Development of an immunotherapy to treat persistent hepatitis C virus infection

2017-02-27T05:18:57Z (GMT) by Latour, Philippe Armand
Hepatitis C Virus (HCV) infection causes liver disease, affecting approximately 200 million people worldwide. In most individuals acute HCV infection is asymptomatic prior to the development of persistent infection in approximately 80% of infected individuals leading to life-long infection. This results in active inflammation, fibrosis and cirrhosis of the liver, in some cases developing into hepatocellular carcinoma. In the remaining 20% of infected patients, spontaneous clearance of the virus is believed to be due to a broad, multi-specific CD4 and CD8 T cell response. Despite the advent of new direct acting antivirals which cure a high proportion of treated patients, many patients will not be cured or will not be treated due to the high cost of therapy. Therefore it is important to investigate immunotherapeutic strategies to combat persistent HCV infection. This thesis focuses primarily on immunotherapeutic methods involving the use of dendritic cells (DC), natural immune sentinels, to illicit an HCV specific immune response. Chapter 3 investigated the anti-HCV immune responses of 6 persistently infected HCV positive patients after administration of autologous monocyte derived DC pulsed with lipopeptides which represent HLA-A2 restricted CTL epitopes. The results of this study showed that the patients developed transient HCV specific responses including responses to HCV peptides not contained within the vaccine. Chapters 4 investigated the use of RNA to endogenously express entire HCV proteins in dendritic cells, using a murine DC cell line as a surrogate to assess different transfection protocols, before application in human monocyte derived DC. Ultimately, although effective protocols for the transfection of the murine cell line were developed, these were inefficient in human monocyte derived DC, highlighting the difficulty in working with these cells. However, the findings of this chapter led to the development of the hypothesis that cells expressing HCV antigens, which are made necrotic, would be powerful immunogens. In chapter 5 the technology explored in chapter 4 was used to transfect the murine DC cell line with an RNA construct encoding the HCV NS3 protein. These cells were then made necrotic and injected into mice. This led to high, specific responses to NS3, highlighting the effective adjuvant activity of necrotic cells. In order to apply this strategy in humans, an appropriate vector to express HCV proteins in monocyte derived DC was required. Consequently, in chapter 6 an effective method involving the pre-treatment of DC with the detergent, polybrene, prior to adenoviral transduction was found to be effective and resulted in high transfection efficiencies. Further experiments in Chapter 6 developed a GMP-like protocol to generate recombinant adenoviruses for large scale applications. Finally in chapter 7, the results of a dose escalation study of HCV antigen- positive, monocyte derived necrotic DC, administered to persistently infected HCV positive patients is presented. Minimal transient HCV-specific immune responses were observed throughout the monitoring period. In summary this thesis presents work resulting in the development of an effective and efficient method of transducing monocyte derived DC with a recombinant adenovirus using lower multiplicities of infection than reported previously by others, the development of a method for the manufacture and purification of recombinant adenoviruses for clinical scale production to GMP standard, and the results from two first in man studies utilising monocyte derived DC for the treatment of HCV.