With 170 million people infected with HCV worldwide, the development of an effective vaccine against HCV is essential. Fortunately, advances in blood screening have led to a decline in the rate of HCV transmission in developed countries. However, in the United States, intravenous drug use still accounts for approximately 60% of HCV infections (see Epidemiology). Furthermore, 10% of the annual HCV infections are not correlated with any clear-cut risk factors, and this number is not likely to decrease (3). These routes of infection, in addition to the one hundred million carriers worldwide, represent a massive reservoir harboring HCV infection.
The need for an effective
vaccine is intensified by the lack of any widely administerable treatment.
Current combination therapy is both expensive and largely ineffective (see
Vaccine
Attributes:
For an HCV vaccine to be
considered effective it would have to display certain key attributes. Since
HCV is primarily a blood borne disease, a vaccine would need to be administerable
to people at high risk for this route of transmission (1).
Such individuals include health care workers, hemodialysis patients, and
injection drug users. Because IV drug abuse begins around early adulthood,
a useful vaccine would likely be targeted at young adolescents before exposure
to drug use.
There has been much discussion
over exactly what type of protection an efficacious HCV vaccine must confer
to recipients. While sterilizing immunity (or the prevention of initial
infection altogether) would be ideal, it is not considered necessary in
the case of HCV. It is believed that a vaccine which allows for transient
infection, while preventing the development of chronic HCV infection, would
prove sufficient (3).
The most daunting task for
designers of an HCV vaccine is the tremendous diversity of wild type strains
(see Virology - Diversity). A
vaccine would have to be active against at least one genotype (3).
Genotype 1 accounts for 60-90% of the HCV strains in the United States,
Europe, and Japan, so an effective vaccine for these areas would have to
protect against this genotype. It is hoped that even a genotype-specific
vaccine would exhibit graded, but clinically measurable, protection from
infections with other genotypes (3).
An obvious but extremely
important characteristic of any potential vaccine is that it confer long
lasting memory in recipients (3). The need for
repeated booster shoots would significantly lower vaccine efficacy, particularly
when targeted at developing countries.
Challenges
to Vaccine Development:
The problems faced in the
development an HCV vaccine have been compared to those faced by HIV vaccinologists.
Both HCV and HIV are long-term, chronic infections. Both also display high
diversity (especially in envelope proteins) which make it difficult to
select an ideal epitope. However, unlike HIV, HCV has been shown to resolve
spontaneously in 15-25% of infections. This suggests that a natural state
of immunity to HCV does exist (1). By determining
what immunological response generates clearance of the hepatitis C virus,
it may be possible to develop a vaccine that mimicks this activity. Also,
because HCV does not integrate into the host genome, cells infected with
HCV display viral antigens (Ags), which can be targeted by the immune system
(1).
Difficulties in growing HCV
in cell culture has limited the assessment of antibody (Ab) response. A
breakthrough has recently been made with the development of a quick assay
to determine the neutralizing ability of an Ab to glycoprotein E2 (gpE2)
expressed on HCV (1). Research has shown that gpE2 has a high affinity
for human cells, and that the binding ligand is the CD81 molecule (see
Virology). The result
has been the neutralization of binding (NOB) assay, otherwise known as
a cytofluorimetric assay. A good correlation has been established between
high titers on the NOB and those individuals who spontaneously clear HCV
infection. Low titers on the NOB have been associated with long term HCV
progressors (1). In the future, similar assays
-- which make use of recombinant receptor- and ligand glycoproteins --
will likely be an invaluable tool for evaluating potential neutralizing
Abs for HCV and other pathogens.
The lack of animal models
has also made vaccine trials difficult (3). The
closest model thus far has been the chimpanzee. Chimpanzees appear to be
susceptible to infection by all major genotypes of HCV, and they demonstrate
primary hepatotropic response with chronic persistent viremia. The key
shortcoming is that chimpanzees do not develop a histologic picture of
active hepatitis. In other words, while HCV is able to replicate
in the chimpanzee, it is unable to progress to full hepatitis infection
(3).
Theoretically, high circulating
titers of neutralizing Ab to immunodominant epitopes of HCV should provide
adequate protection from infiltration to hepatocytes (3).
In practice, the use of neutralizing Ab as a prophylactic agent against
HCV infection has been largely disappointing (1).
Neutralizing protective antibodies have been detected in a small proportion
of infected patients, and have been found to be strain-specific. In fact,
chimpanzees which have recovered from primary infection have been shown
to be vulnerable to re-infection by both identical or different strains.
In 1989, three randomized,
placebo controlled clinical trials were conducted to assess the effectiveness
of immunoglobulin (Ig) adoptive transfer to prevent transfusion-associated
hepatitis C. Unfortunately, the data gathered in these studies provided
no convincing evidence that passive immunization of neutralizing Abs affords
HCV protection (1).
Some experimental studies
have focused on HCV hyperimmune globulin as a means of both prophylactic
and therapeutic passive immunization. Hyperimmune globulin is Ab produced
to a synthetic peptide derived from the hypervariable region one (HVR1)
of gpE2. Abs against this epitope are neutralizing, albeit strain specific.
In chimpanzee models, hyperimmune serum has been shown to be both prophylactic
and therapeutic when administered immediately after virus exposure. Because
these Abs provide no protection against different heterogeneous strains,
and because passive administration induces no memory, hyperimmune serum
has limited utility.
Envelope
Glycoprotein Vaccines:
The most encouraging prototype
HCV vaccine developed to date makes antibodies to HCV envelope glycoproteins
E1 and E2 (3). A recombination vaccinia virus
vector for one strain of HCV is used infect HeLa cells from which glycoproteins
can be then extracted. Combined with adjuvant (MF59), these two glycoproteins
are administered in a series of three booster shots (3).
When challenged with low doses of the identical HCV strain, five of seven
chimpanzees appeared completely resistant to infection (sterilizing immunity).
These five animals all showed very high titers of neutralizing Abs (NOB
assay). The remaining two chimpanzees became acutely infected, but were
able to resolve the infection before it progressed to the chronic stage
(3).
The five animals which completely
resisted infection were later exposed to a different isolate from the same
subtype. While all five became infected, four failed to develop chronic
infection. NOB assay later demonstrated that antiserum containing anti-gpE2
to HCV subtype 1a also was able to cross-neutralize gpE2 from HCV subtype
1b (3). Though these results are somewhat encouraging,
further experiments are needed to determine the extent of cross-neutralization
this vaccine approach provides.
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The company Epimmune (visit
www.epimmune.com)
recently announced that it hopes to take its T-cell epitope based vaccine
into Phase I and II trials. The Epimmune vaccine makes use of a variety
of immunodominant conserved T-cell epitopes, designed to elicit both a
strong CD4+ and CD8+ cellular response (2). Appropriate
peptides are selected using a computer program known as the Rapid Epitope
Identification System (EIS). This program uses a database of sequenced
HCV proteins, from which it selects short peptide sequences, which are
both highly conserved and immunogenic, for use in the preparation of the
vaccine. Often, multiple peptides can be found, each of which react
with up to three superfamilies of HLA class I or II molecules, guaranteeing
a broad coverage (2):
Figure courtesy of Epimmune, www.epimmune.com
The cell-mediated response
is comprised of activated cytotoxic T cells (CTL), and helper T cells (HTL),
which are directed towards epitopes displayed by antigen presenting cells
(APC).
In order to maximize immunogenicity,
these peptides are subsequently conjugated to a carrier complex of Epimmune's
design known as PADRE. This carrier increases the magnitude and duration
of the immune response (2):
Figure courtesy of Epimmune, www.epimmune.com
This epitope based strategy offers a range of potential benefits. Firstly, because epitopes which are conserved can be chosen, it is unlikely that normal mutagenic mechanisms of HCV will allow for viral escape. Also, because Th and CTL epitopes differ (since they bind to different MHC classes), the relative proportions of Th1 and Th2 responses can be carefully controlled (2). As studies described in the section on Pathology indicate, variance in the severity of these responses differentiates long term progressors from non-progressors. The biggest benefit of this technique, however, is that it allows the selection of epitopes that are conserved over various genotypes of HCV (2). There is scope then, for one vaccine that cross-reacts with multiple strains of HCV.
Epitope based approaches do have certain disadvantages, however. Firstly, because peptides must be selected and tested for each HLA type, larger computer databases on MHC reactivity must be created so that predictability power can be increased. Also, a fully effective vaccine would most likely need to include some native protein, so as to be able to induce humoral immunity (2).
The main goal of all nucleic acid based vaccines is to deliver RNA into a target cell where HCV peptides can be expressed through the endogenous MHC class I pathway. MHC class I expression is a good stimulator of CTLs, which are deemed critical for suppression and elimination of HCV following infection. In addition, non-structural core proteins -- the immunodominant epitopes for T-cells -- are normally conserved across genotypes, so a vaccine would likely cross-react with multiple genotypes. Moreover, unlike epitope based vaccines, DNA vaccines co-elicit the humoral immune response (3).
The simplest form of nucleic acid based vaccines are naked DNA plasmids. The recipient is inject intradermally or intramuscularly with bare plasmids; a gene gun is sometimes used (3). Much research has focused on naked DNA vaccines encoding the nucleocapsid (C) gene of HCV (see Virology). The C gene has attracted a good deal of attention because it is largely conserved across HCV genotypes, and immune response to this protein has been correlated with recovery from acute infection (3). Also the C protein has been found to contain both strongly immunogenic MHC class I and II peptides. Mice immunized with C-protein DNA plasmids have produced both humoral and cell-mediated immune responses (3).
A host of experiments have also made use of HCV genes encoding nonstructural (NS) peptides such as NS3, NS4, and NS5 (see Virology). Again, both humoral and cell-mediated immune responses have been detected in mice, including a particularly strong CTL activation (1).
Despite the benefits of naked DNA vaccines, concerns about their safety remain. Exactly how many cells express the foreign DNA, and for how long, is currently unknown. Because the plasmids may or may not integrate into the cell genomes, it is unclear whether dividing cells inherit the foreign genes. Insertion mutations due to plasmid integration could potentially spur cells to transform into carcinomas. In fact, the 5' region of the NS2 gene has been found to be transforming in nude mice and in fibroblast cultures. The HCV C-peptide gene has also been found to inhibit the activity of the p53 promoter in mice, effectively eliminating an important anti-cancer regulator (3).
There is some concern about how constituent expression of foreign DNA could effect the immune response. Negative reactions could include either anergy or autoimmune reaction, directed towards self peptides or towards DNA.
Before an HCV vaccine based on naked DNA plasmids can be widely used, the above concerns need to be addressed, and improvements in immunogenicity need to be made. Despite these obstacles, HCV naked DNA vaccines hold tremendous potential because of their ability to react with a wide range of genotypes, and to stimulate both arms of the immune system. Naked DNA vaccines also are thought to represent a good route for therapeutic treatment -- a method which has thus far been largely unexplored (1).
Like naked DNA vaccines, viral vector vaccines are designed to insert foreign nucleic-acids into a host cell, thereby stimulating the immune system through both the MHC class I and class II pathways. But viral vectors vaccines have potential advantages over naked DNA vaccines; viral vectors allow specific host cells -- such as antigen presenting cells (APCs) -- to be targeted (3).
A viral vector can be chosen which does not integrate its genome into the genetic material of the host cell; instead, its RNA is confined to the cell cytoplasm. As the RNA degrades according to its half-life, the expression of foreign genes by the host cell declines. Thus, problems associated with constituent expression and insertion mutations are reduced. Replication-deficient alphaviruses have been identified as good carriers for potential HCV viral vector based vaccines (3). Few such vaccines have actually been tried. Consequently, little to nothing is known about the experimental efficacy of such vaccines with regard to the hepatitis C virus. Nevertheless, viral vectors represent a cutting-edge approach to HCV vaccine development, which promises to be a powerful tool for the design of an effective vaccine (1).
Clinical Testing for HCV Vaccine Candidates:
Clinical testing for candidate HCV vaccines poses a host of difficulties. Chimpanzees have been helpful as early screeners for vaccine candidates, but considerable discrepancies in immune responses between human beings and chimpanzees have limited the utility of these animals as meaningful models.
Two important factors to consider in designing a vaccine efficiency trial are the efficacy end-points and the identification of a high risk population. To include the possibility that an HCV vaccine may provide either sterilizing immunity or transient immunity, the primary end point of an efficacy trail could be the detection of HCV RNA in blood both during, and at the completion of the trial. The primary efficacy comparison would then be the proportion of individuals in the vaccine- and placebo groups with detectable HCV RNA levels at the end of the follow-up period (1).
Perhaps the most crucial
aspect of a vaccine trial design is finding an appropriate setting, and
a high risk population that can be targeted, recruited, and retained (1).
For HCV trials, such populations would include injection drug users, hemodialysis
patients, health care workers, infants born to HCV-infected mothers, and
recipients of blood products and organs that have not been subject to viral
inactivation (1). The population sample size
in turn depends on the incidence of new infections within the population
being studied; the greater the incidence, the smaller the sample size required,
and vice versa.
1. Hsu H, Abrignani S, Houghton M. Prospects for a Hepatitis C Virus Vaccine. Clinics in Liver Disease 3(4), 1999.
2. The Epimmune Web site on HCV vaccines: http://www.epimmune.com
3. Koff RS. Prevention
of Hepatitis C Virus Infection. Clinics in Liver Disease 1(3), 1997.
Feedback: [Akanksha_Mehta@brown.edu] [David_Hyman@brown.edu]
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