Hepatitis C Vaccines

VIROLOGY

HCV was identified in 1989 as the major cause of transfusion and community-acquired non-A non-B hepatitis. ^, It is an enveloped, single-stranded RNA virus classified as a member of the Hepacivirus genus within the Flaviviridae family. The 9.6-kb positive-sense RNA genome contains a single open reading frame encoding a polyprotein of approximately 3000 amino acids, flanked by highly structured 5′ and 3′ untranslated regions (UTRs), required for RNA replication and translation.

Viral RNA released into the cytoplasm of the host cell is translated via an internal ribosome entry site (IRES) located in the 5′ UTR, giving rise to a polypeptide that is cleaved into 10 different products (core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) ( Fig. 28.1 ). ^{, , ,}

HCV RNA replication takes place in the cytoplasm, in association with a virus-induced intracellular membrane structure termed “membranous web,” and is directed by the error-prone RNA-dependent RNA polymerase encoded by the viral NS5B gene. This is a low-fidelity polymerase that introduces frequent mutations into the viral genome. In the presence of immune selective pressure, this process generates a diverse quasi-species of viruses. The extreme genetic variability of HCV, dwarfing that of HIV, provides a selective advantage for the evasion from immune control and is believed to contribute to the remarkable persistence of HCV in infected individuals. Thus, the 5′ and 3′ UTRs, as well as the core gene are highly conserved, whereas the remaining viral genes and the corresponding proteins exhibit various but substantial levels of heterogeneity. The site of greatest variability is within the N-terminal region of E2 envelope glycoprotein (hypervariable region 1 [HVR1]). This region is a major target of virus-neutralizing antibodies. Recent structural insights into the aminoterminal domain of E1 and the core domain of E2 have revealed unexpected folds not present in glycoproteins from related viruses. ^, In vitro-produced HCV particles have been only recently visualized by electron microscopy. Based on available data, HCV appears to be the most structurally irregular member of the Flaviviridae family: particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 nm to 100 nm in diameter. By analogy to related viruses, HCV virions are thought to consist of an icosahedral lattice of E1/E2 heterodimers bound to a cell-derived lipid bilayer membrane surrounding a nucleocapsid built from multiple copies of the core protein, which, in turn, package the genomic RNA. HCV is invariably found in association with low-density and very-low-density lipoproteins (LDL and VLDL), resulting in the so-called lipo-viro-particles. Infection of the hepatocyte by HCV depends on a complex set of receptors (CD81, scavenger receptor class B type I, claudin-1, and occludin), as well as several auxiliary molecules, facilitating adsorption of the virus to the cell (e.g., glycosaminoglycans, LDL receptor) or virus entry (e.g., epidermal growth factor receptor, Niemann–Pick C1-like cholesterol absorption receptor). Of these, only the tetraspanin cellular protein CD81 and scavenger-receptor B1 (SR-B1) are believed to bind directly to the HCV envelope glycoprotein E2. ^,

For many years, it was impossible to propagate HCV in cell culture. Subgenomic HCV replicons were originally established for genotype 1b. More recently, the panel of available replicons has expanded to include isolates from genotypes 1a, 2a, 3a, 4a, 5a, and 6a. Because of a lack of structural proteins, however, subgenomic replicons could not be used to study immune responses against the virus. A fully permissive cell culture system supporting the production of infectious HCV only became possible with the identification of a genotype 2a isolate cloned from the serum of a Japanese patient with fulminant hepatitis, which was termed JFH1. This breakthrough has allowed the generation of infectious hybrid viruses in vitro, in which the envelope glycoprotein genes of the 2a strain were replaced with equivalent genes from other HCV genotypes. These key developments have opened up the field to investigations into HCV replication, virion structure, and virus-specific immune responses, as well as being valuable in the development of vaccines and new antivirals.

In addition to the above culture systems, a convenient and safe experimental model used to study HCV entry is based on the HCV pseudotype particles (HCVpp). HCVpp are obtained by exploiting a retrovirus in which HCV E1 and E2 substitute for the endogenous retroviral envelope proteins. Because the HCVpp system can express a range of HCV glycoproteins originating from different isolates and genotypes, it represents an excellent platform for the establishment of a virus-neutralizing antibody assay.

HCV can only infect humans and chimpanzees. Chimpanzees are no longer used for HCV vaccine research due to ethical concerns. Even when available, the chimpanzee model had limitations for vaccine research. In general, small numbers of chimpanzees were used for each study, and HCV-infected chimpanzees generally mounted less vigorous anti-envelope antibody responses than infected humans. Historically, attempts to develop small animal models of infection were based on the transplantation of human liver cells into immunodeficient mice. Whereas these have represented useful small animal models to study antiviral agents, they are unsuitable to study the immune response because of the lack of B-cell-mediated and T-cell-mediated immunity. Only very recently was a humanized mouse with a human immune system and liver tissue developed. This was obtained by expressing in Balb/C Rag2(–/–) γC-null mice a fusion protein of the K506 binding protein and caspase 8 under control of a hepatocyte-specific promoter that induces liver cell death. Transplantation of human CD34 ⁺ human hematopoietic stem cells and hepatocyte progenitors into the transgenic mice led to efficient engraftment of human leukocytes and hepatocytes. These animals, termed AFC8-hu HSC/Hep, supported HCV infection in the liver and generated a human immune T-cell response against HCV, and also developed hepatitis and fibrosis. An alternative method to overcome species barriers to HCV infection builds on the observation that CD81 and occludin comprise the minimal set of human factors required to render mouse cells permissive to HCV entry. Thus, transgenic mice were constructed that stably expressed human CD81 and occludin (Rosa26-Fluc mice). These animals support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo. ^, Blunting innate antiviral immunity in these genetically humanized mice infected with HCV, however, resulted in measurable viremia over several weeks. Alternatively, transgenic expression of human CD81 and occludin in an ICR (imprinting control region) mouse background yielded transgenic ICR mice that can support persistent HCV infection, with complete replication cycle and hepatopathological manifestations, without the need for targeting the disruption of interferon-stimulated genes. Future work will assess the usefulness of these models in vaccine research.

HEPATITIS C VIRUS INFECTION

The major site of HCV replication is the hepatocyte. HCV has also been reported to infect other cell types, most notably B cells and dendritic cells, but also cells in the intestine and in the central nervous system. However, the issue of extrahepatic HCV infection remains controversial.

Historically, transmission seemed to occur primarily via direct parenteral exposure to contaminated blood. The frequent transmission of HCV that occurred in transfusion recipients before the discovery of the virus has now disappeared with the advent of serologic tests based on the detection of antibodies to viral proteins and on the direct detection of HCV RNA. As of today, intravenous drug use, unprotected sex with multiple partners, and viral exposure during invasive medical procedures (e.g., surgery, dialysis, and dental treatment) are factors associated with the highest degree of risk for HCV infection. After infection, HCV RNA appears in the plasma within a few days and usually shows a peak within the first few months. The inflammatory processes leading to initial liver injury, as assessed by serum elevation of the liver specific alanine aminotransferase (ALT) enzyme, generally occur within 1–3 months. The absence of correlation between HCV RNA levels and liver damage suggests that HCV does not exert a direct cytopathic effect on the infected cells. At present, liver damage is postulated to be primarily caused by the infiltration of inflammatory cells, which cause extended inflammation that in turn causes hepatocyte damage and death. Although the mechanisms involved in initiating and maintaining an inflammatory response in the HCV-infected liver are not completely understood, the currently accepted model implicates the CD8 ⁺ cytotoxic T-lymphocyte (CTL) T-cell response in mediating most if not all the liver damage that is associated with chronic viral hepatitis. According to this model, hepatocyte injury is initiated by the cytolytic activity of HCV-specific CTLs and CTL-produced cytokines, such as IFN-γ, and IFN-γ-inducible chemokines (e.g., CXCL9 and CXCL10), recruit into the liver parenchyma large numbers of antigen-non-specific inflammatory cells that have the potential to amplify the liver damage. Notably, work in animal models of viral hepatitis shows that platelets play a key role in T-lymphocyte-induced liver damage by facilitating the intrahepatic accumulation of CTLs. If infection is not resolved within the first 6–12 months, patients generally remain infected for life. Chronically infected patients have viral loads that typically range from 10 ⁵ to 10 ⁷ genomes per milliliter of serum. The virions have a rapid turnover, with a half-life of about 3 hours, and up to 10 ¹² viral particles per day are produced in an infected person.

EPIDEMIOLOGY

According to the WHO, approximately 1% of the world’s population is chronically HCV infected. The Eastern Mediterranean region (Middle Eastern countries) is estimated to have the highest prevalence (2.3%); Europe and Africa have moderate prevalence (1.0–1.5%); and the Americas, South-East Asia, and the Western Pacific regions have lowest prevalence (<1%). At the level of individual countries, Egypt is thought to have the highest prevalence of chronic HCV in the world, estimated at approximately 15–20%. What led to an HCV epidemic of this proportions in Egypt is the extensive use of parenteral antischistosomal therapy in a mass treatment setting, a practice discontinued only in the 1980s. Despite increasing availability of DAAs, HCV incidence continues to rise in many regions of the world. In the United States, the incidence of HCV doubled between 2010 and 2014. Seven different major HCV genotypes and many subtypes have been distinguished phylogenetically, with 30–50% sequence variation among viral genotypes and 15–30% among different subtypes. ^, Globally, genotype 1 is the most common (46%), followed by genotype 3 (22%), genotype 2 (13%), genotype 4 (13%), genotype 6 (2%), and genotype 5 (1%). Definitive data regarding the prevalence of genotype 7 are still lacking. Genotype 1b is the most common subtype, accounting for 22% of all infections. Significant regional and countrywide variations in genotype distribution exist. Infections in Europe, North America, and Latin America are predominantly genotype 1. North Africa and the Middle East have large genotype 4 populations, primarily as a consequence of the extraordinarily high prevalence of this genotype in Egypt. Excluding Egypt, genotype 1 is predominant also in this region. Asia is primarily affected by genotype 3, followed by genotype 1, with the former being very common in India, Nepal, and Pakistan. Finally, genotype 1 predominates in Australia, followed by genotype 3.

Transmission of HCV occurs via direct parenteral exposure to contaminated blood. The frequent transmission of HCV that occurred in transfusion recipients before the discovery of the virus has disappeared with the advent of serologic tests based on the detection of HCV-specific antibodies or the direct detection of HCV RNA. At present, a history of intravenous drug use represents the major risk factor for newly acquired hepatitis C infection. Other possible sources of parenteral exposure to HCV are surgical and dental procedures and folk practices such as piercing and tattoos. Although the risk of sexual transmission is low, sexual partners of infected persons still account for a proportion of HCV infections. Vertical transmission also occurs particularly in mothers with high viral load or who are coinfected with HIV. Coinfection with HIV and HCV is common in persons who inject drugs (PWID), and importantly the immunosuppression associated with HIV infection results in higher HCV titers and more rapid progression to cirrhosis and end-stage liver disease.