Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [93]-113).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 64-80).

La Glomérulonéphrite extra-membraneuse est une maladie auto-immune rare mais grave qui conduit dans 30% des cas à une insuffisance rénale chronique terminale nécessitant le recours à la dialyse ou la greffe rénale. Dans les suites d’une greffe, la GEM récidive dans 30 à 40% des cas. En 2009, l’équipe du Pr. Salant en collaboration avec notre équipe a montré que 70% des patients présentant une GEM étaient porteurs d’anticorps (Ac) dirigés contre le récepteur des phospholipases A2 (PLA2R1). Le titre d’anticorps est corrélé à l’activité de la maladie. Il n’existe actuellement aucun biomarqueur permettant de prédire l’évolution de la fonction rénale d’un patient lors de sa prise en charge : dans 30% des cas les patients présentent une rémission spontanée sans traitement immunosuppresseur. Le traitement de la GEM repose sur un traitement symptomatique et une réévaluation après 6 mois. En cas de maladie active persistante, il faut débuter un traitement immunosuppresseur. Dans les formes graves, cette période d’observation de 6 mois peut être à l’origine de lésions irréversibles. Nous avons validé un test ELISA permettant de quantifier les Ac anti-PLA2R1 au cours du suivi de patients porteurs d’une GEM. Ce test nous a permis de montrer sur une cohorte de 15 patients greffés dans les suites d’une GEM qu’un titre d’Ac anti-PLA2R1 persistant après la greffe était associé à un risque de récidive de la maladie sur le greffon. Nous avons ensuite produit dans des cellules HEK les orthologues de PLA2R1 (les récepteurs humain, lapin et murin). / Membranous Nephropathy (MN) is a major cause of nephrotic syndrome in adults. It is a rare but severe kidney disease with different etiologies and outcomes. In most cases (85%), the disease is idiopathic (iMN) and has an autoimmune origin. One third of patients develop end-stage kidney disease and are on kidney transplant waiting list. MN recurred in 30% after transplantation. Another third enter in spontaneous remission under renin-angiotensin system blockade. The treatment of iMN is controversial. KDIGO guidelines recommend a supportive symptomatic treatment with RAS-blockade and diuretics in all patients with iMN, and immunosuppressive therapy in case of renal function deterioration or persistent nephrotic syndrome. Therefore, immunosuppressive treatments are often started only after significant and potentially irreversible complications. No biological markers can predict clinical outcome and orient therapy. A major breakthrough was the discovery of autoantibodies to the phospholipase A2 receptor (PLA2R1, 180 kDa) in 70% of iMN patients in 2009, which has now allowed to develop diagnosis and prognosis tests for better medical care. During my PhD, I have first participated to the development of an ELISA which is now commercially available. I then used this latter to demonstrate that persistent anti-PLA2R1 activity can predict iMN recurrence after transplantation in a retrospective cohort of 15 patients. We then screened 50 patients with iMN on native kidney for their cross-reactivity to human (h), rabbit (rb) and mouse (m) PLA2R1 by western blot (WB) and antigen-specific ELISAs.

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-03Bitstream added on 2014-06-13T20:29:34Z : No. of bitstreams: 1 pinto_jras_me_rcla.pdf: 1057969 bytes, checksum: 2e29026d533c6190983ed253a062779d (MD5) / Os venenos dos insetos da ordem Hymenoptera contêm uma variedade de proteínas alergênicas. As ferroadas desses insetos podem induzir reações alérgicas e ocasionalmente, anafilaxias fatais. Há um crescente interesse nos componentes químicos dos venenos desses insetos, sobretudo no campo da alergia e imunologia clínica. As principais reações desses venenos são as reações inflamatórias e/ou imunológicas em suas vítimas, podendo ocorrer alguns efeitos sistêmicos. Dentre os Hymenoptera sociais, os venenos de abelhas e vespas têm sido extensivamente estudados e muitos de seus componentes moleculares já foram isolados e identificados. Fosfolipase, hialuronidase, antígeno-5 e serinoprotease são proteínas antigênicas de elevada massa molecular que, quando injetadas durante o ato de ferroar, iniciam uma resposta imune peculiar, sensibilizando alguns indivíduos. Porém, poucos estudos têm sido realizados para a identificação e o mapeamento dos epítopos desses alérgenos. O conhecimento sobre a interação molecular dos principais alérgenos destes venenos certamente deverá contribuir para o aperfeiçoamento dos diagnósticos de alergia, bem como para o desenvolvimento de tratamentos mais seletivos de reações alérgicas mediadas por IgE. No presente estudo identificamos e mapeamos os epítopos lineares de células-B do antígeno-5 do veneno da vespa social Polybia paulista. O antígeno-5 é conhecido como um dos principais alérgenos do veneno de Hymenoptera com massa molecular em torno de 23 kDa e função biológica desconhecida, porém muitos estudos demonstram a sua alergenicidade. Tendo conhecimento da sequência primária do antígeno-5, a identificação e o mapeamento dos epítopos foram realizados através da síntese múltipla de peptídeos, utilizando as técnicas do SPOT-Synthesis, imunodetecção e método indireto do ELISA com soro de pacientes sensíveis... / The venom insects of Hymenoptera order contain a variety of allergenic proteins. The stings of these insects can induce allergic reactions and occasionally fatal anaphylaxis. There is a growing interest in the knowledge about the chemical components of the venom of these insects, especially in the field of allergy and clinical immunology. The main reactions of these venoms are inflammation and / or the induction of immune process in the victims; sometimes systemic effects also may be observed. Among the social Hymenoptera, the venoms of bees and wasps have been extensively studied and many of their molecular components have been isolated and identified. Phospholipase, hyaluronidase, antigen-5 and serine protease are antigenic proteins of high molecular weight, which may initiate a peculiar immune response to sensitize some individuals. However, few studies have been performed for the identification and mapping of the epitopes these allergens. The knowledge about the molecular interaction of the major allergens of these venoms with IgG and/or IgE will certainly contribute for improving the diagnosis of allergy, as well as to develop more selective treatments of reactions IgE-mediated allergy. In this study we identified and mapped the linear epitopes of B-cells of the antigen-5 from the venom of the social wasp Polybia paulista. The antigen-5 is known as one of the major allergens from Hymenoptera venoms with molecular weight around 23 kDa and unknown biological function, but many studies show its allergenicity. The aim of this study was to identify and to map the linear epitopes of B-cells of this allergen. Taking into account the knowledge the primary sequence of antigen-5, the identification and mapping of epitopes was performed by using multiple synthesis of peptides with the combination of different protocols: SPOT-Synthesis, immunoblotting and indirect method of ELISA with the sera... (Complete abstract click electronic access below)

Toxoplasma gondii is an obligate intracellular protozoan that belongs to the Apicomplexa phylum and causes toxoplasmosis which is especially severe in immunocompromised individuals and who acquired the infection by congenital transmission. Dense granules are organelles involved in this parasite invasion and replication of the parasite inside the cell. Among the proteins secreted by dense granules, jointly called GRAs, the GRA 2 is particularly immunogenic. In the present work, we aim to study the role of this protein in inducing adaptive immune response by produced and characterized a monoclonal antibody against GRA2. The parasite treated with this antibody showed the lowest rate of replication in intracellular proliferation assay compared to treatment with irrelevant antibody. The epitopes of this protein that binds to the monoclonal antibody have been mapped by phage display. Through bioinformatics analysis, epitopes for B cells and T GRA2 were predicted and based on that and on the results of phage display amino acid sequences were chosen to synthesize two peptides that were tested as potential vaccine. Immunization tests were carried out in C57BL/6 mice with synthetic peptides and these animals were subsequently infected with T. gondii. There was a significant reduction in mortality in the group immunized with the mixture of both peptides. The negative control group, immunized only with BSA, showed the highest mortality and analysis of cytokine present in the sera of these animals there was a predominance of proinflammatory cytokines while antiinflammatory cytokines were shown to be almost nonexistent in the serum of these animals. Given these results, it was found that the group with higher survival showed better Th1/Th2 balance. This demonstrates that immunization with epitopes recognized by both B cells and T cells appear to be more effective. Thus, peptide mimetics GRA2 regions of the protein may be likely candidates for the development of vaccines against toxoplasmosis. / Toxoplasma gondii é um protozoário do filo Apicomplexa intracelular obrigatório causador da toxoplasmose, que é especialmente grave em indivíduos imunocomprometidos e em indivíduos que adquiriram a infecção por transmissão congênita. Grânulos densos são organelas deste parasito envolvidas no processo de invasão e replicação do parasito no interior da célula. Dentre as proteínas secretadas pelos grânulos densos, chamadas conjuntamente de GRAs, a GRA 2 é particularmente imunogênica. No presente trabalho, no sentido de estudar a participação dessa proteína na indução da resposta imune adaptativa, foi produzido e caracterizado um anticorpo monoclonal contra GRA2. O parasito tratado com esse anticorpo apresentou menor taxa de replicação em ensaio de proliferação intracelular comparado ao tratamento com anticorpo irrelevante. Os epítopos de ligação da proteína GRA2 ao anticorpo monoclonal foram mapeados por phage display. Por meio de análises de bioinformática, epítopos para células B e T da GRA2 foram preditos e com base nisso e nos resultados do phage display foram escolhidas seqüências de aminoácidos para sintetizar dois peptídeos que foram testados quanto ao potencial vacinal. Foram feitos ensaios de imunização em camundongos C57BL/6 com estes peptídeos sintéticos e os animais foram posteriormente infectados com T. gondii. Foi observada redução significativa na mortalidade no grupo imunizado com a mistura de ambos os peptídeos. O grupo controle negativo, imunizado somente com BSA, apresentou a maior mortalidade e nas análises de produção de citocinas presentes nos soros destes animais observou-se uma predominância de citocinas pró-inflamatórias enquanto as citocinas anti-inflamatórias mostraram-se quase inexistentes no soro destes animais. Tendo em vista esses resultados, percebeu-se que o grupo com maior sobrevivência apresentou melhor balanço Th1/Th2. Isso demonstra que a imunização com epítopos reconhecidos tanto por células B quanto por células T parece ser mais efetiva. Assim, peptídeos miméticos de regiões da proteína GRA2 podem ser prováveis candidatos no desenvolvimento de vacinas anti Toxoplasmose. / Doutor em Imunologia e Parasitologia Aplicadas

S. pneumoniae pode causar infecções do trato respiratório. Uma alternativa às vacinas conjugadas, que conferem proteção sorotipo-específica, seria uma vacina baseada em epitopos de diferentes variantes de antígenos como PspA e PspC. O objetivo deste trabalho foi a identificação de epitopos de variantes de PspA e PspC, utilizando a técnica peptide array, visando a síntese de um antígeno composto por múltiplos epitopos. Os resultados obtidos indicaram que anticorpos contra epitopos lineares de PspA reconhecem as regiões inicial N-terminal e rica em prolinas, mas estes epitopos parecem ser apenas marcadores de exposição ao pneumococo e epitopos conformacionais seriam os de fato protetores, como observado nos experimentos realizados com os Frags de 100 aminoácidos construídos a partir do PspARx1, especialmente aqueles presentes nas regiões dos Frags 2 e 4. Epitopos lineares de PspC parecem ser importantes na região de ligação à sIgA e FH. Assim, uma vacina proteica de ampla cobertura, poderia conter as regiões do Frag 2 de PspA e de ligação de sIgA e FH de PspC. / S. pneumoniae can cause respiratory tract infections. An alternative to the conjugate vaccines, which confer serotype-specific protection, would be a vaccine based on epitopes of variants of antigens such as PspA and PspC. The objective of this study was to identify epitopes of variants of PspA and PspC, using the peptide array technique, aiming at the synthesis of a multi-epitope antigen. The results obtained with peptide arrays indicated that antibodies against linear epitopes of PspA recognize the initial N-terminal and proline-rich regions, but these epitopes seem to be only markers of exposure to pneumococcus and conformational epitopes would be in fact protective, as observed in the experiments with fragments of 100 amino acids constructed from PspARx1, especially those present in Frags 2 and 4. Linear epitopes of PspC seem to be important in the regions of sIgA and FH binding. Thus, a protein-based vaccine with broad coverage could contain the regions of Frag 2 of PspA and the regions of sIgA and FH binding of PspC.

A origem das doenças autoimunes é multifatorial, sendo que envolve condições ambientais e predisposição genética, dificultando sua identificação. Muitos pesquisadores têm estudado a associação entre agentes infecciosos e autoimunidade, a qual pode ser disparada pelo processo conhecido por mimetismo molecular. Neste caso, respostas imunes cruzadas envolvendo antígenos próprios têm sido documentadas. O presente projeto tem como objetivo a busca in silico por associações entre epítopos de microrganismos e autoantígenos humanos. Iniciaram-se as análises pela identificação de semelhanças de sequências de aminoácidos entre epítopos de microrganismos e autoantígenos humanos por meio do alinhamento local de sequências efetuado pelo programa BLASTP. As sequências de epítopos dos microrganismos e autoantígenos humanos foram previamente adquiridas nos bancos de dados Immune Epitope Database and Analysis Resource (IEDB) e no Genbank, respectivamente. Foram também realizadas modelagens de estruturas proteicas para o antígeno e o autoantígeno que obtiveram melhores valores de alinhamento, com base no valor do E-value, por meio dos programas Modeller e Rosetta. Por fim, a predição de epítopos foi executada, pelo uso dos softwares NetMHC e NetMHCII, para avaliar a possibilidade de epítopos de microrganismos e de autoantígenos humanos se associarem aos mesmos alelos de HLA. Como resultado, foram encontradas similaridades tanto de sequências proteicas quanto de afinidade a 4 tipos de alelos de HLA entre um epítopo do antígeno LSA-1 de Plasmodium falciparum e o autoantígeno de miosina, o que sugere uma associação entre eles, atingindo o objetivo deste trabalho. / The origin of autoimmune diseases is multifactorial. It involves environmental conditions and genetic predisposition that difficulties its identification. Several researchers have studied the association between infectious agents and autoimmunity, which can be initiated by a process named molecular mimicry. In this case, cross immune responses involving self antigens have been documented. This project aims to search in silico for associations between microorganisms epitopes and human autoantigens. The first step was the identification of similarities in amino acid sequences between microorganisms epitopes and human autoantigens by use of sequence local alignment performed by the program blastp. The sequences of the microorganisms epitope and the human autoantigens had been previously acquired in the Immune Epitope Database and Analysis Resource (IEDB) and Genbank, respectively. The modeling of protein structures for the antigen and autoantigen was also carried out to show the best alignment values, based on the E-value, using the programs Modeller and Rosetta. Finally, the prediction of epitopes was performed by use of NetMHC and NetMHCII softwares to evaluate the possibility of microorganisms epitopes and human autoantigens join the same HLA alleles. Similarities of protein sequences was found for both. It was possible to observe affinity of 4 HLA alleles between an epitope from LSA-1 Plasmodium falciparum antigen and the myosin, suggesting an association between them, reaching the goal of this work.

Distinct cellular level of the Ca2+ binding chaperone calreticulin (CRT) is essential for cardiac development and postnatal function. However, CRT is also a potential autoantigen eliciting formation of antibodies (Ab), whose role is not yet clarified. Immunization with CRT leads to cardiac injury, and overexpression of CRT in cardiomyocytes induces dilated cardiomyopathy (DCM) in experimental animals. Hence, we analysed levels of anti-CRT Ab and calreticulin in the sera of patients with idiopatic DCM and hypertrophic cardiomyopathy (HCM). ELISA and immunoblot using human recombinant CRT and Pepscan with synthetic, overlapping decapeptides of CRT were used to detect anti-CRT Ab. Significantly increased levels of anti-CRT Ab of IgA (P<0.001) and IgG (P<0.05) isotypes were found in patients with both DCM (12/34 seropositive for IgA, 7/34 for IgG) and HCM (13/38 seropositive for IgA, 11/38 for IgG) when compared with controls (2/79 for IgA, 1/79 for IgG). Titration analysis in seropositive DCM and HCM patients documented anti-CRT Ab detected at 1/1600 dilution for IgG and 1/800 for IgA (and IgA1) and at least at 1/200 dilution for IgA2, IgG1, IgG2 and IgG3. Pepscan identified several immunogenic CRT epitopes: EVKIDNSQVESGSLED, IDDPTDSKPE, DKAPEHIPDPDA and RKEEEEAEDKEDDAEDKDEDEEDE recognised by IgA and...

As taxas de resposta viral ao tratamento com Interferon- associado com Ribavirina ainda não estão bem definidas. Muitos estudos associam vários fatores virais e do hospedeiro, tais como a idade, sexo, peso, etnia, nível de enzimas hepáticas, estágio de fibrose, genótipo do HCV com os níveis de RNA viral. Outros estudos associam as células CD8+ específicas para epítopos do HCV com o controle da viremia. O objetivo desse trabalho foi verificar se há aumento na pressão seletiva contra o vírus nos pacientes em tratamento com Interferon- associado à Ribavirina respondedores em comparação com os pacientes não respondedores e associar esse aumento com a ação antiviral do tratamento e/ou com o aumento da resposta imune devido à ação imunomoduladora do Interferon-, utilizando-se modelos de máxima verossimilhança, de cálculo da razão dN/dS e realizando o mapeamento dos epítopos das proteínas NS5A. Nossos resultados demonstram que usando modelos par a par não foi detectado evidência de seleção positiva, entretanto utilizando modelos de máxima verossimilhança nós observamos evidência no grupo que não responde a terapia. O mapeamento dos epítopos revelou que o epítopo VLSDFKTWL estava associado com a resposta efetiva do tratamento com suporte estatístico. Estes resultados indicam que a resposta imunológica aumenta durante o período de tratamento, auxiliando na eliminação do vírus. Além das análises, foi desenvolvida uma ferramenta de mapeamento automático dos epítopos do HCV e análise de seleção natural aplicando modelos de máxima verossimilhança / The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Several studies suggest that viral load is closely associated to viral and host factors, such age, sex, body weight, transaminase levels and HCV genotype. Moreover, an strong CD8+ T-cell immunity in acute resolving hepatitis C is matched by strong and sustained CD4+ T-cell proliferation to multiple recombinant structural and non-structural viral proteins is responsible for the control of viraemia in HCV infection. Here, we investigate the differences in CD8 epitopes frequencies from Los Alamos database between groups of patients that showed distinct response to pegylated alpha interpheron with ribavirin therapy, and test for evidence of natural selection on virus in treatment failure group, using five maximum likelihood evolutionary models. Our results indicated no evidence of positive selection by using pairwise models, however we identify evidence of positive selection in non responder group by applying maximum likelihood models. The epitope mapping showed that the epitope VLSDFKTWL was associated with efective therapy outcome, with statistical support. Those results suggest that the immune response increase during the treatment period, allowing the virus clearance. We also developed a tool for automatic mapping of epitopes of HCV that tests for evidence of natural selection by applying maximum likelihood models

Le développement d'un traitement curatif du virus de l’immunodéficience humaine (VIH) est devenu le défi majeur pour le futur mais les réservoirs et une réponse immunitaire insuffisamment efficace constituent des barrières pour l’élimination du virus. La réponse immune contre l’infection VIH dépend en partie de la capacité des cellules hôtes à présenter correctement les épitopes viraux pour induire une réponse spécifique des lymphocytes T CD8+ cytotoxiques (CTL). Cette présentation des épitopes viraux qui pourrait être optimisée par vaccination, dépend du système HLA (Human Leukocyte Antigen) du patient présentant un déterminisme génétique individuel. Correctement pré-stimulés, les CD8 cibleraient et détruiraient efficacement les cellules productrices du virus. Les précédents essais vaccinaux stimulant la réponse CTL n’ont pas montré d’efficacité dans la réponse virologique, probablement parce qu’ils sont composés d’épitopes "génériques" et qu’ils ne prennent pas en compte la variabilité du VIH ni l’immunogénétique des patients.L’objectif de ce travail est d’identifier des épitopes archivés dans l’ADN proviral, susceptibles d’induire une réponse CTL en considérant la variabilité du VIH-1 et la variabilité immunogénétique des patients infectés par le VIH-1 en succès thérapeutique. L’idée, à terme, est d’utiliser les peptides correspondants comme base de vaccin thérapeutique et de permettre au système immunitaire de prendre le relai du traitement antirétroviral pour contrôler l’infection virale.Cent quarante patients infectés par le VIH-1, suivis au CHU de Bordeaux en succès thérapeutique depuis plus de 6 mois ont été inclus dans le projet Provir/Latitude 45 entre 2012 et 2017. Une cartographie de la répartition des sous-types viraux du VIH-1 en Aquitaine a d’abord été réalisée. L’analyse de plus de 3200 génotypes de résistance VIH-1 effectués entre 2012 et 2016 a permis de déterminer que le sous-type viral majoritaire infectant les patients vivants avec le VIH dans la région est le sous-type B, suivi du CRF02_AG qui est majoritaire parmi les sous-types viraux non B. Si l’on se focalise sur les patients inclus dans ce projet on retrouve des répartitions similaires. Suite à cette première analyse, un nouveau virus recombinant composé de CRF06_cpx et de sous-type B a pu être identifié. Il est référencé en tant que CRF98_cpx. Un des patients inclus au sein du projet est d’ailleurs infecté par ce virus. Afin d’identifier des épitopes candidats pouvant servir de base à un vaccin thérapeutique pour l’ensemble de la population ou pour un groupe de la population en fonction de leurs caractéristiques immunogénétiques, nous avons combiné les données des séquences virales avec le typage HLA des patients afin de prédire in silico l’affinité entre un HLA et un peptide via les algorithmes d’IEDB. Pour automatiser l’analyse des données, un logiciel, TutuGenetics, a été développé. Ce logiciel instrumentalise les algorithmes d’IEDB et permet d’étudier la variabilité de la présentation des épitopes par les différents HLA du patient grâce à un score MHC IC50 déterminé pour chaque couple HLA-séquence virale. Ce logiciel a été validé en comparant l’analyse des données issues du séquençage Next Generation Sequencing avec le séquençage Sanger. Finalement, pour les 140 patients inclus dans le projet Provir, TutuGenetics a effectué un découpage des séquences virales par pas de 8 à 10 acides aminés et les valeurs MHC IC50 ont été définies pour toutes les combinaisons HLA-épitope. Une analyse plus fine nous a ensuite permis de déterminer une liste de 15 épitopes avec une forte affinité in silico pour les HLA majoritaires finalement retenue pour une cocktail vaccinal.Ces données in silico vont dans une prochaine phase être confirmées in vitro via des tests d’immunologie fonctionnelle, puis in vivo chez le macaque. / HIV (Human Immunodeficiency Virus) cure is the major challenge of the future but latent reservoir and inefficient immune response do not allow virus elimination. The immune response against HIV infection depends on host cells ability to correctly present viral epitopes to induce specific cytotoxic CD8+ T lymphocytes (CTL) response. This presentation of viral epitopes which could be improved by vaccination depends on the HLA (Human Leukocyte Antigen) system of the patient which is extremely variable. Accurately pre-stimulated, CTL would target and destroy efficiently virus-producing cells. Previous vaccine trials stimulating CTL response haven’t shown efficient virological response, presumably because epitopes used are generic without taking into account HIV-1 or patient’ immunogenetic variability.The aim of this work is to identify epitopes archived in the proviral DNA, considered to induce CTL response according to the HIV-1 and immunogenetic variability of the patients at therapeutic success. The goal is to use these peptides for a therapeutic vaccine and educate the immune system to control the viral replication without any antiretroviral treatment.One hundred and forty patients infected with HIV-1, followed at the University Hospital of Bordeaux, at therapeutic success for more than 6 months have been included in the Provir/Latitude 45 project between 2012 and 2017. A mapping of the distribution of viral subtypes of HIV-1 in Aquitaine was first performed. Analysis of more than 3200 HIV-1 genotypes conducted from 2012 to 2016 determined that the major viral subtype infecting patients living with HIV in the region is subtype B, followed by CRF02_AG which is predominant among the non-B viral subtypes. Focusing on the patients included in this project led us to find similar distributions. Following this initial analysis, a new recombinant virus composed of CRF06_cpx and subtype B could be identified. It is now referenced as CRF98_cpx. One of the patients included in the project is infected with this virus. In order to identify candidate epitopes that can serve as a therapeutic vaccine for the entire population or for a population group based on their immunogenetic characteristics, we combined the viral sequence data with the HLA typing of patients to predict the affinity in silico between an HLA and a peptide via IEDB algorithms. To automate data analysis, a software package, TutuGenetics, has been developed. This software exploits IEDB algorithms and makes it possible to study the variability of the presentation of epitopes by the different HLAs of the patient thanks to an MHC IC50 score determined for each HLA-viral sequence pair. This software has been validated by comparing the analysis of data from Next Generation Sequencing (NGS) with Sanger sequencing. Finally, for 140 patients included in the Provir project, TutuGenetics sliced the viral sequences in steps of 8 to 10 amino acids and MHC IC50 values were defined for all HLA-epitope combinations. A finer analysis allowed us to determine a list of 15 epitopes with high in silico affinity for major HLAs. These epitopes were selected by applying different filters and only HLA-peptide couples with more than 10 patients sequenced per position and by HLA were kept in this "Optimal_Provir" list.These in silico data will in a next phase be confirmed in vitro via functional immunology tests, then in vivo in macaques.