During the course of infection with chronic pathogens such as Hepatitis C virus (HCV), Hepatitis B virus (HBV) and HIV, virus-specific CD8^+ T cells differentiate into heterogeneous dysfunctional subpopulations. Advances in multi-parameter flow cytometry have allowed these subpopulations to be further classified into classes of exhausted T cells, primarily by their expression of multiple inhibitory receptors. However, the exact phenotype of CD8^+ T cells during exhaustion is an area of great interest as many inhibitory receptors are also expressed on functional CD8^+ T cells. Discovering novel and specific markers of T cell exhaustion is fundamental in developing strategies to restore CD8^+ T cell function in chronic viral infections. Recently, genome wide expression profiles have identified broad molecular phenotypes in exhausted T cells that could not have been discovered by flow cytometry alone. I show how similar genomic approaches identify and further characterise the ectonucleotidase CD39 as a novel marker of CD8^+ T cell exhaustion in chronic viral infection. I show that CD39 is highly expressed in HCV and HIV-specific CD8^+ T cells and that CD39^+ CD8^+ T cells are enriched with gene signatures of exhaustion. CD39 is highly co-expressed with multiple inhibitory receptors including PD-1, enzymatically active on CD8^+ T cells in HCV infection and positively correlated with viral load in both HCV and HIV. I also demonstrate the discovery of a novel CD39^High population of cells in the mouse model of chronic Lymphocytic Choriomenigitis Virus (LCMV) infection, which express the highest degrees of PD-1, LAG3 and 2B4 in the CD39^+ fraction. Thus, CD39 is a novel and specific marker of severe CD8^+ T cell exhaustion in human and animal models of chronic viral infection.

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Hepatitis C virus (HCV) is a major global pathogen estimated to infect over 170 million people worldwide. A recent study has shown that vaccination with adenoviral vectors, based on rare human and simian serotypes encoding the non-structural (NS) proteins of HCV, induces highly potent, multi-specific and durable T cell responses in healthy human volunteers. In this thesis I assess the safety and immunogenicity of these vaccines (ChAd3–NSmut and Ad6-NSmut), for the first time in HCV infected patients. This work also explores whether vaccine-induced T cell responses target in vivo circulating HCV antigens and common naturally occurring epitope variants. Patients with treatment naive chronic genotype 1 HCV infection were vaccinated (i.m.) with ChAd3-NSmut and Ad6-NSmut in a heterologous prime boost schedule, either with or without current IFN and ribavirin (IFN/RBV). Epitope-specific T cell responses were defined by fine mapping using HCV peptides. Circulating viral genomic sequence was determined in vaccinated patients at baseline and at any point of viral relapse. Cross-reactivity of vaccine-induced T cell responses was determined in T cell assays, using peptides corresponding to both circulating host virus and common population HCV epitope variants. An in vitro dendritic cell /T cell priming model was used to identify possible candidates for a cross-reactive vaccine immunogen at the most immunodominant epitope, NS3₁₄₀₆. 33 patients were vaccinated. Vaccination was well tolerated. At the highest vaccine dose (2.5 x 10¹⁰vp) vaccine-induced T cell responses were detectable in 11/20 patients receiving concurrent IFN/RBV and 2/4 patients receiving vaccination alone. In total 14 antigenic targets were identified, 2 of which have not previously been described. However, T cell responses were of lower magnitude and more narrowly focused than those observed in healthy volunteers vaccinated with the same regimen. Analysis of viral sequence showed that in many cases vaccine-induced T cells did not target the circulating virus. At the most immunodominant epitope (NS3₁₄₀₆), T cells induced by vaccination failed to target common circulating genotype 1 HCV variants. An in vitro model suggested that in order to target all genotype 1 sequences at this epitope, it would be necessary to insert both a genotype 1a and 1b version of this epitope into a vaccine immunogen. Vaccination with adenoviral vectors induces T cell responses in patients with chronic HCV infection, however immune responses are attenuated compared with healthy volunteers. Ultimately a successful therapeutic or prophylactic vaccine strategy will rely on inducing responses that target conserved or cross-reactive epitopes.

L'infection chronique par le virus de l'hépatite B (VHB) touche 400 millions de personnes dans le monde et conduit à 1 million de décès par an des suites de complications hépatiques. Les traitements actuels permettent de freiner la progression de la maladie mais ne guérissent les patients que dans de très rares cas (3-5%). Le besoin médical pour de nouvelles thérapies est fort et les approches de type immunothérapie semblent aujourd'hui prometteuses dans cette pathologie où des corrélats immunitaires de résolution sont établis. Le but de cette thèse a été l'étude approfondie de candidats d'immunothérapie basés sur un vecteur adénovirus de sérotype 5 humain non réplicatif codant pour plusieurs antigènes du VHB. Des études in vitro et dans un modèle murin, ont montré la capacité : 1/ des antigènes du VHB codés par certains candidats a formé des VLP (microscopie électronique) 2/ des candidats à induire un recrutement de cellules immunitaires sur le site d'injection après administration par voie sous-cutanée. Des études in vivo utilisant un candidat prototype ont visé à caractériser les réponses immunitaires induites et à étudier l'impact de schémas d'immunisation atypiques, tels que ceux qui pourraient être utilisés en clinique. Elles ont montré la capacité du prototype, qu'il soit injecté une ou de multiples fois, à induire des réponses T CD8+ spécifiques du VHB, fortes, multispécifiques et maintenues dans le temps dans des modèles murins naïfs ou tolérants pour le VHB. Les administrations multiples n'ont pas conduit à une augmentation de la proportion de cellules T spécifiques du VHB exprimant des molécules d'inhibition, de type PD1 / Chronic infection with hepatitis B virus (HBV) affects 400 million people worldwide and leads to 1 million of deaths per year as a result of liver complications. Current treatments can slow the progression of the disease but cure patients in very rare cases (3-5%). The medical need for new therapies is obvious, strong and immunotherapy approaches appear promising in this disease, where immune correlates of resolution are established. The aim of this thesis was a comprehensive study of immunotherapeutic candidates, based on a non-replicating human adenovirus serotype 5 vector, encoding several antigens of HBV. Studies in vitro and in a mouse model, showed the ability of: 1 / HBV antigens encoded by some candidates to form VLPs (electron microscopy) 2 / candidates to induce recruitment of immune cells at the injection site after subcutaneous administration. In vivo studies using a prototype candidate aimed at characterizing the induced immune responses and to study the impact of atypical immunization schedules, which could be clinically used. They demonstrated the ability of the prototype, injected once or multiple times, to induce strong, multispecific and sustained over time HBV specific CD8 + T cells, in naive or tolerant to HBV mouse models. Multiple administrations do not increase the proportion of HBV specific T cells expressing inhibitory molecules, such as PD1

Von den acht bekannten HBV-Genotypen sind die Genotypen A und D in Europa vorherrschend, Genotyp A in Nordwesteuropa, Genotyp D im Mittelmeerraum und in Südosteuropa. Dies bestätigte sich auch in der vorliegenden Studie, bei der von 62 genotypisierten Proben 91,9 % diesen beiden Genotypen zugeordnet werden konnten. Genotyp D war mit 64,5 % (40 Patienten) vorherrschend. Es folgten der Genotyp A (17 Patienten) und der Genotyp C (4 Patienten). In einem Fall wurde Genotyp B nachgewiesen. Deutschland als Herkunftsland war bei Patienten mit Genotyp A signifikant häufiger vertreten als bei Patienten mit Genotyp D. Der relativ hohe Genotyp D-Anteil ist möglicherweise darauf zurückzuführen, dass durch zunehmende Immigration das Auftreten verschiedener Genotypen beispielsweise aus dem südosteuropäischen Raum begünstigt wird. Patienten mit Genotyp A sprechen häufig besser auf IFN-alpha an, so dass eine Therapie mit Nukleosid- bzw. Nukleotidanaloga nicht erforderlich ist. Diese Patienten wurden somit à priori nicht in dieser Studie erfasst, was eine mögliche Erklärung dafür ist, dass der Genotyp A-Anteil mit 27,4 % relativ gering ausfiel. Bei der Untersuchung von statistischen Zusammenhängen zwischen HBV-Genotyp und Patientenalter, Geschlecht, Viruslast und Therapiedauer ergaben sich keine signifikanten Ergebnisse. Diese Studie bietet Basisinformationen zur Genotypverteilung in Deutschland. Bezüglich einer Korrelation zwischen den verschiedenen HBV-Genotypen und demographischen, virologischen sowie klinischen Charakteristika wird es künftig weiterer Studien bedürfen. / Among the eight known HBV-genotypes, genotype A and D dominate in Europe. This study established the prevalence of different HBV genotypes in 62 patients from universityhospital Wuerzburg, Germany. The prevalences of genotypes A and D were 27,4% and 64,5%, for genotypes B and C 1,6% and 6,5%. The results indicated that genotypes A and D are the predominat genotypes in German patients with chronic HBV infection.

Student Number : 9903082V - MSc (Med) dissertation - Faculty of Health Sciences / Chronic infection with the hepatitis B virus (HBV) is endemic to sub-Saharan Africa and south-east Asia where it is a major risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). Currently available therapy is only effective in a small subset of chronic carriers. The development of novel treatment modalities for the management of HBV therefore remains an important global medical objective. Sequence plasticity of the HBV genome is limited by its small size and the overlapping nature of its open reading frames (ORFs). These features make HBV an ideal target for therapy based on nucleic acid hybridization. The use of ribozymes (RNA enzymes) and antisense molecules to inhibit gene expression is well documented. The recent discovery of RNA interference (RNAi) has added to the arsenal of therapy based on nucleic acid hybridization. RNAi is the process whereby short RNA duplexes (called short interfering RNA or siRNA) mediate the sequence-specific post-transcriptional silencing of genes homologous in sequence to the siRNA. siRNA function by guiding a protein complex (RNA Induced Silencing Complex or RISC) to target mRNA for degradation or translational repression. The protein X ORF (HBx ORF) is a conserved region of the HBV genome and is common to all viral transcripts. HBx is required for infection by the virus and plays an important role in the establishment of chronic infections in vivo as well as in the development of HCC. RNAi targeted against the HBx ORF may therefore prove useful as treatment of chronic HBV infection. Plasmid based expression cassettes capable of endogenously generating short hairpin RNA (shRNA) targeted to the HBx ORF were constructed. The shRNA function as substrates for the RNAi machinery and are processed into siRNA. The ability of the expression cassettes to knockdown markers of HBV gene expression was tested in a human hepatoma cell line. A panel of 10 U6 promoter-driven shRNA expression vectors was generated. The U6 promoter (an RNA polymerase III promoter) is normally involved in the transcription of small nuclear RNA and as such is ideal for the generation of shRNA of precisely defined length. Three cytomegalovirus (CMV) promoter-driven shRNA expression cassettes incorporating ribozymes that produce defined hairpin sequences were also generated. The CMV promoter (an RNA polymerase II) promoter is involved in the transcription of large messenger RNA. Two hammerhead ribozymes lying 5’ and 3’ of the shRNA encoding sequence were incorporated into the cassette. Cis-cleavage by the ribozymes releases a shRNA of defined length thereby overcoming the limitations imposed by extraneous sequences from CMV promoter-driven transcription. U6 promoter-driven shRNA expression vectors efficiently knocked down markers of HBV replication in liver cells. The CMV promoter-driven expression vectors were incapable of inhibiting HBV gene expression; however shRNA generated in vitro from these vectors mediated efficient knockdown of HBV replication. shRNA-mediated inhibition of gene expression therefore holds promise as a novel treatment strategy for the management of HBV and other mobile genetic elements.

Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the availability of anti-viral agents, there is currently no cure. This double stranded DNA virus is hepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA contains a partially synthesised positive DNA stand and a gap region within the negative strand. After infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An important objective of HBV therapy is the elimination of cccDNA, as its persistence within hepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this replication intermediate is crucial. The objective of this study was to develop a method based on real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap were designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV DNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV transgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse livers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA profile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells, only 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results were validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA pools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA detection as it allows for the rapid simultaneous amplification and quantification of a specific DNA target through either non-specific or specific DNA detection chemistries. In conclusion, this HBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy

Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery

Orientador: Maria Inês de Moura Campos Pardini / Coorientador: Rejane Maria Tommasini Grotto / Banca: Fernando Gomes Romeiro / Banca: João Pessoa Araujo Junior / Resumo: Além de fatores virais e do hospedeiro a progressão da fibrose hepática resultante da infecção pelo Vírus da Hepatite C (VHC) tem sido relacionada a polimorfismos genéticos do hospedeiro. Nesta linha, recentemente polimorfismos dos Antígenos Plaquetários Humanos (HPA) foram associados à progressão para fibrose em pacientes monoinfectados pelo VHC. Alguns destes antígenos HPA residem em proteínas da família das integrinas, cuja expressão, também, já foi associada à progressão da fibrose hepática. No entanto, estudos relacionando polimorfismos genéticos do hospedeiro em pacientes coinfectados com o VHC e o Vírus da Imunodeficiência Humana (HIV) são raros e, não há nenhum estudo relacionando polimorfismos HPA com progressão para fibrose. Assim, o objetivo deste estudo foi avaliar possíveis associações dos polimorfismos dos sistemas HPA-1, -3 e -5, que residem em integrinas, na progressão da fibrose hepática em indivíduos coinfectados VHC/HIV. DNA Genômico de 56 pacientes coinfectados VHC/HIV foi utilizado como fonte para genotipagem dos sistemas HPA -1 e -3 por PCR-SSP, e HPA -5 por PCR-RFLP. Progressão da fibrose foi avaliada utilizando o escore de METAVIR, sendo constituídos dois grupos: Grupo 1 (G1): pacientes coinfectados VHC/HIV com baixo grau de fibrose (F1, fibrose portal sem septos ou F2, com poucos septos) e Grupo 2 (G2): pacientes coinfectados VHC/HIV com fibrose avançada (F3, numerosos septos ou F4, cirrose). Um grupo controle, do estudo de Silva e colaboradores (2012), constituído por pacientes monoinfectados pelo VHC com baixo grau de fibrose (F1 ou F2) e fibrose avançada (F3 ou F4) foi utilizado para as análises realizadas neste estudo. O Teste Exato de Fisher foi utilizado para avaliar possíveis associações entre o polimorfismo dos sistemas HPA -1, -3 e -5 e a progressão para fibrose, utilizando um nível de significância de 5%. Não houve desvio do equilíbrio ... / Abstract: To evaluate the associations of Human Platelet Antigen (HPA) polymorphisms -1, -3 and -5 with HIV/HCV coinfection. In this study were included 60 HIV/HCV-coinfected patients from the Sao Paulo State health service centers. Data reported by Verdichio-Moraes et al (2009) were used as the non-infected and HCV monoinfected groups to evaluate the association of HPA -1, -3 and -5 in HIV/VHC coinfected patients. HPA genotyping was performed in 60 HIV/HCV coinfected patients by PCR-SSP or PCR-RFLP. HIV subtyping and HCV genotyping was performed by RT-PCR followed sequencing. The data analyses were performed using the c2 test or Fisher's Exact Test and the logistic regression model. HIV/HCV coinfected patients presented HCV either genotype 1 (78.3%) or non-1 (21.7%) and HIV either subtype B (85.0%) or non-B (15%). The HPA-1a/1b genotype was more frequent (p<0.05) in HIV/HCV coinfection than in HCV monoinfection and the allelic frequency of HPA-5b in the HIV/HCV coinfected patients was lower (P<0.05) than in HCV monoinfected cases and non-infected individuals. These data suggest that HIV presence may have influenced the interaction of HCV with platelets. On the other hand, HPA-5a/5b was more frequent (p<0.05) in HIV/HCV coinfected and HCV monoinfected groups than in the non-infected individuals, suggesting that this platelet genotype is related to HCV infection, regardless of HIV presence. Results suggest that the HPA profile in HIV/HCV coinfected individuals differs from the one of both HCV monoinfected and non-infected population. So, the HPA polymorphism can be a genetic marker associated with HIV/HCV coinfection / Mestre