Why is it so difficult to develop a hepatitis C virus preventive vaccine?
With an estimated 3% of the world's population chronically infected, hepatitis C virus (HCV) represents a major health problem for which an efficient vaccination strategy would be highly desirable. Indeed, chronic hepatitis C is recognized as one of the major causes of cirrhosis, hepatocarcinoma and liver failure worldwide and it is the most common indication for liver transplantation, accounting for 40–50% of liver transplants. Much progress has been made in the prevention of HCV transmission and in therapeutic intervention. However, even if a new wave of directly acting antivirals promise to overcome the problems of low efficacy and adverse effects observed for the current standard of care, which include interferon-α and ribavirin, an effective vaccine would be the only means to definitively eradicate infection and to diminish the burden of HCV-related diseases at affordable costs. Although there is strong evidence that the goal of a prophylactic vaccine could be achieved, there are huge development issues that have impeded reaching this goal and that still have to be addressed. In this article we address the question of whether an HCV vaccine is needed, whether it will eventually be feasible, and why it is so difficult to produce.
No vaccine for the prevention of hepatitis C virus (HCV) infection is yet available. The current standards of care, based on the combination of interferon-α (IFN-α), ribavirin (RBV) and—for genotype 1 infections—NS3 protease inhibitors, still have suboptimal rates of success, are burdened by considerable side effects, and are unlikely to be widely used because of very high costs . Given that HCV is responsible for a substantial proportion of chronic liver diseases worldwide, including cirrhosis and hepatocellular carcinoma, and that more than 170 million people are infected , a prophylactic vaccine represents an unmet medical need that would be of great public health benefit. Indeed, vaccination has proved to be the most successful and cost-effective strategy to prevent infections and associated diseases.
Among the difficulties that have hampered the development of a vaccine against HCV there are its extreme genetic variability, the lack of small animal models for testing vaccines, and the fact that a cell culture system supporting the production of infectious HCV and allowing studies on virus neutralization in vitro became available only recently [3-5].
Despite these issues, much progress has been made toward the identification of promising prophylactic vaccine candidates, and some of these candidate vaccines have entered clinical trials. However, several issues concerning development and efficacy assessment of HCV vaccines remain to be addressed. Should these be overcome, the control of HCV spread would appear a realistic goal to achieve.
Hepatitis C virus is a small, 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 about 3000 amino acids, which, upon translation, is cleaved into ten different structural (core and envelopes gpE1 and gpE2) and non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins . An impressively high replication rate (1012 virions/day)  and the lack of proof-reading activity by the viral RNA-dependent RNA polymerase account for the high genetic variability of the virus . Seven major genotypes (with 30–35% variability in nucleotide sequence) and many subtypes (15–20% variability) have been distinguished phylogenetically. Moreover, HCV circulates within an infected individual as distinct ‘quasispecies’ .
Acute HCV infection is usually subclinical and 50–85% of infected individuals develop chronic infection, a condition leading to cirrhosis and, in some cases, to liver failure or hepatocellular carcinoma over a period of 10–30 years . Chronic HCV infection is one of the main causes of liver cirrhosis and cancer [12, 13] and >350 000 people die from hepatitis C-related liver diseases every year. Moreover, chronic HCV infection has been associated with several extrahepatic diseases [14, 15] including mixed cryoglobulinaemia  and non-Hodgkin's B-cell lymphoma .
Transmission of HCV occurs via direct parenteral exposure to contaminated blood. HCV epidemiology has changed over the last two decades: transfusion-associated transmission has disappeared with the advent of serological tests based on the detection of antibodies or HCV RNA. At present, a history of intravenous drug injection represents the major risk factor for newly acquired HCV infection (65%) , followed by sexual (15%)  and occupational (4%) risks. Vertical transmission occurs particularly in mothers with high viral load or who are co-infected with human immunodeficiency virus (HIV) (<5%) [20-22].
Do We Need a Preventive HCV Vaccine?
Despite a substantial decline in HCV transmission due to improved prevention strategies and the introduction of new powerful targeted therapies, hepatitis C remains a huge health problem and there are several reasons for continuing to pursue the objective of a vaccine. On the one hand, mortality and morbidity due to HCV are not going to decrease. HCV has surpassed HIV as a cause of mortality in the USA  and the burden of HCV-related liver disease is projected to increase in the next decade . Indeed, the pool of asymptomatic chronic HCV carriers who represent an infectious reservoir will remain substantial for many years and c.50 000 new infections still occur annually in the USA and Europe (CDC data). On the other hand, therapeutic approaches to HCV raise many concerns: current treatments, based on the IFN-α/RBV backbone, are expensive, poorly tolerated and lead to sustained virological response in about half of patients. New directly acting antivirals that target specific HCV proteins are emerging for the treatment of hepatitis C and results of late-stage clinical trials with IFN-free combinations of directly acting antivirals suggest that highly effective therapy to cure HCV infection will be available in the near future . However, the very high costs make new antiviral therapy unlikely to be sustainable for public health systems. Moreover, the treatment rate is very low for HCV-infected patients. Indeed, <30% of patients with chronic hepatitis C are aware of the infection and only about 10% of patients are currently treated [25-27]. Therefore, even if new antivirals could cure 90% of patients there would still be a considerable percentage of patients that would be excluded.
An efficient HCV vaccine would represent the most cost-effective and realistic means to significantly reduce the worldwide mortality and morbidity associated with HCV infection. An HCV vaccine would be initially indicated for persons at risk of coming into contact with contaminated blood, whereas a universal vaccination strategy could be recommended later.
Is a Preventive HCV Vaccine Doable?
The observation that effective immune responses may occur during the natural course of HCV infection supports the feasibility of an effective vaccine. Indeed, in contrast to HIV infection, a sizable proportion (20–35%) of infected persons can clear the virus spontaneously. Generally, this is observed in the first 6–9 months of infection [28, 29], and it has been shown that individuals who have cleared infection are less likely to develop chronic infection when re-exposed [30, 31]. Collectively these data provide evidence that natural immunity to HCV exists and that cross-protective immunity within and between common HCV genotypes can be achieved.
Adaptive immunity is unequivocally mediated by one or more of the following: neutralizing antibodies, CD4+ T-cell-mediated inflammation and CD8+ T-cell-mediated cytotoxicity. In the case of HCV, there is strong evidence that the rapid induction of high-titre cross-neutralizing antibodies targeting HCV envelope proteins correlates with viral clearance and protects from re-infection [31-34]. On the other hand, the importance of broad HCV-specific CD4+ T helper and CD8+ cytotoxic responses in the resolution of infection has been suggested by many studies [28, 29, 35-40]. Therefore, an optimal HCV vaccine probably needs to elicit broad cross-reactive cellular immune responses together with cross-neutralizing antibodies.
As it is the development of chronic infection that causes clinically evident disease, whereas acute infection is mostly silent and without clinical consequences, an HCV vaccine that prevented chronic infection, allowing a self-limited acute infection, would be acceptable.
Several vaccine strategies, listed in Table 1 and reviewed elsewhere [41-43], have been explored to identify suitable candidates, including subunit vaccines and genetic vaccines (plasmid DNA and viral vector vaccines).
Table 1. Clinical-stage vaccine candidates for hepatitis C virus (HCV)
|Chiron/Novartis [46, 62, 63]||Recombinant protein||Recombinant gpE1/gpE2 in oil/water adjuvants||Phase 1 [46, 63]||Prophylactic vaccine|
|Okairos ||Adenoviral vector vaccine||Adenovirus vectors expressing HCV NS 3, 4 and 5||Phase 2||Prophylactic/Immunotherapeutic vaccine|
|Innogenetics/GenImmune ||Recombinant protein||Alum-adjuvanted gpE1 glycoprotein||Phase 2  Programme stopped as no effects on viraemia or fibrosis progression||Immunotherapeutic vaccine|
|Intercell AG (IC41) [67, 68]||Peptide||HCV peptide cocktail with polyarginine||Phase 2 in combination with pegylated interferon [69, 70]||Immunotherapeutic vaccine|
|Globe Immune (GI-5005)||Recombinant yeast cells||Heat-killed yeast expressing Core-NS3 fusion protein ||Phase 2 in combination with pegylated interferon [72, 73]||Immunotherapeutic vaccine|
|Tripep (ChronVac-C)||Plasmid DNA||DNA-based vaccination with NS3/4A-expressing plasmid||Phase 1b||Immunotherapeutic vaccine|
|CIGB-230 ||Plasmid DNA and recombinant protein||Mixture of plasmid-expressing HCV structural antigens with a recombinant HCV core protein||Phase 1b ||Immunotherapeutic vaccine|
|Transgene (TG4040)||Adenoviral vector vaccine||Modified vaccinia Ankara virus expressing NS proteins (NS3-NS5B)||Phase 1||Immunotherapeutic vaccine|
Promising results have been obtained with adjuvanted recombinant gpE1/gpE2 heterodimer vaccine (Tables 2 and 3). When used to immunize naive chimpanzees that were then challenged with a heterologous HCV strain, only one out of nine developed chronic infection , whereas most control animals became chronically infected following viral challenge. Vaccination induced inflammatory CD4+ T-cell responses as well as high titres of anti-gpE1/gpE2 antibodies capable of blocking the binding to CD81, the main HCV receptor on hepatocytes. Moreover, although immunization with polypeptide subunits does not result in direct stimulation of CD8+ T cells, a high number of intrahepatic, HCV-specific CD8+ cytotoxic T lymphocytes secreting IFN-γ was observed in vaccinated chimps 10 weeks after the challenge, and the percentage of these cells was inversely correlated with viral loads . However, not all animals with high anti-E1E2 titres and/or high CD8+ T-cell activity were able to eradicate the infection even though acute viraemia was suppressed. As the result of encouraging preclinical results, the recombinant gpE1/gpE2 heterodimer, formulated in an oil–water adjuvant MF59, was among the earliest preventive vaccine candidates to enter clinical trials. A phase I study has revealed that this vaccine is safe and well tolerated and that it elicits anti-gpE1/gpE2 antibodies able to cross-neutralize major HCV genotypes and CD4+ T-lymphocyte responses to HCV envelope proteins (Table 3) [46, 47].
Table 2. Outcome of chimpanzee vaccination/challenge studies performed with adjuvanted recombinant gpE1/gpE2 formulations
|Acute infections||7/12a||10/10|| |
|Chronic infections (%)||2/12 (17)||7/10 (70)||0.03|
|Acute infections||9/9||14/14|| |
|Chronic infections (%)||1/9 (11)||8/14 (57)||0.04|
|Acute infections||16/21||24/24|| |
|Chronic infections (%)||3/21 (14)||15/24 (63)||0.002|
Table 3. Summary of main results obtained for adjuvanted gpE1/gpE2 heterodimer vaccine and for NS3-5B adenoviral vaccine
|Chiron corp gpE1/gpE2 Heterodimer||Subunit vaccine||Preclinical||Prophylactic efficacy in Chimpanzee model||Completed||Protection from chronic hepatitis after challenge with homologous and heterologous strain||High titres of anti-E1E2 nAb, which correlate inversely with viral titres; hepatitis C virus (HCV) specific CD4+ T-cell response|
|Phase I||Blinded randomized dose-ranging study in 60 healthy volunteers||Completed||Satisfactory safety and tolerability||Anti-E1E2 antibodies able to cross-neutralize major HCV genotypes with titres between 100 and 2000 (protected chimps range); very strong CD4+ T-cell responses to E1E2|
|Phase II||Proof-of-concept field trial in high-risk subjects||Stopped at planning stage|| || |
|Okairos NS3-5B||Adenoviral vector vaccine||Pre-clinical||Prophylactic efficacy in Chimpanzee model||Completed||Amelioration of acute hepatitis and acute viraemia in vaccinees after challenge with heterologous strain. No significant difference in carrier rates between vaccinees and controls||Broad CD4+ and CD8+ T lymphocytes|
|Phase I||Blinded randomized study in 40 healthy volunteers||Completed||Safe and highly immunogenic||Broad CD4+ and CD8+ T lymphocytes secreting multiple cytokines (interleukin-2, interferon-γ, tumour necrosis factor-α) targeting multiple epitopes and recognizing heterologous strains. Presence of polyfunctional and proliferative long-term memory population after 1 year|
|Phase I/II||Randomized, double-blind, placebo control in intravenous drug user population; (Phase I: 68 volunteers; Phase II: 276 additional volunteers)||Ongoing (results expected in 2015/2016)|| || |
It should be pointed out that the relevance of a humoral response in resolving HCV infection is still controversial. Anti-envelope antibodies are detected in nearly all chronically infected patients but do not always have neutralizing properties. However, the advancements in design and selection of immunogens may greatly improve the efficacy of a vaccine based on the elicitation of antibody response. For instance, it is now possible to interrogate in great detail the B-cell repertoire of HCV-infected patients, in particular those patients that have cleared acute HCV infection, and to identifiy several HCV cross-neutralizing antibodies that in turn could lead to the identification of E1E2 broadly neutralizing epitopes. This approach may guide the structural design of novel E1E2 antigens with improved efficacy. The same approach has been extensively adopted in the HIV field with impressive results, and a few HCV broadly neutralizing antibodies have already been described and reviewed .
T-cell-based vaccines have also been proposed as vaccine candidates [43, 49]. Adenoviral vectors are particularly attractive for this approach because they are the most potent at T-cell priming in primates and humans . The main problem of adenoviral vectors is pre-existing immunity to the vector, which can lead to its clearance before a response is elicited to the inserted immunogen. However, this issue has been overcome by using chimpanzee adenovirus as the gene vehicle . Good results in terms of elicitation of broad CD4+ and CD8+ T-cell response in preclinical studies have been obtained for one of these vaccines, consisting of a chimpanzee adenoviral vector delivering non-structural genes NS3–NS5B, which contain many well-defined CD4+ and CD8+ epitopes (Table 3). After heterologous viral challenge, all animals became HCV-infected but those that were vaccinated presented a 100 times lower average peak of viraemia than controls. Moreover, three out of four chimpanzees presented viraemia of short duration . This T-cell-based vaccine has been shown to be safe, well tolerated and highly immunogenic in phase I studies and has now moved to a phase II trial .
Overall, these results suggest that the goal of an effective vaccine, protecting from different HCV strains and preventing chronic infection, should be achievable and could be obtained by strategies that combine the elicitation of broadly cross-neutralizing antibodies with the induction of strong HCV-specific T-cell responses.
Why Is It So Difficult to Make One?
In contrast to another epidemic hepatotropic virus, hepatitis B virus (HBV), for which a vaccine has been successfully developed, a vaccine against HCV represents a significant challenge. Many viral and non-viral factors can be taken into account to explain why it is so difficult to develop an HCV vaccine (Table 4).
Table 4. Summary of the main obstacles to the development of an effective hepatitis C virus (HCV) vaccine as compared to the successfully developed hepatitis B virus (HBV) vaccine
|Viral factors||High rate of development of chronic infection following asymptomatic acute infection in most immunocompetent hosts; High genetic variability between different genotypes (30–35%); High variability and heavy N-glycosylation of external proteins; Several mechanisms of evasion from host innate and adaptive immune system evolved||Asymptomatic or clinical acute infection with rare chronic infection development in most immunocompetent hosts; Low genetic variability between different genotypes (8%); The envelope protein (hepatitis B surface antigen) is very conserved and have minor post translational modifications|
|Efficacy assessment factors||No clear correlate of protection identified; No simple cohort available for efficacy trials; No immunocompetent small animal model available; Prevention of chronic infection is technically difficult to assess||Neutralizing antibodies represent correlate of protection; Efficacy trials relatively easy and doable in high incidence populations at time of vaccine development|
|Technical issues||The glycoproteins E1/E2 are very complex to scale up, because they have to be purified as intracellular material from the endoplasmic reticulum of mammalian cells||Recombinant hepatitis B surface antigen is easily produced in yeast cells|
|Other factors||The myth has been spread that HCV will disappear in a few years and that in any case new antiviral agents will finish the job; as a result there private or public investments in the HCV vaccine field are scarce|| |
| ||Vaccine still under development||Highly efficacious vaccine available (estimated >1 000 000 deaths prevented worldwide)|
Hepatitis C virus displays extremely high genetic diversity (30% of nucleotide divergence between genotypes) compared with HBV (8% divergence between different genotypes) . HCV has evolved several effective immune escape strategies, leading to a very high rate of chronic hepatitis following acute infection (50–80%). Conversely, only 5–10% of immunocompetent adults develop chronic hepatitis after acute infection with HBV . The mechanisms of HCV evasion from the host immune system affect both innate and adaptive immune responses. Evasion from the innate immune system includes impairment of dendritic and natural killer cell responses and reduction of type-I IFN induction [56-59]. Among the mechanisms used to escape from the adaptive immune response, of crucial importance are the continuous generation of new viral variants occurring during HCV infection, allowing the virus to escape the action of HCV-specific CD8+ T cells and neutralizing antibodies, and the exhaustion of CD8+ T cells observed during chronic infections, which leads to a reduced ability both to secrete antiviral cytokines (including IFN-γ) and to proliferate in response to antigen stimulation . Another evasion mechanism is the ability of HCV to be transferred to naive cells through direct cell–cell contact in a CD81-independent way, so avoiding the effect of neutralizing antibodies . Finally, single point mutations, glycosylation site modifications and conformational changes occur in HCV glycoproteins during HCV evolution and contribute to the virus' adaptation to humoral pressure. Conversely, the HBV envelope protein (i.e. the hepatitis B surface antigen, or HBsAg), is highly conserved and undergoes minor post-translational modification.
Apart from these intrinsic obstacles, the exact correlate of immune protection against HCV infection has not been identified, possibly because of the heterogeneity contained within small study populations of patients together with the limitations in terms of animal models of HCV infection.
The planning and execution of clinical trials for efficacy assessment pose many questions as well. Because of the relatively low incidence of new infections in the developed world, it is not trivial to identify the appropriate at-risk population to enrol. Post-transfusion infection risk has been eliminated by donor screening, and other high-risk groups suitable for efficacy testing can have inherent difficulties such as lack of compliance (in intravenous drug users), low incidence of infection (healthcare workers), lack of supporting infrastructure (in many developing countries where incidence of infection is high) and ethical issues (in prisoner populations where prevalence and incidence of infection are both high). However, some of these cohorts have been used successfully for testing HBV vaccine, and so these obstacles should not be insuperable.
As discussed above, in the case of HCV, a vaccine that allowed only a ‘transient infection’, while preventing the development of chronic HCV infection, would be as beneficial as one that provided sterilizing immunity. However, the design of an efficacy trial intended to measure prevention of chronic infection is not straightforward, in particular in the absence of a strong correlate of immunity. In fact, studies on patients with acute hepatitis C have shown that HCV infections treated with pegylated IFN-α during the acute phase are eradicated in virtually all patients . As the chronic state of the infection is defined starting from 6 months after onset, it becomes very difficult to conclude an efficacy trial. When an infected vaccinee is detected, IFN treatment will have to be started without delay and it will be impossible to establish whether the infection will become chronic. For this reason, for example, a phase II, proof-of-concept trial with adjuvanted E1/E2 heterodimer was stopped at planning stage.
Finally, developmental issues exist for gpE1/gpE2 vaccine. Indeed, the heterodimer has to be purified as intracellular material from endoplasmic reticulum of mammalian cells, making it very difficult to scale up its production.
Only a few years ago, the prospects for effective vaccination against HCV were considered remote. The situation today is more optimistic for several reasons. First, we now know that the spontaneous eradication of virus occurs in a consistent fraction of acute infections and is associated with specific immune responses to the virus. Second, clear evidence indicates that, despite the high percentage of chronic HCV infections, the immune system is capable of a response that leads to HCV clearance and protection against re-infection.
In light of this, several efforts have been made to identify and develop vaccine candidates recapitulating these immune responses. Some of these candidates have given promising results in the chimpanzee model of infection and have subsequently moved to clinical trials. However, many issues still remain to be resolved before a vaccine against HCV can be achieved. A clear correlate of protection has not yet been identified. Moreover, not only is it difficult to identify the appropriate at-risk population to enrol in an efficacy trial for a preventive HCV vaccine, but it may also be very difficult to conclude an efficacy trial designed to measure prevention of chronic infection. If these obstacles are removed, the prospect of a vaccine appears a realistic objective for the near future. Considering that HCV is the first cause of primary liver cancer, this would mean that an important cause of global morbidity and mortality would be controlled.
The authors declare no conflicts of interest.