Course Authors
Raymond S. Koff, M.D.
Dr. Koff is Professor of Medicine, University of Massachusetts Medical School, and attending physician, University of Massachusetts Memorial Medical Center, Worcester, Massachusetts, USA.
During the last three years, Dr. Koff has received grant/research support from Schering-Plough, GlaxoSmithKline and Roche. He has been a consultant to GlaxoSmithKline and has also served on the Speakers' Bureau for Roche and Schering-Plough.
Estimated course time: 1 hour(s).
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In support of improving patient care, this activity has been planned and implemented by Albert Einstein College of Medicine-Montefiore Medical Center and InterMDnet. Albert Einstein College of Medicine – Montefiore Medical Center is jointly accredited by the Accreditation Council for Continuing Medical Education (ACCME), the Accreditation Council for Pharmacy Education (ACPE), and the American Nurses Credentialing Center (ANCC), to provide continuing education for the healthcare team.
Upon completion of this Cyberounds®, you should be able to:
Describe the rationale for attempts to develop a HCV vaccine
Discuss the heterogeneity of HCV and likely mechanisms of viral persistence
Define the obstacles to HCV vaccine development.
Hepatitis C virus (HCV) infection is now the predominant cause of chronic viral hepatitis in the United States and in many developed countries. Although the incidence of acute hepatitis because of HCV infection has fallen in recent years to no more than 40,000 cases annually, current estimates, nonetheless, suggest that over 4 million individuals have been infected and close to 3 million are chronically infected.(1)
While the natural history of HCV infection has yet to be fully defined, the fact that persistent infection occurs in the majority of infected individuals remains undisputed. Indeed, chronic infection occurs in 55% to 85% of acutely infected patients regardless of whether or not the initial infection is clinically recognized. Serious, life-threatening sequelae of chronic HCV infection, resulting from the development of hepatic fibrosis, include cirrhosis, end-stage liver disease and hepatocellular carcinoma. These occur in a substantial proportion of infected patients in the decades that follow acquisition of infection. As a consequence, liver failure because of hepatitis C-related cirrhosis has become the single most common indication for liver transplantation in the United States. Moreover, hepatocellular carcinoma developing in those with HCV-related cirrhosis has been increasing in frequency.(2)
One of the central unanswered research questions about HCV infection is the nature of the determinants of chronic infection. What are the viral or host factors that mediate or permit persistent infection? In the interval since HCV was identified by molecular cloning in 1989, considerable information about the molecular biology of the virus has been elucidated and it seems possible that specific features of the infection may play a critical role in the failure of the human host to clear the virus following the initiation of infection.
A full understanding of the biology of HCV infection and the host response may be critically important for the development of an effective HCV vaccine that can prevent chronic infection, even if it fails to induce sterilizing immunity (see below). In this focused Cyberounds® review, current knowledge of selected aspects of the molecular biology of HCV will be described and progress in vaccine development will be highlighted.
Characteristics of HCV
HCV is currently classified as a separate, third genus in the Flaviviridae, which includes both the Flavivirus genus and the Pestivirus genus. HCV is a spherical, lipoprotein-enveloped, RNA-containing agent with a probable particle diameter of between 38 and 60 nm and an inner, nucleocapsid core estimated to be about 30 to 35 nm in diameter.(3) The HCV envelope contains two distinct glycoproteins - E1 and E2 - with antigenic domains that elicit antibodies, some of which have been reported to have virus neutralizing activity.
The positive-sense, single-stranded RNA genome has a length of about 9400 nucleotides and contains a 5'-nontranslated region which is well conserved and a single, open reading frame that encodes a polyprotein of about 3000 amino acids. Within the 5'- nontranslated region, an internal ribosomal entry site that regulates HCV translation has been identified.(4),(5),(6) After the large open reading frame, a short but variable 3'-nontranslated region contains poly(U) or poly(A) sequences. The 3'-nontranslated region also may contain elements essential to viral replication.(7)
The large polyprotein encoded by the HCV open reading frame is subjected to post-translational cleavage through the action of cellular and viral proteinases that yield both structural and non-structural proteins. Thus far, four structural and six non-structural proteins have been identified.
Genetic Variability of HCV
Extreme variability in nucleotide sequence has been identified in HCV; six different clades (a new term for closely related viruses) encompassing at least 11 different genotypes are now recognized. HCV genotype 1 is the most prevalent in North America and accounts for about 70% of all infections: the remainder of North American infections result largely from genotypes 2 and 3. Genotyping is performed by sequence homology studies, restriction fragment length polymorphisms (RFLP) of amplified polymerase chain reaction products, polymerase chain reaction amplification with subtype-specific primers, or line-probe hybridization assays.
During infection, HCV mutations accrue at the rate of 1-2 x 103 per base per year. Minor variants in nucleotide sequence result in the presence of a population of closely related viruses, so-called "quasispecies," which are often present simultaneously in any patient.
The genetic variability in HCV envelope proteins and genetic changes elsewhere in the genome have long been thought to be responsible, at least in part, for the ability of HCV to evade the cell-mediated and humoral immune mechanisms that mediate resolution of acute infection and thereby permit the virus to continue to replicate and infect additional hepatocytes.(8) Additional factors that may play an important role in the development of persistent HCV infection are reviewed below.
Mechanisms of HCV Persistence
There is no evidence that the HCV genome can be integrated into the host genome. Hence, in the absence of integration, viral persistence requires other explanations. Following host-mediated proteolytic degradation, HCV proteins and their antigenic domains are presented on the cell surface by several major histocompatibilty complex (MHC) Class I and II haplotypes. Available information suggests that both antigen processing and presentation are unimpaired in HCV infection, although further work to confirm this concept seems necessary.
Processing and presentation permit recognition of these antigens by immunoglobulins and T-cell receptors(9) and lead to a humoral and cell-mediated cytotoxic T-lymphocyte (CTL) immune response against the viral antigens. In patients in whom infection becomes persistent, the humoral and T-cell immune response appear to be insufficient to clear the virus. A large body of data indicate that the humoral immune response in HCV infection is modest at best and the T-cell-mediated response is a limited one. The mechanisms resulting in this failure are still incompletely understood. In a study of chimpanzees that had recovered from an initial HCV infection and rapidly cleared viremia when rechallenged with homologous or heterologous HCV genotypes, a strong T-cell proliferative response was identified.(10) While there may be important differences in the biology of HCV infection in chimpanzees compared to human beings, a more thorough understanding of the determinants of this T-cell response may prove beneficial in the design of an HCV vaccine.
Although the development of resistant HCV escape mutants and quasispecies has been thought to be a consequence of immune-mediated selection leading to persistent infection, and mutations in immunodominant CTL epitopes (antigen binding site) or the development of CTL inhibitory substances also have been suggested to play a role, evidence that these are the principal mechanisms of HCV persistence remains sketchy. Recent studies suggest that immune-mediated selection of escape variants is not essential for the development of chronic infection.(11)
Persistence of infection may be a consequence not so much of immune evasion but may result from the occurrence of very high rates of viral replication which, in turn, may lead to a very high proportion of infected hepatocytes. In this setting, the number of infected hepatocytes, perhaps approaching 30% of all hepatocytes, may simply be too large a target for the available effector T-cells and, therefore, replication continues in those hepatocytes spared from the T-cell response.
Other potential mechanisms permitting persistence of infection involve an impaired hepatocyte HCV CTL response because of an absence or reduction of essential intracellular costimulatory signals in hepatocytes or failure of the hepatocyte to produce the appropriate cytokines or other mediators of cytotoxicity. Definitive understanding of the responsible mechanisms remains a goal.
The Need for a HCV Vaccine
The major rationale for the development of a HCV vaccine is the need to prevent persistent HCV infection, since it is chronic infection that leads to the important morbidity and mortality associated with HCV infection. A vaccine that prevented acute HCV infection, as well as chronic infection, would, of course, be welcome. However, in its absence, a vaccine that failed to induce so-called sterilizing immunity but nonetheless resulted in self-limited infection only would be highly desirable.
While the incidence of acute hepatitis from HCV has been declining in the United States, no major reductions have been reported for nations of the less affluent, developing world. In the developing world, HCV infection continues to occur at high rates because of re-use of injection or other tissue-penetrating equipment in the practice of medicine and folk-medicine, failure to develop or utilize adequate blood donor screening technology and the continuing spread of injection drug use among the population. For these nations, the incorporation of an effective and safe HCV vaccine into childhood immunization programs will be essential. In the United States, based on experience with other hepatitis vaccines, HCV vaccine would be initially targeted to high-risk groups and only later recommended for childhood immunization.
Obstacles to HCV Vaccine Development
The heterogeneity of HCV, as reflected by the existence of six distinct clades, multiple genotype subtypes and quasispecies, together with evidence for superinfection by one genotype in individuals infected by another, has been viewed as potential impediments to vaccine development. If cross-protection between the recognized genotypes is non-existent, an effective HCV vaccine would need to incorporate antigenic domains from the commonest genotypes. Other obstacles are the absence of small animals susceptible to HCV and the limited availability and high cost of the only other susceptible primate -- the chimpanzee.
Another obstacle to vaccine development has been the lack of a tissue culture system supporting high-level HCV replication. However, the cultivation of RNA replicons, permitting high- level RNA replication, has been reported recently.(12),(13) Of course, once a vaccine is developed, protective efficacy and safety need to be established in trials, the duration of protection and need for boosters must be determined and the presence or absence of an influence of this vaccine on the immune response to other commonly used vaccines must be assessed.
HCV Neutralizing Antibodies
HCV infection elicits neutralizing antibodies but their titer in the circulation and their potency are believed to be less than optimal. The humoral response is restricted, isolate-specific and, over time, changes. In a number of studies, in vitro incubation of HCV with plasma containing HCV antibodies appeared to prevent infection when the material was used to inoculate chimpanzees. In other chimpanzee studies, antibodies induced by inoculation of a synthetic HCV peptide were shown to neutralize HCV when mixed with HCV in vitro.(14) Commercially available preparations of conventional human immune globulin for intramuscular or intravenous administration provide no protection against HCV infection.
Whether a hyperimmune globulin with high titers of neutralizing antibodies against HCV could be utilized for post-exposure prophylaxis remains uncertain. In limited studies, a human plasma-derived HCV "hyperimmune" globulin did not induce sterilizing immunity but may have restricted the extent of intrahepatic HCV replication in a study in chimpanzees.(15)
Approaches to Vaccine Development
In the absence of a current technology permitting the attenutation of HCV by serial passage in tissue culture, prospects for a live, attenuated whole-virus vaccine are extremely limited. Furthermore, because of the possibility of reversion to wild-type and the development of chronic infection, even if a live, attenuated HCV vaccine could be developed, it is unlikely to ever be tested or used in human subjects. More feasible approaches to HCV vaccine development have included the use of purified recombinant HCV antigens, recombinant viruses incorporating HCV antigenic material and the use of genetic, DNA-based immunization.
Envelope Glycoprotein Prototype HCV Vaccine
In initial studies published in 1994, chimpanzees immunized with a prototype vaccine consisting of the envelope glycoproteins (E1/E2) of HCV and containing both MF-59 (a proprietary adjuvant) and muramyl dipeptide adjuvants were reported to demonstrate high circulating titers of neutralizing antibodies in the post-inoculation period.(16) Challenges with low doses of the same strain of live HCV when given at the time that peak antibody levels were reached resulted in complete protection (sterilizing immunity) in five of the 12 immunized animals. Importantly, in five chimpanzees in whom acute infection occurred despite immunization, none developed evidence of chronic infection.
Although work on this prototype HCV vaccine continues, published information about protection with heterologous challenges and studies about the duration of protection following immunization are very limited.(17) Nonetheless, this vaccine is currently being assessed for both immunogenicity and safety. It has been suggested that so-called immune-stimulating complexes, containing the well-conserved (among the various HCV genotypes and subtypes) HCV core protein, might serve as an effective adjuvant for the envelope glycoprotein vaccine.(18)
DNA-Based HCV Vaccine
DNA complementary to the RNA of HCV (cDNA) has been used to inoculate mice in an attempt to develop genetic immunization. Both humoral and cell-mediated immune responses to the HCV envelope and core proteins were demonstrated but multiple inoculations usually have been required and the responses generally have been weak.(19)
Genetic immunization, utilizing synthetic epitopes of the hypervariable region 1 (HVR1 ) of the envelope glycoprotein (E2), induced cross-reacting antibodies against many naturally occurring HVR1 variants in mice and rabbits.(20) In a challenge study in the chimpanzee, immunization with a plasmid DNA, encoding the cell-surface E2 glycoprotein, was shown to induce both antibodies and cell-mediated responses to E2. On challenge with homologous HCV, sterilizing immunity could not be achieved. However, liver injury appeared earlier and resolved completely in the immunized chimpanzees, whereas the infection became persistent in the one and only non-immunized, control chimpanzee.(21) While the number of chimpanzees in this study limits interpretation, the available data are promising and suggest that this vaccine might indeed prevent chronic infection, the major goal of HCV vaccine development.
Other Novel Approaches
In the search for an HCV vaccine, a considerable body of innovative research has been undertaken. For example, a tobacco plant-derived vaccine, expressing the HVR1 region of E2, has been reported to induce antibodies binding HCV after intranasal immunization of mice.(22) Recombinant virus-like particles of the hepatitis B surface antigen (HBsAg) of the hepatitis B virus, containing sequences from the HCV E2 glycoprotein, also have been reported to induce antibodies in mice.(23) Synthesis of HCV-like particles in insect cells through use of a recombinant baculovirus containing the cDNA of the structural proteins of HCV is another potential source of material for use in vaccine development.(24) HCV recombinant viral vector systems, using a number of agents such as adenovirus,(25) pox viruses(26) and sindbis virus,(27) also are under study.
Summary
Persistent infection by HCV is responsible for most of the morbidity and mortality associated with hepatitis C. A considerable body of research on the molecular biology and immunology of HCV infection is being directed toward increased understanding of the factors responsible for persistence of infection. While immune evasion because of selection of resistant strains has received considerable attention and is supported by the known genomic heterogeneity of HCV, an inadequate immune response may also play a role.
Application of the concepts learned from further studies of the molecular biology of HCV may be useful for the design of immunoprotective vaccines. Development of a vaccine that completely prevented HCV infection would certainly be an outstanding achievement; development of a vaccine that reduced the risk of chronic infection would have a major impact on global health.