In beiden Gruppen reagierten Milzzellen nach Restimulation mit Chitinase, wobei die Proliferationswerte der STP/Chitinase-Gruppe über denen der Alum/Chitinase-Gruppe lagen. Durch den Einsatz von Salmonellen, die Chitinase exprimierten, konnten die T-Zell-Antworten verstärkt werden. Es konnten jedoch keine antigen-spezifischen Antikörper gefunden werden. Mit Hilfe von drei verschiedenen T-Zell-Algorithmen (Algorithmus nach Rothbard und Taylor, nach Humphreys und die MHC-II-Bindungs-Motive nach Rammensee) wurden T-Zell-Epitope innerhalb der OvL3-Chitinase identifiziert. Alle vorhergesagten Epitope wurden als synthetische Peptide hergestellt und in T-Zell-Proliferationstests eingesetzt. Hierzu wurden Milzzellen von Mäusen verwendet, die dreimal mit rekombinanter Chitinase und dem Adjuvans STP immunisiert worden waren. Um möglichst viele T-Zell-Epitope innerhalb der Chitinase zu ermitteln, wurden überlappende Peptide (Pepscan), die die Gesamtheit der Chitinase umfaßten, in T-Zell-Tests eingesetzt. Die verwendeten Algorithmen wurde auf ihre Sensitivität und Spezifität überprüft, indem die vorhergesagten Epitope mit den ermittelten T-Zell-Epitopen des Pepscans verglichen wurden. Dabei konnte der Algorithmus von Rammensee den besten Index an Sensitivität (0,47) und Spezifität (0,7) erzielen. Eine Kombination der Algorithmen ergab, daß die Verknüpfung des Algorithmus nach Rothbard und Taylor mit den MHC-Bindungs-Motiven nach Rammensee die Sensitivität auf 0,67 erhöhen konnte, doch die Spezifität sank durch die hohe Anzahl der vorhergesagten Epitope auf 0,33. Die Anwendung der Algorithmen auf die Sequenz der OvL3-Chitinase führte zu der Identifizierung von fünf Bereichen, die von allen Algorithmen vorhergesagt wurden, und von denen vier in T-Zell-Proliferationstests deutliche Proliferationswerte erzielten. Zusätzlich wurde die OvL3-Chitinase und die OvL3-CBD in Kamerun hinsichtlich ihrer Fähigkeit getestet, PBMC von Onchozerkose-Patienten zu restimulieren, die aus einem hyperendemischen Onchozerkose-Gebiet stammten. Diese Untersuchungen bestätigten die Ergebnisse aus den Immunisierungsstudien mit BALB/c-Mäusen, in denen Chitinase eine TH2-Immunantwort hervorrief. Die PBMC der Onchozerkose-Patienten zeigten nur eine sehr geringe Proliferation nach Stimulation mit OvL3-Chitinase und OvL3-CBD. Begleitet wurde diese Proliferation von Ausschüttung typischer TH2-Zytokine wie IL-10 (Chitinase) bzw. IL-4, IL-5 und IL-10 (CBD). Die Untersuchung der Antikörperantworten der Onchozerkose-Patienten zeigte, daß bei 77% der untersuchten Patienten IgG4-Antikörper gegen die Chitinase und bei 20% gegen die OvL3-CBD im Serum nachgewiesen werden konnten. / Chemotherapeutic treatment of filarial infections has rendered difficult and still insufficient. Therefore, the identification of potentially protective molecules which can be used for vaccine development is desirable. The chitinase of larvae stage three of Onchocerca volvulus constitutes a promising antigen. Immunological characterization of this protein and the identification of immunodominat regions was performed in this study. The complete sequence (OvL3-chitinase) and the C-terminal end responsible for the binding of chitin (OvL3-CBD) were cloned in an expression vector, overexpressed in E. coli and subsequently purified. Recombinant chitinase was enzymatically active. The OvL3-chitinase and the OvL3-CBD were used for immunization studies using the rodent filaria A. viteae in the animal model M. unguiculatus. No decreased number of adult worms was observed after immunization with OvL3-CBD in the present of Alum as adjuvans, while chitinase together with STP resulted in the reduction the worm burden to 40 % (first trial) and to 17,7 % (second trial). Additional immunization studies using BALB/c-mice were performed with OvL3-chitinase in the absence or the presence of two different adjuvans. Spleen cells isolated from mice immunized with chitinase in the absence of adjuvans were devoid of proliferative capacity after in vitro antigenic restimulation. Antigen-specific IgG1 antibodies were the only subtype detectable in sera from mice. These results suggested that a Th2 response was induced after immunization with chitinase under these conditions. Including STP or Alum in the immunization protocol a polarization of the obtained immune response was found. Immunization with chitinase together with Alum elicited strong IgG1 response followed by slightly increase of IgG2a and IgG2b. Co-administration of chitinase and STP evoked increased IgG2a and IgG2b antibodies in addition to the observed IgG1 response. Good proliferative responses were observed for spleen cells from mice immunized with chitinase in the present of both adjuvans after in vitro restimulation with chitinase. Furthermore stronger T-cell reactivity was found in the group immunized with chitinase/STP. Also chitinase was expressed in Salmonella and oral immunization of mice with this construct enforced the T-cell reactivity, however antigen specific antibodies were undetectable. To further characterize T cell reactivity against OvL3-chitinase T cell epitopes using the Rothbard and Taylor algorithm (1988), Humphreys prediction and the MHC-II-binding motifs from Rammmensee (1995) were identified. All predicted epitopes were synthesized and tested in T-cell proliferation assays. Additional synthetic L3-chitinase-derived overlapping peptides were also used in T-cell proliferation assays. The sensitivity and specificity of the algorithms was evaluated by comparing the predicted epitopes with the epitopes determined by overlapping peptides. Using this approach, prediction based on MHC-II-binding motifs showed the highest sensitivity (0,47) and specificity (0,7). A further increase of the predictive power was obtained by a combining MHC-II-binding motifs and Rothbard and Taylor algorithm, resulting in increased sensitivity (0,67) but lower specificity (0,33). Five immunodominant regions were identified by all algorithm and four of them were confirmed as T-cell epitopes in T-cell assays. PBMCs isolated from patients affected with Onchocerca volvulus from Cameroon were also tested for their reactivity against OvL3-chitinase and the OvL3 CBD in T-cell proliferation assays. After in vitro restimulation with OvL3-chitinase and CBD only marginal T-cell response was observed accompanied by release of Th2-like cytokines such as IL-10 when stimulated with chitinase and IL-4, IL-5 and IL-10 when stimulated with CBD. Strong antibody responses of the IgG4 isotype were detected in the serum of 77% of the patients against chitinase and only in 20% of the patients against CBD.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-03-10T13:11:37Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Rafael de Freitas e Silva.pdf: 13510018 bytes, checksum: 080b7ee10e7a8be555cb7a7ca29c10fb (MD5) / Made available in DSpace on 2017-03-10T13:11:37Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Rafael de Freitas e Silva.pdf: 13510018 bytes, checksum: 080b7ee10e7a8be555cb7a7ca29c10fb (MD5) Previous issue date: 2016-09-06 / As leishmanioses são doenças causadas por protozoários do gênero Leishmania e estão presentes em 98 países e territórios e possuem incidência anual de 2 milhões de casos. A Leishmania (Viannia) braziliensis (L.V. braziliensis) é uma das principais espécies causadoras da leishmaniose cutânea (LC) no Brasil. Apesar disso, ainda não há uma vacina segura e eficaz para ser utilizada em seres humanos. Nesse sentido, o objetivo deste trabalho foi identificar no proteoma predito de L.V. braziliensis, potenciais epítopos de linfócitos T e avaliá-los por meio de ensaios imunológicos. No primeiro capítulo, o proteoma predito de L. braziliensis foi comparado ao de outras espécies e analisado quanto a presença de epítopos. Nessa etapa foram encontrados epítopos derivados de mais de 8 mil proteínas conservadas entre diferentes espécies de Leishmania. Os epítopos foram clusterizados e então utilizados para etapa de docagem molecular com estruturas de MHC I e MHC II depositadas no Protein Data Bank. A docagem molecular resultou em epítopos peptídicos de 15 aminoácidos com alta afinidade de ligação às moléculas de MHC I e MHC II. Os 10 melhores resultados foram então sintetizados e avaliados, in vitro, quanto à capacidade de estimular a proliferação de células mononucleares do sangue periférico (PBMC) de indivíduos com LC após o tratamento (PT). Os resultados indicaram que 50% das moléculas testadas apresentaram capacidade de estimular, significativamente (p<0,05), a proliferação celular quando comparado às células de indivíduos saudáveis que não vivem em região endêmica para LC. No segundo capítulo, os peptídeos foram avaliados quanto à capacidade de estimular a proliferação de PBMC de indivíduos com LC em sua fase ativa (AD) e indivíduos moradores de área endêmica para LC resistentes à infecção (RT). Em paralelo, quantificou-se a expressão do fator de transcrição T-bet em PBMC de indivíduos PT, e citocinas dos perfis Th1, Th2 e Th17 foram mensuradas no sobrenadante de cultura das células de indivíduos PT e AD. Os resultados demonstraram altos níveis de proliferação nas células do grupo RT para todos os peptídeos testados. Além disso, níveis significativos de Tbet foram observados em linfócitos T CD4+ e CD8+ após estímulo com seis peptídeos. Níveis significativos de IFN-γ, TNF e IL-6 foram observados no sobrenadante das células do grupo PT com quatro dos peptídeos testados. Altos níveis dessas citocinas também foram encontrados no sobrenadante do grupo AD. No terceiro capítulo, avaliou-se o efeito dos peptídeos sobre células dendríticas de medula (BMDC) murinas, produção de citocinas de sobrenadante, e células dendríticas esplênicas murinas após estímulo com os peptídeos. Verificou-se altos níveis de MHC II e CD40 em uma subpopulação de BMDC estimuladas com as moléculas e altos níves de TNF e IL-6 após 48h de estímulo. Para as células esplênicas, foram observados altos níveis de subpopulações celulares expressando CD11b+, IL-12p70+, CD205+ e CD11b+ após estímulo com o peptídeo que teve o melhor resultado in silico. Por fim, os resultados indicam o grande potencial imunogênico que os epítopos identificados apresentam, o que dá suporte ao desenvolvimento futuro de abordagens vacinais. / The leishmaniasis are diseases caused by protozoans from the genus Leishmania which are present in 98 countries and territories, with an annual incidence of 2 million cases. Among the other species, Leishmania (Viannia) braziliensis is the main specie implicated with cutaneous leishmaniasis (CL) in Brazil. Besides that, there is no safe and effective vaccine against leishmaniasis to be applied in humans. In this context, the aim of this work was to identify in the predicted proteome of L. braziliensis potential CD4+ and CD8+ T cell epitopes and evaluate them by immunological assays. In the first chapter, the predicted proteome of L. braziliensis was compared with other species and analyzed for the presence of epitopes. In this step, epitopes from more than 8,000 conserved proteins were found among other species of Leishmania. The epitopes were clustered and then used for the molecular docking with MHC I and MHC II structures deposited in the Protein Data Bank. This approach resulted in 15 aminoacids peptide epitopes with high binding affinity for MHC I and MHC II. The 10 best results were synthesized and evaluated in vitro for their capacity to stimulate the proliferation of peripheral blood mononuclear cells (PBMC) of individuals with CL post treatment (PT). The results have shown that 50% of the tested molecules had the capacity to stimulate, significantly (p<0.05), cell proliferation when compared with cells of healthy individuals living in non-endemic regions. For the second chapter, the peptides were evaluated for their capacity to stimulate the proliferation of PBMC from CL individuals with active disease (AD) and of individuals resistant to infection (RT) living in endemic region. In parallel, the T-bet transcription factor expression was quantified in PBMC of PT individuals, and cytokines from the Th1, Th2 and Th17 profiles were measured in culture supernatant of PT and AD groups. High levels of cell proliferation in the RT group were demonstrated for all peptides tested. Moreover, significant levels of T-bet in CD4+ and CD8+ T cells were verified after stimulation with six peptides. For IFN-γ, TNF and IL-6, significant levels were detected in the supernatant of cultures from the PT group with four peptides tested. High levels of these same cytokines were also present in the supernatant of AD group. In the third chapter, the peptide effects over murine bone marrow dendritic cells (BMDC), the production of cytokines in the supernatant and murine spleen dendritic cell subsets were evaluated after peptide stimuli. High levels of MHC II and CD40 were verified for stimulated BMDC and high levels of TNF and IL-6 after 48h of stimuli. For spleen cells, high levels of cells expressing CD11b+, IL-12p70+, CD205+ e CD11b+ were observed after stimulation with the peptide which showed the best in silico result. In conclusion, the results indicate the great immunogenic potential of the identified peptides and support the further development of vaccine approaches using those molecules.

HINTERGRUND: Die Reaktivierung des Humanen Cytomegalievirus (HCMV) ist immer noch eine häufige Ursache für Morbidität und Mortalität unter immunsupprimierten Patienten. Eine effiziente T-Zell-Antwort vermag die unkontrollierte Ausbreitung des Virus zu verhindern. Vieles über diese T-Zell-Antwort ist aber noch unklar. Im Rahmen dieser Studie wurden daher bei HCMV-seropositiven herz- (n = 17) und lungentransplantierten (n = 3) Patienten Epitope in zwei wichtigen T-Zell-Zielen, den HCMV-Proteinen IE-1 (UL123) und pp65 (UL83), identifiziert, die Frequenzen der für diese Epitope spezifischen T-Zellen gemessen und die Klonalität ausgewählter starker CD8+ T-Zell-Antworten untersucht. METHODEN: Dazu wurden Pentadecapeptide, die die gesamte Aminosäure-Sequenz von IE-1 bzw. pp65 umfassten und sich um jeweils 11 Aminosäurereste überlappten, in Pools von 25 bis 30 Peptiden so zusammengefasst, dass jedes Peptid in einer einzigartigen Kombination von drei Pools enthalten war. PBMC der Patienten wurden dann mit den Peptid-Pools stimuliert und die resultierenden T-Zell-Reaktionen durch Färbung von intrazellulär zurückgehaltenem Interferon-gamma durchflusszytometrisch sichtbar gemacht. Immunogene Peptide konnten anhand der jeweiligen drei Pools, die zu IFN-gamma-Produktion führten, eindeutig identifiziert werden. Einige dieser T-Zell-Populationen wurden durch einen IFN-gamma-Sekretions-Assay, magnetische Zellseparation und durchflusszytometrische Feinsortierung aus PBMC isoliert und ihre Klonalität mit Hilfe einer Polymerase-Kettenreaktion zum Nachweis klonal expandierter gamma-T-Zell-Rezeptor-Rearrangements (TCR-PCR) und anschliessender Fragmentanalyse fluoreszenzmarkierter PCR-Amplifikate untersucht. ERGEBNISSE: Bei den Patienten bestanden grosse Unterschiede hinsichtlich des jeweils immundominanten Proteins, der Dominanz von CD4+ bzw. CD8+ T-Zell-Subpopulation, der antigenen Determinanten, der gemessenen Peptid-spezifischen T-Zell-Frequenzen sowie der Anzahl der identifizierten Epitope. Zehn zuvor noch nicht beschriebene Epitope wurden eben-falls identifiziert und die präsentierenden HLA-Allele der meisten in der Patientengruppe identifizierten Epitope bestimmt. Die mittels TCR-PCR untersuchten CD8+ T-Zell-Reaktionen waren auf einen oder wenige Klone fokussiert. Die Korrelation der experimentellen Daten mit den klinischen Verläufen der Patienten hinsichtlich HCMV-Reaktivierung und -Erkrankung erbrachte jedoch keine Hinweise auf einen konkreten Zusammenhang. SCHLUSSFOLGERUNGEN: Zusammenfassend ermöglichen die hier vorgestellten Methoden die Untersuchung des Langzeitverlaufes der CD4+ und CD8+ T-Zell-Antwort gegen immundominante Proteine auf Epitop-Ebene nach initialer Identifizierung der antigenen Determinanten, die direkte Bestimmung der Frequenzen der Epitop-spezifischen T-Zellen sowie die Untersuchung der Klonalität dieser Reaktionen aus ca. 2 x 20 ml Blut. Die Langzeit-Untersuchung von Patienten mit hohem Risiko für HCMV-Reaktivierung und -Erkrankung kann so zu einem besseren Verständnis der komplexen HCMV-spezifischen T-Zell-Anwort und damit möglicherweise auch zur Verbesserung von Diagnose, Prophylaxe und Therapie dieser Patienten beitragen. / BACKGROUND: Human cytomegalovirus (HCMV) reactivation is still a leading cause of morbidity and mortality among immunosuppressed patients. Uncontrolled viral spread is prevented by an efficient T-cell response. However, little is known about the nature of this T-cell response. In this study we identified epitopes in two immunodominant HCMV-proteins, IE-1 (UL123) and pp65 (UL83), measured the frequencies of T-cells specific for these, and studied the clonotypic composition of selected T-cell responses in a group of HCMV-seropositive heart (n = 17) and lung (n = 3) transplant patients. METHODS: For both proteins overlapping pentadecapeptides covering the entire respective amino acid sequences were arranged in pools of 25 peptides each in such a way that every peptide was contained in exactly 3 pools. PBMC were stimulated with the resulting 15 pools for IE-1 or 16 pools or pp65, respectively, as well as with pools containing all peptides of the corresponding protein. Individual peptides leading to a positive T-cell response were identified by flow cytometric detection of intracellular interferon-gamma, each single peptide corresponding to a unique combination of 3 peptide pools. Selected T-cell populations specific for the previously identified single peptides were purified by performing an IFN-gamma secretion assay prior to magnetic cell separation and subsequent fluorescence-activated cell sorting. The clonality of these highly purified peptide-specific T-cell populations was then investigated by a T-cell receptor-gamma rearrangement-PCR and subsequent fragment analysis of fluorescence-labelled PCR amplificates. RESULTS: We observed broad heterogeneity among the patients in terms of the immunodominant protein, number of epitopes, predominance of CD4 or CD8 T-cell responses, and epitope-specific T-cell frequencies. 10 previously unknown epitopes were identified, and the HLA-restriction of most of the identified epitopes could be determined. The investigated T-cell responses showed a high degree of clonal focussing. These data were correlated to the patients episodes of HCMV reactivation, but a correlation between differences in the T-cell responses and a different clinical outcome in terms of HCMV-reactivation could not be established. CONCLUSIONS: In summary, this novel approach allows the rapid identification of epitopes contained in a given protein, direct determination of T-cell frequencies, and investigation of the T-cell clonality in the CD4 and CD8 T-cell subsets from as little as 2 times 20 ml of blood. Long-term follow-up of patients at risk for HCMV reactivation and disease may thus allow a more detailed insight into the complexity of the T-cell response to HCMV and may thus lead to improved diagnosis, prophylaxis and therapy.

De nombreux antigènes de tumeur ont été identifiés depuis la découverte du premier antigène de tumeur humain il y a une vingtaine d’années, et plusieurs d’entre eux ont été utilisés comme antigène cible pour l’élaboration de vaccins thérapeutiques anti-Cancer. Cependant, les résultats des essais cliniques visant à évaluer l’efficacité de ces vaccins se sont la plupart du temps révélés décevants. Aussi, il reste indispensable d’identifier de nouveaux antigènes de tumeur cibles capables d’induire des réponses anti-Tumorales fortes et durables. Parmi les antigènes de tumeur considérés comme cibles potentielles pour un vaccin anti-Tumoral se trouve la Cycline B1, une protéine endogène impliquée dans la régulation du cycle cellulaire. Normalement exprimée de façon transitoire dans les cellules saines en division, cette protéine est surexprimée dans diverses tumeurs et est indispensable au développement tumoral. De plus, des réponses immunitaires spontanées spécifiques de cette protéine ont été observées chez des patients atteints de cancer. L’objectif de ma thèse était de caractériser la réponse en lymphocytes T CD4+, qui jouent un rôle capital dans la réponse immunitaire anti-Tumorale, spécifique de la Cycline B1 humaine chez des sujets sains et chez des patients atteints de cancer. Nous avons mis en évidence l’existence, chez des individus sains, de deux populations de lymphocytes T CD4+ préexistants spécifiques de cette protéine, à savoir des lymphocytes T CD4+ naïfs et des lymphocytes T CD4+ mémoires, cette seconde population lymphocytaire se retrouvant également chez des patients atteints de cancer. De multiples épitopes T CD4+ ont été identifiés dans cette protéine, et étaient différemment reconnus par ces deux populations de lymphocytes T CD4+. En outre, des anticorps IgG anti-Cycline B1 ont été détectés chez des patients atteints de cancer comme chez des individus sains, sans différence significative dans les taux d’anticorps entre ces deux catégories de sujets. Ainsi, cette étude montre que la Cycline B1 est un antigène de tumeur caractérisé par un profil singulier de réponses immunitaires, et confirme le potentiel vaccinal de cette protéine pour l’élaboration d’un vaccin anti-Cancer. / Many tumor antigens have been identified since the discovery of the first human antigen about twenty years ago, and some of them have been used as targets for the development of therapeutic cancer vaccines. However, most of the time, the results of clinical trials designed to assess the efficacy of these vaccines proved to be disappointing. Thus, it is still necessary to identify new tumor antigens able to induce strong and long-Lasting anti-Tumor responses that could be used as targets for cancer vaccine. Cyclin B1, an endogenous protein involved in cell cycle regulation, is one of the tumor antigens which are currently considered as potential targets for a cancer vaccine. Usually expressed transiently in healthy dividing cells, this protein is overexpressed in numerous tumors and is necessary for tumor development. Moreover, Cyclin B1 specific spontaneaous immune responses have been observed in cancer patients. My PhD work aimed at characterizing the response of CD4+ T cells, which play a major role in anti-Tumor immune responses, specific to human Cyclin B1 both in healthy individuals and cancer patients. We showed that, in healthy individuals, there exists two pre-Existing Cyclin B1 specific CD4+ T cell populations, namely naive CD4+ T cells and memory CD4+ T cells, the latter lymphocyte population being also found in cancer patients. Multiple CD4+ T cell epitopes have been identified in this protein, and were differently recognized by these two CD4+ T cell populations. Besides, anti-Cyclin B1 IgG antibodies have been detected both in healthy individuals and in cancer patients, without significant differences in antibody levels between these two groups of donors. Therefore, this work shows that Cyclin B1 is a tumor antigen characterized by a singular pattern of immune responses, and confirms the potential of this protein as a target for a cancer vaccine.

One-third of the world’s population is latently infected with Mycobacterium tuberculosis, which causes over 2 million deaths every year. The current live attenuated vaccine, Bacille Calmette-Guerin (BCG), protects against miliary tuberculosis in children, but fails to consistently protect against pulmonary tuberculosis in adults. The global resurgence of tuberculosis, together with the HIV pandemic and emerging multi-drug resistance, has heightened the need for an effective vaccine. Completion of the M. tuberculosis genome sequence paved way for identification of many new candidate antigens for protective vaccine against tuberculosis. This includes the discovery of two multigene families of proteins PE and PPE which constitute 10% of the coding capacity of the M. tuberculosis genome. Members of the PE and PPE protein families are characterized by highly conserved N-terminal domains and the C-terminus, however, exhibit considerable variation in the number of residues as well as in the sequence. Till date, little is known about the functional role of the proteins of PPE or PE family in the biology of M.tuberculosis. Some of the PE_PGRS proteins have been found to be associated with the cell wall and influence interactions with other cells. PE and PPE family of proteins are of potential interest from the point of view of immune response, since they show antigenic variation which may play a role in immune evasion. Very little is known about the immunogenecity of these two classes of proteins and only few proteins have been shown to be potent B or T cell antigens, like Rv3873, Mtb39 and Rv0915c. Two proteins from PE_PGRS subfamily, Rv1759c and Rv3367 are expressed during infection and show antibody response in humans and rabbits, respectively. Rv1196 and Rv0915c from PPE family have been shown to be good T cell antigens. Another study has shown that the PE domain of PE_PGRS protein Rv1818c upon immunization into mice induces good cell mediated immune response in mice, whereas the PGRS domain is responsible for good humoral response. In humans there is increasing evidence to suggest that CD8+ T cells are elicited in response to infection with mycobacteria. CD8+ CTL may play an important role through several mechanisms. They produce potent anti-bacterial cytokines such as IFN-γ and TNF-α in response to antigenic stimulation and IFN-γ is critical for immunity to TB. Thus, identification of antigens and peptides that induce T cell responses could be useful for designing new vaccines to protect against TB. Relatively few epitopes in mycobacterial antigens have so far been identified for human CD8 T cells. In this regard, release of genome sequences of M. tuberculosis has provided an opportunity to identify proteins with vaccine potential that could give immune protection in individuals with different HLA backgrounds. Objectives and scope of the present work 1. Prediction of putative T cell antigens in PE and PPE family of proteins of Mycobacterium tuberculosis through immuno-informatics approach 2. Evaluation of immune response to three of the PE and PPE proteins in mouse model. 3. Evaluation of immune response against chosen PE and PPE proteins of Mycobacterium tuberculosis with Human Peripheral Blood Mononuclear Cells (PBMCs) from PPD positive healthy donors and TB patients. 4. Immune response to multi-epitope DNA vaccine construct for Mycobacterium tuberculosis. Prediction of MHC class I peptides from PE and PPE proteins. In an effort to identify potential T cell antigens from PE and PPE family of proteins, we have carried out a systematic in silico analysis of the 167 different PE and PPE proteins. Employing immuno-informatics approach, a set of HLA class I binding peptides have been identified from these proteins. Further, their binding abilities have been ascertained using independent methods such as molecular modeling and structural analysis methods. The nonameric sequences from PE and PPE families of proteins were predicted to contain high percentage of binding peptides to human class I HLA, whereas PE_PGRS proteins show relatively low level of binding. This difference is seen in spite of PE and PE_PGRS being Sub-families of the same family, PE. Seventy-one high- as well as low-affinity peptides from both PE and PPE proteins have been analyzed for structural compatibility with crystal structures of HLA in terms of intermolecular energies and were found to correlate well with the corresponding affinities predicted by the BIMAS algorithm. Most of the peptides binding to HLA are specific with very few promiscuous binders. Identification of T cell epitopes from three of the PE/PPE proteins using DNA immunization This work describes the evaluation of immune responses to three of the PE and PPE proteins in mouse model. Three of PE and PPE proteins, coded by Rv1818c, Rv3812 and Rv3018c genes were chosen based on immuno-informatics approach. They were cloned, expressed in prokaryotic and mammalian expression vectors and recombinant protein expressing stable cell lines were made. T lymphocytes from DNA immunized mice recognize synthetic peptides from chosen proteins in vitro, indicating that these peptides are being processed and presented by MHC molecules to T cells. By MHC stabilization assay, 5 of the synthetic peptides were found to stabilize the MHC class I molecules on the cell surface for more than 6 hrs, validating the computational prediction. Recognition of T cell epitopes derived from PE/PPE proteins by human PBMCs This work describes the evaluation of immune response against three of PE and PPE proteins of Mycobacterium tuberculosis with Human Peripheral Blood Mononuclear Cells (PBMCs) from PPD positive Healthy donors and TB patients. Proliferation response of PBMCs from ten PPD positive healthy donors as well as from ten TB patients, indicated that the peptides from PE and PPE proteins of Mtb can sensitize naive T cells and induce peptide specific IFN-γ and also the T cell response to the chosen peptides was both HLA class I restricted and CD8 mediated. After the peptide specific expansion, significant percentage of CD8+ T cells were shown to secrete IFN-γ and stained positive for perforin. Antigen specific CD8+ T cells were found to have cytolytic potential in addition to their cytokine function. Immune response to a multiepitope DNA vaccine in mouse model Minigene poly-epitope vaccine constructs coding for nine peptides derived from identified T cell antigens of PE and PPE proteins and three of the experimentally mapped epitopes from M tuberculosis was designed and constructed. The minigene was used to immunize mice and the immune response was tested. The DNA primed splenocytes recognized the full length poly-epitope protein as well as the individual peptides. T cell response to epitopes was enhanced by mere presence in multi-epitope construct compared to full length antigens. Human PBMCs derived from both PPD+ve and TB patients also recognized the peptides in vitro. It is thus obvious that a large cocktail of proteins are required to achieve reasonable population coverage. Besides, this work suggests the feasibility of designing haplotype specific subunit vaccine, which can be given to individuals with known HLA haplotype. The haplotype specific vaccines can be combined to target a population where the distribution of HLA alleles is known. This work also indicates that use of single or limited number of genes in a DNA vaccine may not be suitable to cover a given population.