Hepatitis C virus is an infectious disease affecting millions of people worldwide and causing chronic liver disease. The current standard of care is not only long but causes numerous side effects. Due to incomplete virological response and poor tolerability, only 50% of the patients are cured, with variability by genotype. Despite the development of non-enzymatic viral protein inhibitors, new therapies target mainly enzymes responsible for viral replication or translation. Being commonly used for antiviral and anticancer therapy, nucleosides analogues have played an important role as anti-HCV agents. Despite their potency and selectivity, nucleoside analogues appear to be poor substrates for metabolic enzymes. In particular, the first essential phosphorylation step is often rate-limiting thus, resulting in poor bioactivation to the active triphosphate form. Hence, monophosphate prodrug strategies have been applied to efficiently deliver intracellularly the key monophosphate derivatives. Such strategies have been successfully used for anti-HCV therapy and the phosphoramidate ProTide INX-08189, discovered in our lab, is one such example. Aiming at developing back-up molecules of INX-08189, we report in the present work, the synthetic strategies to obtain several modified β-2’-C-methyl-6-O-methyl guanosine and other modified β-2’-C-methyl purine nucleoside analogues. The phosphoramidate ProTide approach and the phosphorodiamidate approach were applied to these modified nucleosides. In-vitro, and sometimes in-vivo evaluation against HCV replication is reported, and the mechanism of bioactivation to their corresponding monophosphate species is discussed. Enzymatic experiments using carboxypeptidase Y and Huh-7 cell lysates were carried out to investigate the release of the monophosphate forms. We also investigated the hydrolysis of the 6-O-methyl group at the nucleoside level with adenosine deaminase enzyme, and at the monophosphate level using molecular docking in adenosine deaminase like protein-1. Eventually, the intracellular putative mechanism of activation of the ProTides was studied using molecular modeling with cathepsin A enzyme and human Hint-1 phosphoramidase.

Hepatitis C virus (HCV) infection is one of the major causes of liver morbidity and mortality worldwide. While effective therapies are now available, if eradication of this virus is to be achieved globally, an effective vaccine is still necessary. During hepatitis C virus (HCV) infection, broadly neutralizing antibody (bNAb) responses targeting E1E2 envelope glycoproteins are generated in many individuals. It is unclear if these antibodies play a protective or a pathogenic role during chronic infection or if they could prevent infection or reinfection with the virus. I investigated the presence and clinical associations of bNAb responses in three cohorts of individuals infected with or exposed to HCV infection. One with chronic HCV infection at differing disease states, one with chronic HCV infection at an early disease state and one group of individuals at high risk of HCV exposure who remained uninfected by conventional testing. I also studied bNAb responses in an individual from a HCV-HIV co-infected cohort who experienced spontaneous clearance of HCV after a post-therapy relapse (‘secondary spontaneous clearance’). I found a proportion of individuals when exposed to or infected with HCV produce a polyclonal bNAb response which may contribute to viral clearance in some cases. Host genetics and the ability to target multiple neutralising epitopes on the envelope protein are associated with such responses, although resistance mutations to bNAbs do exist in vivo. The presence of bNAbs is associated with lower levels of liver fibrosis. Using next generation sequencing technology in the study of B cell receptors in HCV infection revealed subtle changes in the B cell repertoire on HCV infection, this technology may be used in future to gain insight into the generation of bNAb responses.

Feline immunodeficiency virus (FIV) is a naturally occurring lentivirus of domestic cats, which shares many similarities with its human counterpart, human immunodeficiency virus (HIV). FIV infects its main target cell, the CD4+ T lymphocyte, via interactions with its primary receptor CD134 (an activation marker expressed on activated CD4+ T lymphocytes), and, the chemokine receptor CXCR4. According to the different ways in which FIV isolates interact with CD134, FIV may be categorised into two groups. The first group contains strains that tend to dominate during the earlier phase of infection, such as GL8 and CPG41. These strains are characterized by their requirement for an additional interaction with the second cysteine rich domain (CRD2) of the CD134 molecule and are classified as “CRD2-dependent” strains. The second group, on the other hand, contains either laboratory-adapted isolates or isolates that emerge after several years of infection, such as PPR or the GL8 variants that emerged in cats 6 years post experimental infection and were studied in this thesis. These isolates are designated “CRD2-independent” as they can infect target cells without interacting with CRD2 of the CD134 molecule. This study provides the first evidence that FIV compartmentalisation is related to FIV-CD134 usage and the tissue availability of CD134+ target cells. In tissue compartments containing high levels of CD134+ cells such as peripheral blood and lymph nodes, CRD2-dependent viruses predominated, whereas CRD2-independent viruses predominated in compartments with fewer CD134+ cells, such as the thymus. The dynamics of CD4+CD134+ T lymphocytes at different stages of FIV infection were also described. The levels of CD4+CD134+ T lymphocytes, which were very high in the early phase, gradually decreased in the later phase of infection. The dynamics of CD4+CD134+ T lymphocyte numbers appeared to correlate with FIV tropism switching, as more CRD2-independent viruses were isolated from cats in the late phase of infection. Moreover, it was observed that pseudotypes bearing Envs of CRD2-dependent variants infected CD134+ target cells more efficiently than pseudotypes bearing Envs of CRD2-independent variants, confirming the selective advantage of CRD2-dependent variants in environments with high levels of CD134+ target cells. In conclusion, this study demonstrated that target cell types and numbers, as well as their dynamics, play important roles in the selection and expansion of FIV variants within the viral quasispecies. Improved understanding of the roles of target cells in FIV transmission and pathogenesis will provide important information required for the development of an improved, more successful protective FIV vaccine and will provide insight into the development of effective vaccines against other lentiviral infections such as HIV.

Dendritic cells are efficient antigen-presenting-cells that can be used in tumour-antigen specific vaccination for malignant disease. Melanoma patients were recently treated with a dendritic cell vaccine expressing gp100 and Melan-A antigens after non-viral (CL22 peptide) transfection. Although clinical and immunological responses were noted, there was no correlation between responses and whole antigen expression levels in the vaccine cells that varied widely. Here, it is established that patient cells expressed detectable levels of Class I restricted epitopes from both antigens, although there was no correlation with whole antigen detection. CL22 transfected dendritic cells could simultaneously present a viral antigen (EBNA1) to CD8 and CD4 T-cells, which had not previously been demonstrated. Using RNA transfection, it was demonstrated that early after transfection cells are whole Melan-A positive yet negative for Class I epitopes and with time Melan-A antigen levels fall whilst Class I epitopes are generated. Loss of whole antigen expression seemed related to lysosomal function and, unlike the viral antigen EBNA1, Class I presentation from Melan-A was lysosome-dependent. For Class II presentation of EBNA1, cellular localisation seems to determine access to the Class II pathway although this depends on the time-scale over which epitope presentation is assessed.