Sofosbuvir is a uridine based nucleotide inhibitor of the hepatitis C viral (HCV) polymerase that is the backbone of many treatment regimens. In combination with drugs targeting other viral enzymes (including the poorly potent guanosine analogue ribavirin or highly potent inhibitors of viral NS5A or protease) most patients clear virus and resistance to sofosbuvir is rare, allowing effective retreatment with sofosbuvir. Patients with Genotype 3 HCV respond less well than other genotypes and response is reduced in those previously exposed to interferon. Here we show that patientderived virus from patients with Genotype 3 HCV who relapse to sofosbuvir-based therapies have a reduced sensitivity to SOF in an in-vitro phenotyping assay. Analysis of viral sequencing data revealed two distinct polymorphisms (A150V and K206E) in the HCV polymerase that are associated with treatment failure and in-vitro; they reduce sofosbuvir sensitivity against genotype 3 hepatitis C virions. However both polymorphisms modify the cellular response to type I interferon and in cells lacking response to interferon the impact on sofosbuvir sensitivity is minimal. The A150V polymorphism reduces the response to interferon 70 fold whereas the K206E substitution has minimal effects on interferon in isolation but in combination with A150V reduces the response 100 fold. Preliminary data indicates that the A150V polymorphism interferes with the late response to type I interferons enabling the virus to overcome the induction of interferon-stimulated genes. These data indicate a complex interaction between direct acting antiviral drugs and the innate antiviral response.

Hepatitis C virus (HCV) is a major global pathogen that infects an estimated 170 million people worldwide, and for which currently no vaccine is available. HCV is a highly diverse viral pathogen and exists as 6 major genotypes sharing only 75% sequence homology; developing a vaccine that is cross-reactive between genotypes is a major challenge. Defining immune responses that target different HCV genotypes will facilitate pan-genotypic T cell vaccine development. HCV genotype 3 (gt3) is now the most common infecting genotype in the United Kingdom and large parts of Asia; however, data regarding the T cell antigenic targets of this genotype is very limited. In this thesis, HCV gt3 specific T cell targets were defined in acute, chronic and spontaneously resolved infection: in chronic gt3 infection, T cell responses were low in magnitude and narrowly focused in specificity, similar to those previously reported for gt1; in contrast, resolved infection was associated with a higher magnitude and broader specificity of CD4+ and CD8+ T cell responses across the genome. Overall, T cell specificity in gt3 infection was markedly different to that previously described for gt1, confirming that sequence differences between genotypes result in distinct immunological profiles. Previous work from our laboratory demonstrated that, though T cell responses induced by a potent T cell vaccine containing HCV gt1b non-structural regions do target epitopes dominant in natural infection, induced T cells show limited cross-reactivity against other genotypes. In this thesis, it was assessed whether T cells primed in natural gt3 infection are able to recognize viral sequence variants at dominant epitopes, which would make these potential targets in cross-reactive vaccine design. For seven gt3-specific T cell epitopes identified here as dominant, major sequence variability was observed within and between genotypes, and limited T cell cross-reactivity observed against identified viral variants. This suggests that regions frequently targeted in natural infection may not serve as attractive targets for cross-reactive vaccine design. These results informed the subsequent design of a cross-reactive vaccine based on fragments of HCV that are conserved between genotypes. A generic algorithm was developed to define viral regions conserved between major HCV genotypes (for 1a/1b, 1/3a, 1-6), and these were joined to form immunogens between 819 and 1543 AA long. Possible artificial, non-HCV epitopes formed by junctions were identified using online epitope prediction servers, and abrogated through the insertion of 2-6 amino acid linkers. To address the concern that conserved regions may not be immunogenic, epitopes described in natural HCV infection were mapped on HCV sequences, showing that conserved segments are well populated with epitopes; additionally, strong binding peptides were predicted for conserved segments using online epitope prediction programs, suggesting potential in vivo immunogenicity. In conclusion, HCV T cell specificity is distinct between genotypes, with limited T cell cross-reactivity between viral variants. Leading from this result, vaccine immunogens were designed entirely based on conserved viral regions. This work paves the way for future studies of novel HCV immunogens based on conserved viral segments between genotypes.

Submitted by ANA CLAUDIA SILVA BRAGA null (anabragga@gmail.com) on 2017-08-31T16:15:53Z No. of bitstreams: 1 Tese Doutorado Ana Claudia Silva Braga.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-01T14:16:37Z (GMT) No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Made available in DSpace on 2017-09-01T14:16:37Z (GMT). No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) Previous issue date: 2017-08-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O vírus da hepatite C (HCV) causa a doença da Hepatite C e estima-se que cerca de 3% da população mundial esteja infectada com o vírus. A infecção por HCV promove a alteração na expressão de várias proteínas celulares. Estudos têm demonstrado que muitas proteínas de choque térmico (HSPs) possuem um perfil de expressão alterado na presença do vírus e algumas HSPs interagem diretamente com proteínas do HCV. Assim, o presente estudo teve como objetivo avaliar in vitro os níveis de expressão de proteínas de choque térmico na presença e ausência de HCV. Com este propósito, células de hepatoma humano Huh7.5 e células Huh7.5 infectadas com o vírus (HCV JFH-1) foram submetidas à extração de RNA e síntese de cDNA. A expressão diferencial de 84 HSPs e chaperonas foi avaliada por qPCR Array. Os resultados demonstram que cinco genes apresentaram expressão aumentada (em Log2 2), enquanto outros cinco apresentaram expressão reduzida. Para validar estes resultados os 10 genes diferencialmente expressos foram testados por qPCR em três modelos celulares para o HCV: células contendo replicon subgenômico do HCV (SGR-JFH-1), células infectadas com JFH-1 (ambos do genótipo 2a) e células contendo o replicon subgenômico S52 (genótipo 3). O gene HSPB8 mostrou expressão aumentada nos três modelos testados, condizente com os resultados obtidos por qPCR Array. Em seguida, promovemos o silenciamento de HSPB8 e foi observado um aumento na replicação viral. Em contraste, quando aumentamos a expressão de HSPB8, o HCV teve uma diminuição na taxa de replicação. O mesmo procedimento foi adotado para o gene DNAJC5B, validado no modelo viral genótipo 3, e o HCV mostrou padrão de replicação semelhante ao observado para o gene anterior. Esses resultados sugerem que HSPB8 pode atuar como um fator intracelular contra a replicação do vírus da hepatite C e DNAJC5B apresenta a mesma função, mas específico para o genótipo 3. Também avaliamos interações diretas com proteínas do HCV e os resultados demonstraram uma interação física entre a proteína NS4B de HCV e HSPB8. Esses resultados podem contribuir para uma melhor compreensão dos mecanismos envolvidos na replicação do HCV. / Hepatitis C virus (HCV) causes Hepatitis C disease and it is estimated that about 3% of world population are infected with the virus. HCV infection promotes alteration in the expression of several cellular proteins. Studies have shown that many heat shock proteins (HSPs) have an altered expression profile in the presence of the virus and some HSPs interact directly with HCV proteins. Thus, the present study aimed to evaluate in vitro the expression levels of heat shock proteins in the presence and absence of HCV. With this purpose, human hepatoma Huh7.5 cells and Huh7.5 cells infected with the virus (HCV JFH-1) were subjected to RNA extraction and cDNA synthesis. The differential expression of 84 HSPs and chaperones was assessed by qPCR Array. The results demonstrate that five genes showed increased expression (over Log2 2), while five other presented reduced expression. To validate these results, the 10 differentially expressed genes were tested by real-time PCR in three different HCV cell culture models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all of three tested models, consistent with qPCR Array results. Then we promoted the silencing of HSPB8 and observed an increase in viral replication. In contrast, when we increased an expression of HSPB8, HCV had a decrease in replication rate. The same procedure was adopted for the DNAJC5B, validated in the viral model genotype 3, and HCV showed replication pattern similar to that observed for the previous gene. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and DNAJC5B have the same function, but genotype 3 specific. We also evaluated direct interactions with HCV proteins and the results demonstrated a physical interaction between the HCV NS4B protein with HSPB8. These results can contribute for a better understanding of the mechanisms involved in HCV replication. / FAPESP: 2013/17253-9