Vaccination for Hepatitis C Virus

Closing in on an Evasive Target

John Halliday; Paul Klenerman; Eleanor Barnes

Expert Rev Vaccines. 2011;10(5):659-672.

Vaccine Approaches: Current Status

Over the last decade numerous HCV vaccine approaches have been assessed in mice and primates. Only a small fraction of animal HCV vaccine studies have progressed to human trials. The majority of these trials have evaluated potential therapeutic vaccines in HCV-infected patients. A smaller number have assessed vaccines in healthy volunteers; either with the aim of developing a prophylactic HCV vaccine or as a bridge to evaluating vaccine in HCV-infected patients.

The question as to which HCV antigen a vaccine should target is a key one. The envelope region, which is essential for viral cell entry, may seem the obvious target for a prophylactic HCV antibody-inducing vaccine – but as discussed previously, the major antigenic determinants of the envelope protein are hypervariable both between, and within, infected individuals. Chimpanzee data have demonstrated that the induction of HCV envelope antibodies will afford protection with challenge with homologous viral strains only.^[65] This view has been recently challenged using collated chimpanzee data that have demonstrated protection from heterologous viral strains (both genotype-1a),^[66,67] and cross-genotype neutralizing antibodies have been demonstrated using a SCID mouse model transplanted with human hepatocytes.^[68] A prophylactic vaccine that induces anti-envelope immunity and attenuates the course of primary infection either alone, or in combination with other approaches, remains an attractive goal.

The HCV core protein might seem the obvious candidate for a therapeutic T-cell vaccine, since this is the most highly conserved region of the translated HCV genome both within, and between, different HCV genotypes. However, studies have shown that the core protein can interfere with innate and adaptive anti-HCV immune responses.^[69,70] Furthermore, our own data suggests that in persistent infection, anticore T-cell responses are frequently detected in the absence of viral escape, suggesting that these responses in particular are unable to control viral replication.^[71] Many core-based DNA vaccines have been tested in small animal models, and clearly robust anticore cellular responses can be generated. However, a heterologous prime–boost (plasmid-encoding DNA, boost with recombinant protein) vaccination strategy in chimpanzees vaccinated with core, E1, E2 and NS3 failed to induce any anticore responses.^[72]

For these reasons, the most recent strategies have focused on inducing T-cell responses to the NS HCV antigens, which are genetically conserved compared with the HCV envelope, and which are known to contain multiple CD4⁺ and CD8⁺ T-cell epitopes.^[73]

Four main vaccine strategies have been investigated in human clinical studies: recombinant protein vaccines, peptide vaccines, DNA vaccines and vector vaccines. The advantages and limitations of each of these approaches, in combination with a summary of human trials in HCV vaccine development, will be outlined in the following sections.

Recombinant Protein Vaccines

The use of recombinant proteins as potential vaccine candidates assumes that inducing an immune response to a limited number of viral epitopes is sufficient to develop protective immunity. The principle of this approach is to isolate the gene(s) encoding the appropriate protein and clone it in bacteria, yeast or mammalian cells. Recombinant proteins are prepared either from culture medium or transfected cells. While some recombinant proteins are sufficient in isolation to elicit a strong immune response, others require adjuvant therapy. Generally, protein-based approaches induce antibody and CD4⁺ T-cell responses.

Envelope Protein Vaccines

The hepatitis B vaccine was the first successful recombinant protein vaccine used in humans. This vaccine employs a conserved hepatitis B surface antigen and is effective in preventing hepatitis B infection through the production of antibodies. By contrast, the genetic variability of the HCV viral envelope, which is the main target for anti-HCV antibodies, makes such an approach for a HCV vaccine challenging. Nevertheless, recognition that the presence of pre-existing antibodies to HCV envelope proteins is associated with a better response to PEG-IFN therapy,^[74] and that anti-envelope antibodies can lead to an attenuated course of primary infection,^[75] has led to therapeutic and prophylactic vaccine studies, respectively, which aim to induce anti-envelope antibodies.

Prophylactic Vaccine The only published clinical trial of a prophylactic vaccine for HCV utilized a recombinant E1/E2 heterodimer adjuvanted with adjuvant MF59C (an oil-in-water emulsion).^[76] This placebo-controlled, dose-escalation Phase I study evaluated the vaccine in 60 healthy subjects. All subjects developed neutralizing antibodies and T-cell lymphocyte proliferation responses to E1/E2 and an inverse response to increasing amounts of antigen was noted. The vaccine was well tolerated. The study authors suggest that larger clinical trials to evaluate vaccine efficacy are indicated.

Therapeutic Vaccines The first candidate therapeutic vaccine for HCV was administered to humans in 2003 (Table 1).^[77,78] The vaccine consisted of a recombinant HCV-E1 protein in alum adjuvant. It was administered over 6 months via multiple injections to 20 healthy, and 34 chronically infected, treatment-naive patients. The vaccine induced HCV-specific antibody and T-cell responses in both patient groups (50 out of 54). Assessment of efficacy showed no change in HCV RNA levels but, in some subjects, improvements in liver histology were seen; a total of 24 HCV-infected patients underwent liver biopsies before and after vaccination. In nine of these patients there was histological improvement after 17 months. The observed increase in anti-E1 antibody levels correlated with improvement in liver histological scores and reduction in serum alanine transaminase levels (a measure of liver inflammation).

As a result, this work progressed to a placebo-controlled, multicenter trial (presented in abstract form in 2008^[79]) that evaluated 122 patients who received four courses of six injections over 3 years. Humoral and cellular immune responses to the E1 protein were induced but vaccination did not prevent histological progression of liver disease.^[79] Innogenetics, the company investigating this vaccine, ceased its program in 2008 and no further work has been published.

Core Proteins

Heat-killed yeast cells (Saccharomyces cervisiae) expressing conserved core–NS3 fusion protein have been trialed as a therapeutic vaccine candidate (GI5005).^[80] In a Phase II, placebo-controlled trial, GI5005 was combined with standard therapy (PEG-IFN/ribavirin) in 66 chronic HCV-1 patients. The protocol consisted of a 12-week run-in of standard therapy, followed by weekly doses for 5 weeks followed by monthly doses for 2 months of GI5005 vaccine, administered subcutaneously. Prior nonresponders received 72 weeks of standard therapy while treatment-naive patients received 48 weeks. No data on immunological response have been published. The investigators report an increase in SVR rates in patients homozygous for the IFN-λ3 risk alleles.^[81] Published peer-reviewed data are awaited.

A vaccine using conserved HCV core protein with an adjuvant composed of saponin, cholesterol and phospholipid, called ISCOMATRIX®, has been evaluated in a Phase I trial of 30 healthy volunteers.^[82] The vaccine was safe and all eight volunteers who received the highest dose (50 µg) developed a specific humoral response to the core protein. However, HCV-specific CD8⁺ T cells could only be detected in two patients. Further studies are planned by the same investigators to evaluate this approach as a therapeutic vaccine in HCV-infected patients.

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Table 1. Recombinant protein hepatitis C vaccine studies.
Structure (Investigator)	Phase (year)	Subjects	Outcome	Ref.
HCV E1 + alum adjuvant (Innogenetics/GenImmune)	I (2003)	20 healthy volunteers + 34 HCV-infected, treatment-naive patients	Well tolerated. No HCV viral load change. 50 out of 54 patient T-cell response. Humoral response correlated with improvement in ALT and liver histology	[77,78]
	II (2008)	122 HCV-infected patients randomized (3:1)	Well tolerated. Induced humoral and cellular immune responses. No change in histological progression of liver disease over 3 years	[79]
HCV E1/E2 glycoproteins/MF59C adjuvant (Novartis)	I (2010)	60 healthy volunteers	Well tolerated. Induced antibody and E1/E2-specific T-cell responses in all patients independent of vaccine dose	[76]
GI5005: recombinant yeast transfected with HCV NS3–core fusion protein (Globeimmune)	II (2009)	66 patients with chronic HCV-G1 (treatment-naive and nonresponders)	17% SVR with vaccine + standard care, compared with standard therapy response of 5% in prior nonresponders	[80]
HCV core protein/ISCOMATRIX® (CSL Ltd)	I/IIa (2009)	30 healthy volunteers	Well tolerated, mild local redness; all developed antibody response	[82]

Recombinant protein hepatitis C vaccine advantages: well tolerated with low toxicity; induces cross-neutralizing antibodies; proof-of-concept (HBV vaccine).
Disadvantages: generally only weak T-cell responses elicited.
ALT: Alanine transaminase; HCV: Hepatitis C virus; SVR: Sustained virological response.

Table 2. Peptide hepatitis C vaccine studies.
Structure (Investigator)	Phase (year)	Subjects	Outcome	Ref.
IC41: five peptides from core, NS3 and NS4 + poly-l-arginine adjuvant (Intercell AG)	II (2008)	60 chronic HLA A2 HCV-infected nonresponders	Well tolerated. 67% T-cell response. Transient serum HCV RNA decline (>1 log) in three patients	[84]
Peptide derived from core protein (C35–44) (Karume University)	I (2009)	26 patients with chronic HCV (23 nonresponders, three untreated)	Well tolerated. 15 out of 25 responded, two out of 25 had a 1 log decline in HCV RNA	[90]
Personalized peptide approach CD8+ A24 peptides + Freund's adjuvant (Karume University)	I (2007)	12 chronic HCV-1b-infected nonresponders	Well tolerated. Majority developed peptide-specific T-cell responses. Five patients reduced ALT and three patients had a HCV RNA decline	[91]
Autologous dendritic cell delivered HLA A2 epitopes (Burnet Institute + others)	I (2010)	Six chronic HCV-infected nonresponders	Well tolerated. Transient T-cell responses	[92]
NS3/Virosome (Pevion Biotech)	N/A	30 healthy volunteers	Results pending

Peptide hepatitis C vaccine advantages: well tolerated with low toxicity; induces both cellular and humoral responses.
Disadvantages: limited immunogenicity (few epitopes only); need to tailor to patient HLA type; concern regarding appropriate peptide choice (e.g., induce Tregs rather than CD8⁺ cells).
ALT: Alanine transaminase; HCV: Hepatitis C virus; N/A: Not available.

Table 3. DNA hepatitis C vaccine studies†.
Structure/vector (Investigator)	Phase (year)	Subjects	Outcome	Ref.
CICGB-230: plasmid core/E1/E2 + recombinant core protein (University of Montreal + others)	I (2009)	15 genotype-1- infected nonresponders	Well tolerated, no viral clearance, 11 out of 15 – weak specific T-cell responses (ELISpot and proliferation). Six out of 15 – weak humoral response	[106]
ChonVac-C: plasmid NS3/4a + electroporation (Tripep)	I/IIa (2009)	12 HCV-1-infected, treatment-naive patients	Well tolerated. A total of four out of six who received higher doses: viral load reduction >0.5 log with corresponding T-cell response in three patients	[105]

†Ideal genes should represent part of the genome with limited genetic variability and maximal immunogenicity.
DNA hepatitis C vaccine advantages: presents a broad range of epitopes.
Disadvantage: electroporation is painful.
ELISpot: Enzyme-linked immunosorbent spot; HCV: Hepatitis C virus.

Table 4. Viral-vectored hepatitis C vaccine studies.
Structure/vector (Investigator)	Phase (year)	Subjects	Outcome	Ref.
TG4040: MVA vector expressing NS3/4/5B proteins (Transgene)	I (2009)	15 chronically infected HCV patients	A total of six out of 15 declined HCV viral load (0.5– 1.4 log) associated with T-cell response (IFN-g ELISpot)	[107]
Adenovirus vector (Ad6 and AdCh3) expressing NS3–5B proteins (Okairos and Oxford University).	I (2009)	36 healthy volunteers	Highly immunogenic and well tolerated	[73]

Viral-vectored hepatitis C vaccine advantages: efficient induction of cellular immune responses; presentation of a broader range of viral epitopes.
Disadvantages: pre-existing immunity to viral vector (adenovirus; may be overcome by the use of rare serotypes); limited experience in humans.
Ad: Adenovirus; ELISpot: Enzyme-linked immunosorbent spot; HCV: Hepatitis C virus; MVA: Modified vaccinia Ankara.

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