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Active immunotherapy against Hepatitis C virus infection
 Hepatitis C virus (HCV) infection is a serious cause of liver disease worldwide, with
more than 170 million people now chronically infected and at risk of developing liver cirrhosis and
hepatocellular carcinoma. Our fundamental knowledge of HCV replication and pathogenesis still remains
poor, however, primarily due to a lack of in vitro culture systems and experimental animal models,
with the exception of the chimpanzee. Although there are existing treatments using interferon and
chemical agents, the effectiveness of these classical therapies is limited. Hence, the development
of effective vaccines is urgently needed.
In the case of HCV vaccine development, the classical approaches using either live attenuated or
whole inactivated virus may not be feasible due to the absence of effective culture systems for
producing viral particles. In addition, a live attenuated approach against HCV is not realistic
because of its tendency to cause chronic liver disease. Thus, our attempts have been primarily
focused on recombinant proteins-,
DNA- and epitope
-based immunization.
Recombinant protein-based approach
Envelope proteins are regarded as important targets for HCV vaccine development as they are likely to be
involved in virus-host recognition and antibodies directed against these proteins have been shown to
neutralize the virus. Although there have been several attempts to utilize a recombinant subunit vaccine
against HCV infection, some serious problems still exist. For example, there are technical difficulties
involved in the production and purification of recombinant HCV envelope proteins from mammalian cells,
and these envelope proteins have also been shown to have low immunogenicity.
 To overcome
these obstacles, we postulated that the virus-like particle of HCV might be expressed efficiently and
purified successfully in a yeast expression system by utilizing the technology with which we previously
developed the yeast-derived recombinant Hepatitis B vaccine at MBRI. By testing a series of constructs
generated by domain shuffling, we found that the virus-like particle of HCV could indeed be expressed
and purified as efficiently as HBsAg in yeast. More importantly, these purified particles have been shown
to elicit both the humoral and cellular immune responses, specific to HCV glycoproteins, in small animal
systems. Currently we are examining the nature of these immune responses elicited by recombinant HCV
particles using surrogate challenge systems, such as recombinant vaccinia viruses or pseudotype viruses.
We can therefore confirm the validity of this candidate vaccine against HCV.
DNA-based approach
DNA vaccines now represent a novel method for expressing antigens in vivo to generate both the
humoral and cellular immune responses, and have been previously shown to elicit protective immunity in
a number of disease models. Since cellular immunity appears to be relevant for HCV clearance, DNA vaccines
therefore have a theoretical advantage over protein-based vaccines, which in general are less efficient
in generating cellular immune responses. Based on this hypothesis, we have now selected a DNA vaccine as
a potential candidate for the development of a therapeutic or prophylactic vaccine against HCV.
Currently, we are developing an efficient HCV DNA vaccine which can be treated with the virus-like particle
of HCV by prime/boost regimen. To this end, several approaches have now been attempted to maximize the
efficacy of this vaccine: 1) the development of an antigen presenting cell-specific vector system; 2) the
manipulation of the HCV envelope gene to either change its cellular location or to increase its immunogenicity
and 3) the evaluation of several co-stimulatory molecules as potential DNA vaccine adjutants. The efficacy
of these DNA vaccine constructs is presently under investigation in terms of the induction of cellular and
humoral immune responses in small animal models.
Epitope-based approach
Cytotoxic T lymphocytes (CTLs) are activated by specific antigen fragments known as antigen-specific epitopes,
and they are thought to play an important role in viral clearance of HCV. Accordingly, amplification of the
CTL response by HCV-specific epitopes could be an effective strategy for therapeutic vaccine development.
An epitope-based vaccine would also offer several potential benefits over traditional vaccines. It is unlikely
that the typical genetic conversion of HCV will allow for viral escape because the epitopes are selected from
the conserved regions of the HCV. Another benefit of using epitope-based vaccines is that any possible
pathological side effects caused by whole HCV antigens could be minimized.
Our research in this area has been focused on the identification of supertype-restricted epitopes from
conserved regions of HCV polyproteins. These supertype-restricted epitopes have a broad range of binding
affinities for several different types of MHC and can induce antigen-specific CTL responses against HCV.
We have identified proprietary epitopes by ex vivo screening of predicted epitopes using PBMC cultures
from HCV patients and ELISPOT assays. Moreover, the efficacy of the selected epitopes is subsequently
evaluated in HLA-transgenic mice using an ELISPOT assay and surrogate challenge systems for the development
of an epitope-based therapeutic HCV vaccine. |
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