Hepatitis C Virus Antigenic Convergence

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development

A study of novel biomarkers of hepatitis C related liver injury

Chronic hepatitis C virus (HCV) is the most frequent indication for liver transplantation. Unfortunately HCV recurrence is universal following transplantation and many patients will experience aggressive disease recurrence. This thesis comprises 3 related studies with the main focus being an assessment of factors which may impact on the course of HCV recurrence post-transplant. The first study was a retrospective analysis of 118 consecutive HCV-positive liver transplant patients with a median duration of follow-up of 32.4 months. Peak viral RNA ≥ 107 in the first year post-transplant was shown to be an independent predictor of diminished patient survival. The second study was a cross-sectional pilot study using a recently developed CD antibody microarray in patients with various causes for liver disease. This demonstrated disease-specific consensus patterns of expression of CD antigens for patients with chronic liver disease and in particular, the ability to separate major stages of liver disease. In the third study serial CD antigen expression profiles were performed on patients undergoing liver transplantation for HCV infection. Differential antibody expression was most significant in the pre-transplant phase suggesting a significant impact of pre-transplant factors on disease severity post-liver transplantation. The findings in this thesis have demonstrated utility of the CD antibody microarray in the study of human liver disease and have shed light on the importance of post-transplant viral loads in determining the severity of post-transplant HCV recurrence

IMPACT OF NONSTRUCTURAL HEPATITIS C VIRUS ANTIGENS AND TOLL-LIKE RECEPTOR AGONISTS ON DENDRITIC CELL IMMUNOGENICITY

Dendritic cells (DCs) function mainly as antigen presenting cells (APCs) and as such they play a significant role in activating the adaptive immune system. Dendritic cells express toll-like receptors (TLR), and when these receptors are engaged by their cognate agonists, they promote DC maturation, which is critical in the induction of potent T helper (Th) cell -1 responses. Due to the multifunctional abilities of DCs, they have been explored as vaccine carriers, largely in cancer immunotherapy and some infectious diseases including hepatitis C. Previous studies showed that DCs loaded with mRNA of hepatitis C virus (HCV) antigen(s) induced strong immune responses but immune protection was not complete. Therefore, I expected that adoptive transfer of DCs transfected with HCV NS3/4A and/or NS5A mRNA and further treated with TLR agonist(s) ex vivo would induce HCV-specific immunity in vivo. Bone marrow-derived DCs generated with Flt3L (FL-DCs) or GM-CSF (GM-DCs), and loaded with HCV NS3/4A and/or NS5A mRNA showed maturation characteristics and produced substantial amounts of IL-12 after ex vivo activation with CpG ODN or CpG ODN plus Poly I:C, when compared to their untreated counterparts. Treatment with a combination of CpG ODN and Poly I:C synergized to augment IL-12 production in comparison with stimulation with CpG ODN alone. IL-12 secretion by DCs is pivotal in directing immune responses towards a Th1-bias response, which is needed to eliminate HCV. However, the ex vivo responses of stimulated DCs bearing HCV antigen(s) were not efficiently translated in mice to potentiate vigorous antigen-specific T cell responses. This resulted in a lack of protection after challenge with recombinant vaccinia virus expressing HCV NS3/NS4/NS5 in immunized mice. In contrast, both antigen-specific humoral and cell-mediated immune responses were induced in mice vaccinated with HCV recombinant NS3 or NS5A protein co-formulated with CpG ODN, host defense peptide and polyphosphazene. These responses, however, did not mediate viral clearance, as vaccinated mice remained unprotected from infection with recombinant vaccinia virus expressing HCV antigens. Taken together, these results suggest HCV recombinant protein co-formulated with triple adjuvant to be a better vaccine candidate than the DC-based vaccine

Comparison of the Mechanisms of Drug Resistance among HIV, Hepatitis B, and Hepatitis C

Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) are the most prevalent deadly chronic viral diseases. HIV is treated by small molecule inhibitors. HBV is treated by immunomodulation and small molecule inhibitors. HCV is currently treated primarily by immunomodulation but many small molecules are in clinical development. Although HIV is a retrovirus, HBV is a double-stranded DNA virus, and HCV is a single-stranded RNA virus, antiviral drug resistance complicates the development of drugs and the successful treatment of each of these viruses. Although their replication cycles, therapeutic targets, and evolutionary mechanisms are different, the fundamental approaches to identifying and characterizing HIV, HBV, and HCV drug resistance are similar. This review describes the evolution of HIV, HBV, and HCV within individuals and populations and the genetic mechanisms associated with drug resistance to each of the antiviral drug classes used for their treatment

Molecular Aspects of Hepatitis C Virus Infection and Associated Mitochondrial DNA Damage

Hepatitis C virus (HCV) is the main cause of viral hepatitis in the UK and leads to chronic liver disease in many infected individuals. There is a substantial burden on diagnostic laboratories to provide rapid, cost-effective tests for monitoring HCV infection. Commercial assays are expensive and so the development of validated in-house methods is beneficial. This thesis describes the development and implementation of rapid and inexpensive real-time PCR assays for HCV quantitation and genotyping to support clinical practice. Additionally, the development of methods for defining HCV isolates at the subtype level, important in epidemiological and transmission studies, is described. These assays were utilised in a study on spontaneous HCV clearance, the results of which suggest that HCV type 1 infection and younger age at infection are factors which are associated with spontaneous viral clearance. Chronic HCV infection is linked to oxidative stress with numerous deleterious cellular effects. Mitochondrial DNA (mtDNA) is more susceptible to oxidative damage than nuclear DNA making it an ideal marker to assess the overall level of cellular DNA damage - including deletions and mutations. This thesis illustrates the development of real-time PCR assays to detect and quantify two major mtDNA deletions. The D-loop region of mtDNA is particularly prone to damage - with two well recognised 'hotspots of mutation'. The creation of an RSCA (heteroduplex-based) method using capillary electrophoresis, to detect and quantify damage in this region, is described. To evaluate the clinical utility of these assays, a study of mtDNA damage in patients with liver disease was undertaken. The aim of this study was to identify whether chronic HCV infection results in increased levels of mtDNA damage compared to other liver pathologies. Low levels of mtDNA deletions were detected in the majority of liver biopsy specimens and there was no correlation between liver aetiology and quantity of deletions. The RSCA method identified numerous D-loop mtDNA species within the liver tissue of several individuals. There was no correlation between liver aetiology and the presence of multiple D-loop species

The role of humoral immune response in hepatitis C virus infection

The World Health Organization (WHO) has estimated that about 3% of the world’s population is currently infected with Hepatitis C Virus (HCV). Although spontaneous clearance of HCV infections occurs in 10-20% of patients, nearly 185 million people are still chronically infected by HCV, which is one of the leading risk factors for developing liver cirrhosis and/or liver cancers. In the HIV-1 infected population, chronic HCV infection has become one of the leading causes of non-AIDS specific morbidity and mortality. In the United States about 25% of HIV-1 positive individuals have co-infections with HCV. It has been observed that HIV-1 infection reduces the rate of spontaneous clearance and exacerbates the clinical course of HCV infection in HIV/HCV co-infected individuals, but the mechanisms driving this remain unknown. One of the major hurdles of studying the mechanisms of HCV infection over the last two decades has been the inability to grow and study this virus in a laboratory setting, as well as, the inability to develop reliable animal models. The development of the HCV pseudotyped viral particles (HCVpp) system was ground-breaking and became one of the most important in vitro study tools to test the neutralizing antibodies against virus entry and vaccine studies. Therefore, we used HCVpp to evaluate the role of neutralizing antibodies in an HIV-infected subject who spontaneously cleared HCV, but subsequently was re-infected with a different genotype of HCV. In this study, we examined the role of HCV specific neutralizing antibodies in HCV clearance and chronicity in a HIV/HCV co-infected subject who was enrolled in Pittsburgh portion of the Multicenter AIDS Cohort Study (MACS). The subject’s records showed a 10year history of HIV infection and treatment with antiretroviral drugs for 2 years prior to HCV infection. According to our previous studies, this person was consecutively infected with two different genotypes of HCV. By sequence analysis, the initial infecting HCV genotype was 3a and the subsequent infecting genotype, 1.5 years later, was HCV 1a. Sequence analysis of HCV with longitudinal samples from this individual shows that the initial infecting HCV 3a was cleared spontaneously after one and half year infection, but after the subsequent infecting HCV 1a established its chronic infection in this individual. We hypothesized that neutralizing antibodies played an important role in controlling and clearing the HCV 3a infection, but had minimal impact on the HCV 1a infection. Using HCVpp containing autologous envelope proteins of HCV subtype 3a or 1a, we measured the neutralization activity of antibodies present in the subject’s serum. We found that after pre-incubating genotype 3a specific HCVpp with the subject’s serum collected 6 months after HCV 3a infection, there was a tendency of reduction in the genotype 3a specific HCVpp infectivity. However, we observed no reduction in infectivity of genotype 1a specific HCVpp, which were pre-incubated with the subject’s serum collected after HCV 1a infection. Therefore, our results suggest that the development and presence of neutralizing antibodies may be important for the spontaneous clearance of HCV 3a in HIV co-infected individuals. This study contributes to our understanding of immune control of HCV, which may lead to the development of preventive vaccine against HCV and have a great Public Health impact in preventing HCV infection

Short interval change in hepatitis C hypervariable region 1 in chronic infection. Are there treatment windows in the envelope?

Hepatitis C Virus (HCV), an RNA virus, is one of the leading causes of cirrhosis worldwide and, remains the leading indication for orthoptic liver transplantation in the United States. Dual treatment with pegylated interferon and ribavirin has until 2010 been the mainstay of treatment. The emergence of newer agents with direct activity against specific virus proteins has revolutionised HCV treatment but, the high cost of these medications are likely to prevent universal access, particularly in developing countries and, strategies to optimise response to cheaper combination treatments are required. The Irish Hepatitis C outcomes research network (ICORN) has proposed a target of 2025 for the complete eradication of Hepatitis C from Ireland. HCV replicates in an error prone fashion resulting in mutant progeny known as quasispecies(QS), thought to form an important mechanism of host immune evasion in the establishment and maintenance of chronic infection, which develops in 50-80% of those acutely infected. HCV has three hypervariable regions (sections of the virus genome that appear to tolerate higher substitution rates) and one of these, Hypervariable region 1 (HVR1) has been recognised as a major target of the adaptive immune response. HVR1 quasispecies complexity and diversity have been implicated as predictive of response to dual therapy. Little, however, is known about the natural history of these parameters in chronic infection. We discuss evolutionary concepts and how they apply to quasispecies and hypothesise how viruses might select a setting appropriate mutation rate in order to optimise adaptation, advancing the theory of replicative homeostasis. We prospectively study 23 patients with chronic HCV infections and, differing degrees of liver fibrosis fortnightly for a 16 week period prior to commencement of treatment. Using amplicon sequencing, cloning and next generation sequencing we explore the behaviour of HVR1 QS, establishing the utility of each technique in describing QS change. We identify variable and unpredictable HVR1 change in our cloning data which precludes the use of these metrics in pre treatment prediction models. HVR1 change is far greater in non cirrhotic patients and the transition to cirrhosis appears to be associated with a change from positive to purifying selection. Using molecular clock techniques we illustrate differing substitution rates within HVR1 among cirrhotic and non cirrhotic patients. We identify, by including an additional retrospective sample, that the patterns we describe are sustained over prolonged periods and further clarify the mode and tempo of HVR1 change by estimating the substitution rates. Using next generation sequencing techniques we identify similar patterns of HCV change when compared with our cloning data. However, the sequence depth provided permits the description of time specific network of HVR1 clones, all connected by a single amino acid substitution to a central node. By separating our samples into immunoglobulin bound and free fractions we describe the importance of host immune mediated change driving the changes seen in our pyrosequencing and cloning data. Finally, using known viral and host molecular markers predictive of treatment response we explore unsuccessfully for models predictive of treatment response

Investigating virus entry using cell-culture adapted hepatitis C virus

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Present estimates predict that between 120-130 million people worldwide are infected with HCV with the majority of all infections progressing to chronicity, ultimately leading to fibrosis, cirrhosis and hepatocellular carcinoma. The virus, which belongs to the family Flaviviridae, has a single-stranded RNA genome of positive polarity that codes for a unique polyprotein of approximately 3000 amino acids. The structural proteins E1 and E2 constitute the viral envelope glycoproteins. These glycoproteins have multiple functions in the viral life cycle such as promoting viral entry and fusion, assembly of infectious virions and aid in viral persistence through immune escape. Numerous cell culture-adaptive mutations have been reported within the HCV glycoproteins. The value of such mutations in understanding the virus interaction(s) with cellular receptors and neutralizing antibodies was first recognised from studies characterizing the E2 cell culture adaptive mutation G451R. This single mutation altered the affinity of HCV to the cell surface receptors CD81 and SRB1 as well as increasing its sensitivity to neutralizing antibodies targeting the viral glycoproteins. A striking observation from previously reported E2 cell culture adaptive mutations is their frequent occurrence within a highly conserved region of E2, spanning residues 412-423. Indeed, the long-term passaging of JFH1 infected cells here in this study also created an adapted virus with a substitution at residue 415. The aim of this study was to determine the phenotypic changes to viral entry caused by mutations in this region. To do this, four JFH1 viruses containing the mutations N415D, T416A, N417S and I422L were constructed and characterized. These mutant viruses were found to have very similar phenotypes to the G451R virus, suggesting all E2 adaptive mutations are selected to alter a specific function in viral entry. Residues 412-423 of HCV E2 also constitute the epitope of the in-house generated broadly neutralizing antibody AP33. ELISA binding and virus infection inhibition assays using AP33 with the E2 mutant viruses provided important information regarding the E2 contact residues of this antibody. In a separate study, intergenotypic chimeric JFH1 viruses were generated and characterised. Viable intra- and intergenotypic JFH1 chimeric viruses have previously been generated by different research groups by replacing the core to NS2 genes of JFH1 with those from different genotypes. Many of these chimeric viruses required numerous cell culture adaptive mutations to permit efficient infectious virus production. In the present study, 5 intergenotypic viruses were constructed by replacing the JFH1 envelope genes with those from other HCV genotypes. Despite these chimeric genomes replicating efficiently, none were capable of producing infectious virus. These viral genomes also failed to acquire infectivity during pro-longed cell passaging, suggesting that replacing the JFH1 envelope glycoproteins with those from other genotypes may confer total incompatibility for virus assembly. In addition to this work, the infectivity of a previously generated genotype 4a/JFH1 chimera was improved by repeatedly passaging the virus infected cells. The chimeric virus contained the core to NS2 genes of a genotype 4a strain in place of the those from the original JFH1 sequence. A total of six-adaptive mutations were identified throughout the adapted genome that enhanced infectivity by more than 100-fold. Achieving higher titers with this chimera permitted studies on its viral entry properties as well as its sensitivity to neutralizing antibodies. The ability of the adapted virus 4a/JFH1 virus to spread during multiple rounds of infection was greatly reduced compared to WT/JFH1 due to its inefficient cell-to-cell spread. The 4a/JFH1 virions were also highly sensitive to neutralizing antibodies targeting both linear and conformational E2 epitopes, suggesting that the glycoproteins are more exposed on the surface of this virus. In its totality, this study has provided key insights into the viral entry and antibodymediated neutralization properties of cell-culture adapted and intergenotypic chimeric forms of the JFH1 virus