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HCV Vaccine Article-Marty

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  • 2byteme@bellsouth.net
    Vaccine Development for Hepatitis C Martin Lechmann, Ph.D. and T. Jake Liang, M.D., Liver Diseases Section, NIDDK, National Institutes of Health, Bethesda,
    Message 1 of 1 , Aug 26 5:50 PM
      Vaccine Development for Hepatitis C

      Martin Lechmann, Ph.D. and T. Jake Liang, M.D., Liver
      Diseases Section, NIDDK, National Institutes of Health,
      Bethesda, Maryland



      Abstract

      Given the global disease burden and public health impact of
      hepatitis C, the development of an effective vaccine is of
      paramount importance. However, many challenging obstacles
      loom ahead of this goal. The hepatitis C virus (HCV), being an
      RNA virus, can mutate rapidly in adaptation to the environment,
      thus contributing to the high sequence divergence of multiple viral
      isolates in the world. The highest heterogeneity has been found in
      the hypervariable region of the envelope glycoprotein 2, which
      contains a principal neutralization epitope. HCV also causes
      persistent infection in a high percentage of immunocompetent
      hosts despite active immune response. The lack of an efficient
      tissue culture system for propagating HCV and testing
      neutralizing antibodies adds further complexity to the task of
      vaccine development. The immunologic correlates associated
      with disease progression or protection are yet to be defined, but
      recent studies suggest that a vigorous multispecific cellular
      immune response is important in the resolution of infection.
      Induction of high-titer, long-lasting, and cross-reactive
      antienvelope antibodies and a vigorous multispecific cellular
      immune response that includes both helper and cytotoxic T
      lymphocytes may be necessary for an effective vaccine. Several
      promising approaches have been used to develop an HCV
      vaccine. Novel vaccine candidates based on molecular
      technology such as recombinant proteins, peptides, viruslike
      particles, naked DNA, and recombinant viruses are being
      explored. The final vaccine product may require multiple
      components that target various aspects of protective immunity.
      Finally, sterilizing immunity may not be necessary if a vaccine can
      be developed to prevent chronic infection, which is the major
      cause of morbidity and mortality from this disease. [Sem Liver
      Disease 20(2):211-226, 2000. © 2000 Thieme Medical
      Publishers, Inc.]



      Introduction

      One of the greatest achievements in medical science can be
      attributed to Edward Jenner's initial description of a smallpox
      vaccine in 1776, when he inoculated an 8-year-old boy with
      cowpox (vaccinia) that protected the child against subsequent
      challenge with the virulent smallpox. This monumental discovery
      had lapsed for more than 150 years until the accomplishments of
      vaccine pioneers, such as Koch, von Behring, Ehrlich and
      Pasteur, that led to successful vaccines against smallpox, rabies,
      typhoid fever, cholera, and plague. This concept of using benign
      materials derived from virulent pathogens to induce protective
      immunity against the same pathogens has resulted in some of the
      greatest human triumphs against the infectious scourges that
      plagued much of human history. Yet, there is still a great deal that
      we do not understand about how vaccines work. Such
      knowledge can be applied to improve on existing vaccines with
      respect to better efficacy and less adverse effects and to develop
      vaccines against viruses for which there are none today, like HIV
      and hepatitis C virus (HCV). In addition, the classic concept of
      vaccination has been extended beyond protection of infection to
      therapy of existing infection and cancer.

      Figure 1 illustrates the various traditional and newer approaches
      in vaccine development. Most vaccines today are
      whole-organism vaccines containing inactivated whole or live
      attenuated bacteria or viruses. Live attenuated vaccines are
      rather potent in inducing a long-lasting cell-mediated and humoral
      immunity. These vaccines have been very successful because
      they resemble the natural infection closely. However, the
      potential risk, especially in immunocompromised hosts, that
      attenuated viruses or bacteria may mutate to virulent wild-type
      strains exists. On the other hand, inactivated organisms or
      inactivated toxins are noninfectious but are less immunogenic
      than attenuated viruses. Therefore, adjuvants and booster
      injections are necessary to enhance the immunogenicity for
      induction of protective immunity.


      Figure 1. (click image to zoom) Various
      strategies in vaccine development. The traditional
      vaccines, shown on the left side, are usually
      whole-organism vaccines containing inactivated
      whole or live attenuated bacteria or viruses.
      Because no cell culture system is available so far
      to propagate HCV, this strategy cannot be
      adapted for the development of an HCV vaccine.
      On the right side are illustrated the strategies of
      the newer types of vaccines that are being
      pursued for an HCV vaccine. These approaches
      evolve around the concept that one or several
      genes of the pathogen are incorporated into the
      genome of a normally nonpathogenic organism for
      amplification of the immunogens.


      The principles of the newer types of vaccines evolve around the
      concept that one or several genes of the pathogen are
      incorporated into the genome of a normally nonpathogenic
      organism for amplification of the immunogens. The subunit
      vaccine is then generated from the heterologous organism by
      purifying the immunogen (protein immunogen), isolating the
      naked DNA in the form of a plasmid carrying the gene encoding
      for the immunogen (DNA vaccine), or using the entire host as a
      live vector (recombinant viruses or bacteria). In addition,
      chemically synthesized peptides have also been explored as
      subunit vaccines. Several recombinant subunit vaccines have
      been successfully developed, including the yeast-derived
      hepatitis B vaccine. The recombinant subunit vaccines offer many
      advantages over the traditional approaches. First, the pathogen
      can be excluded from the vaccine and is therefore not infectious.
      Second, the vaccine can be specifically designed and optimized
      for a particular pathogen to exclude toxins or to induce specific
      arms of the immune response known to be important for
      protection. Third, this approach can be applied for viruses like
      HCV that cannot be grown in cell culture. Therefore, a variety of
      approaches based on this concept is being investigated to
      improve on existing vaccines and to develop vaccines against
      more challenging diseases such as hepatitis C or AIDS, for
      which the traditional approaches have failed or cannot be
      adapted. However, many new approaches are limited by the
      poor immunogenicity of recombinant viral proteins when they are
      administered alone. Thus, the effort to develop new and more
      potent adjuvants has intensified in the past years. There is now a
      variety of promising novel compounds, such as
      lipopolysaccharide-derived monophosphoryl lipid A, saponin
      derivative QS21, microemulsion MF59, lipid-particle
      immune-stimulating complex (Iscoms), and CpG
      oligo-nucleotides (for review, see Ref. 1) that can provide
      substantial enhancement to the efficacy of subunit vaccines.
      Clinical trials are currently underway to determine their efficacy
      and safety in humans.

      Approximately 200 million persons are chronically infected with
      HCV worldwide, and this large reservoir of infected persons
      constitutes a daunting source of potential new infections.
      Therefore, there is a compelling need to develop an effective
      vaccine. However, many obstacles to the development of a
      successful vaccine against hepatitis C exist. This review
      summarizes the current status and highlights novel promising
      strategies in HCV vaccine development. Because another article
      in this volume provides extensive coverage of the immune
      responses of hepatitis C, we limit our discussion of immune
      responses to those that are pertinent to vaccine development.
      Humoral Immunity

      B cells play an important role in protection against viral
      infections. During primary infection, antibodies, cell-mediated
      immunity, or both are crucial. However, during secondary
      infections, antibodies are the critical mediators and are often
      essential for the control of viral spread. This observation is
      reflected by the fact that many successful antiviral vaccines are
      based on the induction of neutralizing antibodies. In infections
      with other flaviviruses such as yellow fever,[2] dengue,[3] and
      tickborne encephalitis virus,[4] antibodies against the envelope
      glycoproteins have neutralizing capacity and protect against lethal
      flavivirus challenge. In addition, it has been reported that
      antibodies against a nonstructural protein of the tickborne
      encephalitis virus are also able to protect against virus
      challenge.[5] The complete identification of neutralizing or
      protective epitopes in HCV has not yet been accomplished. The
      envelope protein E2 of HCV has been of particular interest
      because it contains highly variable sequences within the
      N-terminal region (HVR1) that encodes neutralizing B-cell
      epitopes. [6] In addition, the E2 protein binds to CD81, which is
      thought to be a receptor for HCV,[7] although the CD81 binding
      region of E2 is probably not located within the HVR1 site.[8] The
      hypervariability of this region has been suggested as a possible
      mechanism through which the virus evades the immune
      response.[6,9,10]

      Mutations within the N-terminus of HVR1 have been shown to
      occur rapidly in infected individuals and coincide with the
      disappearance of preexisting anti-HVR1 antibodies.[11] Farci et
      al.[9] reported in vitro neutralization of HCV with a rabbit
      hyperimmune serum raised against a homologous synthetic
      peptide derived from the HVR1 region. The anti-HVR1 serum
      induced protection against the homologous HCV strain in
      chimpanzees but not against the mutants that cannot be
      neutralized by the antiserum. However, the antibody response
      against E2 and its role in viral clearance is still controversial.
      Early development of anti-E2 or anti-HVR1 has been suggested
      to be associated with recovery from acute HCV infection in
      humans.[12,13] However, other studies found no correlation
      between anti-E2 and self-limited infection by HCV in humans
      and in chimpanzees. [14-17] Because protection against HCV
      infection did not correlate with anti-HVR1 levels in a chimpanzee
      immunization experiment,[18] neutralization determinants other
      than the HVR1 site likely exist. Finally, antibody responses to the
      envelope proteins develop slowly and achieve only modest titers
      during primary infection.[19] Therefore, neutralizing antibodies
      may emerge too late to prevent chronic infection. In addition,
      antienvelope antibodies tend to be short-lived and disappear
      gradually after viral clearance.[19]
      Cell-Mediated Immunity

      There is accumulating evidence that failure to generate an
      effective immune response against HCV in the acute phase of
      infection is responsible for the high rate of chronicity. Most HCV
      proteins have been shown to be targets of helper T-cell
      responses and cytotoxic T lymphocyte (CTL) activities. Strong
      T-cell proliferative responses against HCV core,[20-24] E2, [22]
      NS3, [22,24,25] NS4, [21-24] and NS5 [21-23] proteins have been
      found to be associated with self-limited infection. The identified
      immunodominant epitopes are highly conserved among the
      known HCV isolates and can be presented by different human
      histocompatibility leukocyte (HLA) class II molecules.[26,27]
      Among these epitopes, several highly conserved CD4 + T-cell
      immunodominant epitopes within the NS3 protein have been
      particularly linked to viral clearance in acute hepatitis C.[27] In
      addition, the ability to generate anti-HCV multispecific T-cell
      proliferative responses has been shown to correlate with
      response to interferon treatment.[28-30] Thus, broadly directed and
      vigorous proliferative responses against structural and
      nonstructural proteins seem to be important in controlling HCV
      infection. Analysis of the cytokine profiles of HCV-specific T
      cells revealed that persons displaying a T helper type I profile
      (antigen-dependent production of interleukin [IL]-2 and
      interferon-gamma) that promotes cellular effector mechanisms
      rather than humoral immune responses are more likely to
      experience viral clearance.[25,28,31,32]

      HLA class I-restricted CTLs can directly kill virus-infected cells
      and produce potent antiviral cytokines and therefore are crucial
      in clearing viral infections. However, CTL-mediated lysis of
      virus-infected host cells, if inefficient, can result in persistent
      infection and chronic tissue injury. In HCV-infected patients,
      CD8 + T-cell responses are directed against structural and
      non-structural proteins in the context of different HLA
      molecules.[33-40] Chronic hepatitis C occurs despite a polyclonal
      and multispecific HCV-specific CTL activity that can be found in
      the peripheral blood and in the liver.[33-35,39,41-43] CTL escape
      mutants, including CTL antagonists, may contribute to the
      manifestation of chronic infection.[44,45] On the other hand,
      studies showed an inverse correlation between levels of
      HCV-specific CTL activity and viral loads, suggesting that HCV
      can be controlled to some extent by CTLs.[46-48] This observation
      is confirmed by studies in chimpanzees showing that during acute
      infection, CD8 + CTL activities correlated better with protection
      than the antibodies.[49] Additional support for this evidence
      comes from studies in agammaglobulinemic children in whom
      resolution of HCV infection can occur independently of
      antibodies. [50-52] Thus, the vigor and character of CTL responses
      in the early phase of infection are probably crucial in clearing the
      virus, whereas in the later phase insufficient viral-specific CTL
      responses may contribute to hepato-cellular injury.
      Obstacles In Developing An HCV Vaccine

      The development of an effective vaccine against HCV faces
      many challenges (Table 1). First, substantial sequence diversity
      exists among HCV strains isolated within and between
      geographic areas. There are at least 6 HCV genotypes and more
      than 50 subtypes. This makes the development of a global HCV
      vaccine rather complex. Second, even within an infected person,
      HCV isolates with rather divergent sequences in certain region of
      viral genome (quasispecies) are present and mutations occur
      frequently during the course of infection. In particular, the
      N-terminus of the E2 protein contains a hypervariable region of
      about 30 amino acids (HVR1), which shows extensive variation
      among all known isolates. The genetic variability within this
      region is thought to allow the virus to escape immune
      surveillance. Third, immunologic correlates that are associated
      with protection or disease progression are still being defined. The
      knowledge of immunogenic epitopes and their relevance to viral
      clearance and the existence of conserved cross-reacting epitopes
      are still unclear. These problems are further complicated by the
      lack of a reliable infectious tissue culture system for testing
      neutralizing antibodies or passage and expanding of the virus.
      The availability of such tissue culture systems has been invaluable
      in the successful development of other vaccines. For HCV, a
      surrogate assay for the determination of possible neutralizing
      antibodies has been developed. In this assay, antibodies are
      tested for their ability to neutralize the binding of highly purified
      recombinant E2 protein (NOB assay)[53] or antibody-captured
      HCV derived from high-titer sera 54 onto susceptible cells such
      as MOL-4 cells. This assay measures only inhibition of binding
      to target cells, which does not necessarily reflect neutralization of
      infectious virus in vivo. The only reliable model for HCV
      infection is the chimpanzee, which as an endangered species is
      not only costly but also difficult to study. Furthermore, the course
      of HCV infection in chimpanzee may not necessarily represent
      that in humans. Earlier experiments in chimpanzees in which
      challenge of apparently recovered chimpanzees with a
      homologous or heterologous strain of HCV resulted in
      reinfection suggest an absence of protective immunity from
      natural infection. In addition, HCV manages to persist in
      chronically infected persons despite the presence of broad
      antibody and T-cell responses. The viral and host factors that
      lead to persistence are not fully understood and remain to be
      elucidated in the future. Because the availability of small animal
      models would have greatly facilitated the development of HCV
      vaccine, intense effort has been under way to search for such
      models. Tupaia belangeri, a small primatelike animal, has been
      shown to be infectable by hepatitis B virus (HBV)[55] and is now
      being evaluated as a small animal model for HCV.[56] However,
      the robustness and reproducibility of this model remain to be fully
      confirmed. Alternatively, mouse models for HCV has been
      developed by either establishing HCV transgenic mice or
      transplanting human hepatocytes into immunodeficient mice.
      These models may prove to be useful in certain aspects of HCV
      vaccine development.
      DNA Vaccine

      Nucleic acid immunization is the most recent approach in vaccine
      development. The efficacy of DNA vaccines to protect against
      challenge with pathogens has been demonstrated in animal
      models of influenza virus,[57] malaria,[58] mycobacterium,[59]
      HIV,[60] and Ebola.[61] A DNA-based vaccine usually consists of
      purified plasmid DNA carrying sequences encoding for an
      antigen of interest under the control of eucaryotic promoter.
      After injection of the plasmid into the muscle or skin, the host
      cells take up the plasmid and express the antigen intracellularly.
      The expression of the encoded antigens by the host cells is one
      of the major advantages of this approach because it mimics
      natural infection. Furthermore, DNA immunization offers several
      other advantages, including the ease to generate and manipulate
      DNA and its potency in priming different arms of the immune
      response such as CTL, T helper cell, and antibody responses.
      Many studies have been published on the development of
      DNA-based vaccines against HCV. The DNA immunization
      approaches for HCV are summarized in Table 2.

      Antibody Responses

      The HCV core protein is highly conserved among various
      genotypes and therefore an attractive target for a DNA-based
      vaccine. However, studies in which mice were immunized with
      HCV core DNA alone showed no or only weak antibody
      responses.[62-64] To enhance the humoral immune responses
      against this nonsecreted viral protein, DNA encoding IL-4, IL-2,
      or granulocyte-macrophage colony-stimulating factor
      (GM-CSF) was coadministered along with the HCV core
      DNA.[62] Coimmunization with each of the cytokine genes
      substantially increases the seroconversion rate from 40% to
      80%. Similarly, in another study, a boost with a recombinant
      core protein after priming with HCV DNA induced anticore
      antibodies, whereas core gene immunization alone could not
      generate IgG response in mice.[65] Other strategies for enhancing
      the immunogenicity of a core DNA-based vaccine included the
      construction of various HBV envelope-HCV chimeras designed
      to express secreted forms of the core protein.[64,66] In two
      independent studies, the chimeric expression plasmids induced
      anticore antibodies in all immunized mice as compared with
      0%64 and 40%66 response rates in mice immunized with the
      HCV core plasmid alone. However, in only one of the two
      studies could the secretion of the fusion proteins be
      demonstrated.[64]

      Because HCV is an enveloped virus and neutralizing
      determinants likely reside on the surface of the envelope, the
      major focus for developing a DNA-based HCV vaccine has
      been the E2 protein. Immunization studies in mice using plasmids
      coding for the full- length E1 or E2 protein showed only low
      antibody responses, probably because the intact E2 glycoprotein
      expressed alone or together with E1 is retained in the
      endoplasmic reticulum.[67] Therefore, various DNA constructs
      have been designed to enhance the expression and secretion of
      the envelope glycoproteins. Mice and macaques immunized with
      a plasmid in which the E2 protein was targeted to the cell surface
      by replacing the C-terminus with a transmembrane domain
      showed an antibody response against E2 that occurred earlier
      and had higher titers than animals immunized with a plasmid
      expressing the full-length E2.[68] Based on these results, two
      chimpanzees were immunized three times with this construct.
      Only one animal developed anti-E2 antibodies, and preliminary
      data from challenge studies showed no protection against viral
      challenge.[69] The codelivery of cytokines was also explored to
      enhance the immune responses against the HCV envelope
      proteins. Lee et al.[70] constructed various DNA vaccine vectors
      carrying E1 or E2 genes with or without GM-CSF. To optimize
      the secretion of the E1 and E2 proteins, the authors replaced the
      signal sequence of the E1 and the E2 proteins with the signal
      sequence of the herpes simplex virus type 1 (HSV 1)
      glycoprotein D and additionally truncated the C-terminal
      hydrophobic regions of the envelope proteins. The antibody
      responses could be greatly enhanced (4-fold higher for E1 and
      over 10-fold higher for E2) in buffalo rats by codelivery of a
      bicistronic plasmid that expressed the GM-CSF and the
      engineered envelope genes. A combined vaccine regimen,
      consistent of priming with E2 DNA and boosting with
      recombinant E2 protein, could also enhance antibody
      (immunoglobulin G2a) responses. [71] The mode of DNA
      delivery has also been suggested to influence the strength of
      antibody responses. Intradermal injection of plasmids expressing
      different immunogenic domains of E2 as fusion proteins with the
      HBV surface antigen induced up to 100- fold higher titers of
      antibodies compared to intramuscularly injection in mice.[72] The
      combination of both delivery routes may be more efficient in
      inducing broad antibody responses.[73]

      Two studies on the DNA immunization using plasmids encoding
      NS3, NS4, and NS5 proteins individually or together
      demonstrated the successful induction of HCV-specific
      antibodies against NS3, NS4, and NS5 in mice and buffalo
      rats.[74,75] Surprisingly, the codelivery of GM-CSF did not
      enhance the antibody titers to HCV NS3, NS4, and NS5.[75]

      Lymphoproliferative and Cytokine Responses

      DNAimmunization can induce lymphoproliferative responses
      against the structural proteins core,[62,65,66,76] E1, 77 E2, [77] and
      the nonstructural proteins NS3, NS4, and NS5 in mice 74 and
      buffalo rats.[75] However, the T-cell proliferative responses
      against the structural proteins are typically weak. Various
      approaches have been used to enhance the CD4 + T-cell
      responses. The codelivery of GM-CSF, IL-2, or IL-4 genes can
      increase significantly the lymphoproliferative responses.[62,75]
      Additionally, boosting with a recombinant HCV core protein
      after HCV core DNA immunization appears to enhance the T
      helper cell proliferation.[65] Spleen cells from mice immunized
      with an HBV envelope/HCV core chimeric construct showed
      higher levels of proliferative activities than those from mice
      immunized with the nonchimeric core construct.[66] Intramuscular
      DNA immunization induced predominantly interferon-gammabut
      not IL-4 production, suggesting a Th1 response.[62,74,78] Analysis
      of the IgG subtypes showed an almost exclusive IgG 2a and 2b
      antibody production,[72,73,77] which is also consistent with a
      Th1-like response.

      Cytotoxic T-cell Responses

      Several studies have demonstrated the generation of CTL
      activities in mice immunized with HCV core plasmids.[65,66,76,79-81]
      Core-specific CTL activities were highest in mice coimmunized
      with an IL-2 expressing plasmid, whereas GM-CSF did not
      significantly augment CTL activities.[62] In addition, immunization
      with HBV and HCV chimeric proteins 66 or combined
      DNA-protein immunization 65 did not alter the generation of
      core-specific CTL responses. In contrast, coadministration with
      an IL-4 construct suppressed HCV core-specific CTL
      activity.[62] Furthermore, mice injected with an HCV core
      construct truncated at amino acid 69, which removes a known
      CTL epitope, showed almost no induction of CTL activities in
      BALB/c mice.[66]

      Several studies have also addressed the CTL responses of DNA
      immunization against the envelope proteins E1 and E2. [71,81-83] In
      one of these studies, six recombinant plasmids were
      constructed.[81] These plasmids included the structural proteins
      either individually or together, E1 and E2 together, and a
      truncated E2 in which the N-terminal hypervariable region was
      deleted. In this study, CTL activities were tested against target
      cells infected with a recombinant vaccina virus expressing core,
      E1 and E2. Specific CTL responses were detected only in mice
      injected with plasmid constructs encoding core alone or together
      with E1 and E2. The authors suggested from these data that the
      core region might have the strongest CTL epitope in the
      structural region for BALB/c mice. However, studies in our
      laboratory (J. Satoi, personal communication) showed that in
      BALB/c and FBV/n mice, immunization with a plasmid
      expressing core alone generated core-specific cytolytic activities,
      whereas injection of a core/E1/E2 expressing plasmid induced
      CTL responses only against E2 but not against core or E1.
      These data suggest that the coexpression of a particular protein
      in the context of other proteins may generate a different CTL
      responses. In another study, the authors constructed plasmids
      encoding core, E1 and E2 individually or together, under the
      control of a cytomegalovirus promoter.[82] The authors compared
      the efficacy of these constructs with a plasmid containing coding
      sequences for all three structural proteins under the control of the
      human elongation factor 1(EF-1a) promoter. BALB/c mice were
      immunized only once with these constructs, and the site of
      injection was given an electric pulse to enhance the efficiency of
      DNA uptake in cells. Spleen cells obtained from the
      DNA-immunized mice were assessed for their ability to lyse
      major histocompatibility complex (MHC)-matched target cells
      either pulsed with core or E1 peptide or infected with
      E2-expressing vaccinia virus. Only mice inoculated with the
      plasmid expressing core, E1, and E2 under the EF-1promoter
      generated HCV-specific effector responses against all three
      proteins after a single immunization. Furthermore, E2 DNA
      immunization followed by a boost with a recombinant HSV 1
      glycoprotein D HCV-E2 fusion protein enhanced the CTL
      responses in mice, which is closely associated with the protection
      of mice against challenge with a E2 expressing tumor cell line.[71]
      CD8 + CTL activities have also been demonstrated for the
      nonstructural proteins NS3 and NS5 after three intramuscular
      injections with NS3- and NS5-encoding plasmids.[74] In
      addition, a tumor model in which syngeneic cells stably
      transfected with an NS5 expression plasmid was used to assess
      CTL activity in vivo.[74] About 60% of immunized mice were
      protected against tumor formation, and in those who developed
      tumors, the tumor weight was significantly reduced as compared
      with the unimmunized mice.

      To better approximate HCV infection in humans, a transgenic
      mouse model expressing the human HLA-A2.1 has been
      developed.[80] This transgenic mouse model can be used to study
      the generation of humanlike HLA-A2.1 restricted CTL
      responses. HLA trangsenic mice were immunized three times
      with a plasmid vaccine encoding the entire core protein. At 2, 6,
      and 14 months after the last boost, mice were challenged with
      recombinant vaccinia virus expressing the HCV core protein or a
      control vaccinia virus expressing hemag-glutinin. Mice were then
      killed and vaccinia virus titers determined in the ovaries.
      DNA-immunized mice showed a reduction of vaccinia titer 6-12
      logs at month 2, 7 logs at month 6, and 5 logs even at month 14
      after the last immunization, as compared with the corresponding
      mock-immunized controls. These results suggest that the HCV
      core DNA vaccine generated a long-lasting protection against
      infection with recombinant vaccinia virus expressing HCV core in
      vivo, despite a weak anticore CTL response requiring at least
      three stimulations with peptides to detect the HCV core-specific
      CTL activities in the standard 51 Cr release assay. The authors
      also showed that the protection was mediated by CD8 + cells.

      The nucleic acid-based vaccine studies demonstrated the
      potency of DNA-based vaccines to induce both humoral and
      cellular immune responses against various HCV proteins.
      However, most of these studies focused on either the humoral or
      cellular immune responses. Therefore, more potent vectors need
      to be designed to generate both strong humoral and cellular
      immune responses against multiple epitopes within the structural
      and nonstructural proteins. The codelivery of cytokines has been
      shown to enhance the immune responses against DNA-based
      HCV vaccines. Thus, the benefit of other immunomodulating
      molecules such as B7, 84 CD40 ligand,[85,86] or CTLA4 87 that
      has been shown to enhance nucleic acid immunization should be
      explored for HCV DNAvaccination. The delivery of DNA is
      also a crucial step that should be studied in more detail; factors
      such as the route(s) (e.g., im) and methods of delivery and the
      delivery systems should be optimized. Furthermore, the
      prime-boost combination of DNAand protein vaccines should be
      carefully evaluated to establish an immunization protocol that
      maximizes the potency of both approaches.
      Recombinant Virus

      Recombinant viruses are an efficient vehicle for DNA delivery
      that can result in high level of recombinant protein expression in
      host cells. In a number of experimental models, infection of
      animals with recombinant viruses encoding foreign viral proteins
      induce protective immunity to a variety of viruses.[88-90] Several
      recombinant viral vectors are being evaluated for HCV vaccine
      development (Table 3). The defective recombinant adenovirus is
      an attractive candidate because of its hepatotropism, its potency
      to induce both humoral and cell-mediated immunities, and its
      ability to be administered parenterally or orally. Recombinant
      adenoviruses that are defective in their replication and lack the
      E1 and E3 regions of the genome have been used to increase the
      amount of foreign sequences that can be inserted. Studies in mice
      showed that a recombinant adenovirus containing genes for the
      structural proteins of HCV-induced antibody responses to each
      of the three structural proteins. [91] In addition, strong cytotoxic
      T-cell responses against core and E1 could be detected in
      splenocytes from mice immunized with an adenovirus carrying
      core and E1 genes.[92] The cytotoxic T-cell responses lasted for
      at least 100 days. Coadministration of a recombinant adenovirus
      expressing IL-12 led to a marked increase in cellular immune
      responses when administered at a dose of 10 7 plaque-forming
      units.[93] The cellular immunity was abolished when higher doses
      of the IL-12 expressing adenovirus were used. However, the
      recent tragedy of death in a gene therapy trial using adenovirus
      has severely dampened the enthusiasm for the use of this viral
      vector in humans.

      Spleen cells from mice immunized with a recombinant vaccinia
      virus expressing the HCV core gene exhibited strong
      core-specific CTL activities.[94] In addition, studies in our
      laboratory showed that immunization with a recombinant vaccinia
      virus carrying sequences for the structural proteins generated
      strong CTL and T helper cell responses against all structural
      proteins in BALB/c mice. Interestingly, studies using HCV
      vaccinia recombinants revealed that vaccinia-specific CTL
      responses were greatly suppressed by vaccinia recombinants
      expressing the core protein, suggesting that HCV core may alter
      the immunogenicity of a vaccine and may play a role in the
      persistent of HCV infection.[95] Furthermore, a nonreplicating
      canary-pox virus encoding polycistronic core/E1/E2/NS2/NS3
      genes are being used to potentiate the immune response to HCV
      DNA immunization. Preliminary data suggest that a booster
      injection with this recombinant canarypox virus enhances the
      HCV-specific immune response and generates broader T-cell
      activity (P. Pancholi, et al., presented at the 6th international
      symposium on hepatitis C and related viruses).

      There are some promising new studies on recombinant virus
      vectors approaches in the HIV field that can be also adopted for
      the development of an HCV vaccine. Strategies using new
      poxvirus vectors such as canarypox, fowlpoxvirus,[96] attenuated
      vaccinia strains NYVAC 96 or modified vaccinia virus strain
      Ankara have been developed.[97] Alphavirus vectors such as
      Venezuelan equine encephalitis virus [98,99] or Semiliki Forest
      virus 100 have also been explored as a vehicle for HIV DNA
      immunization. An attractive approach is the use of alphavirus
      replicon vector as a DNA vehicle. These replicons have the
      advantage that they are self-replicating, express foreign genes in
      infected cells, but lack the viral structural proteins and can be
      administered as naked DNA vaccine. Live attenuated salmonella
      (Salmonella typhimurium) is an alternative vehicle of DNA
      delivery.[101] Oral or nasal immunization with this bacterial vector
      has been shown to induce both mucosal and systemic immune
      responses against DNA encoded antigens. Although all these
      recombinant virus approaches are very promising, safety and
      regulatory issues may be of concern with implementation.
      Peptide Vaccine

      Peptide vaccines follow the basic principle that T lymphocytes
      recognize antigens only as peptide fragments that are generated
      intracellularly and bound to MHC class I or II molecules on the
      surface of the antigen presenting cells. Helper T cells recognize
      antigenic peptides that are bound to the MHC class II molecules,
      whereas CD8 + cytotoxic T cells are bound to the MHC class I
      molecules. Therefore, small peptides that are present in the
      extracellular milieu can bind directly to MHC class I or II
      molecules without undergoing the antigen processing pathway.
      Consequently, chemically synthesized peptides that are potent
      immunogenic antigens are being pursued as vaccine candidate for
      HCV (Table 4).

      The rationale of this approach is based on the knowledge that
      certain T-cell epitopes on the HCV polyprotein may be
      important for viral clearance. Using amino acid motifs to predict
      the binding of peptides to MHC class I and II molecules,
      MHC-peptide binding assays, and CTL and T helper assays,
      several CTL and T helper epitopes on the HCV polyprotein that
      may be important for the design of a peptide vaccine have been
      identified.

      Peptides containing epitopes from the core,[102-104] NS4, 104 and
      NS5 102 regions have been shown to induce strong CTL
      responses in BALB/c and HLA-A2.1 transgenic mice. The
      covalent attachment of the CTL peptide to a T helper peptide
      seems to be crucial for generating a strong CTL response.[102-104]
      In addition, enhancement of the immunogenicity of a
      core-specific CTL epitope has been achieved by substitution of
      one amino acid on the native peptide.[105] Furthermore,
      covalently linked T helper and CTL epitopes were more potent
      immunogens when delivered as lipidated peptides.[104]

      Other strategies for developing peptide vaccines are using
      peptides to generate antibodies against linear epitopes. Because
      HVR1 contains a neutralizing epitope, it is an attractive target for
      peptide-based vaccine. A chimpanzee that was immunized with
      recombinant E1 and E2 glycoproteins together with HVR1
      peptides derived from a different isolate was protected against
      inoculation of the isolate from which the peptide sequence was
      derived.[106] In addition, antiserum from this protected
      chimpanzee was shown to neutralize the homologous strain by
      inoculation of this mixture into another chimpanzee. Similarly,
      rabbits that were immunized with a series of synthetic HVR1
      peptides 107 produced high titers of broadly cross-reactive
      antibodies to HCV that could block the binding of
      antibody-captured HCV to MOLT-4 cells.

      The most difficult problem of choosing the HVR1 as the target
      for a HCV vaccine is the existence of quasi-species in this region
      of HCV genome. The screening of phage displayed peptide
      libraries has been used to identify a consensus profile from over
      200 HVR1 sequences of different viral isolates. HVR1
      sequences most commonly recognized by patient sera and able
      to bind anti-bodies that cross-react with a large panel of HVR1
      were identified (Table 5).[108] A sequence pattern within these
      so-called mimotopes that was responsible for the detected
      cross-reactivity could be developed. Mice immunized with a
      mixture of the mimotopes shown in Table 5 could generate
      antibodies that recognized 95% of the same panel of natural
      HVR1 variants. This finding was confirmed by another study that
      among the 25 different HVR1 proteins derived from genotypes
      1b, the protein that contains the sequence similar to the reported
      consensus sequence was the most frequently recognized protein
      by patient's sera.[109]

      The major obstacles for a peptide-based approach lie in the
      observation that single peptide without helper function may be a
      poor immunogen, and many effective vaccines are typically
      multivalent in generating a broad immunity against several
      different antigens. However, this limitation can be overcome by
      the coadministration of potent adjuvants or the use of multiple
      epitopes vaccine that contains a mixture of peptides.
      Recombinant Protein Subunit Vaccine

      The initial attempt to develop an HCV vaccine was directed
      toward generating a recombinant protein subunit vaccine.
      Because it has been shown for several fla-viviruses that
      antibodies to the envelope protein can provide protection,
      recombinant HCV E1 and E2 proteins were used in early
      vaccination studies from Chiron (Table 4).[110] However, the
      success was limited. In their initial effort, the E1 and E2 proteins
      were purified from HeLa cells infected with a recombinant
      vaccinia virus. The purified recombinant envelope HCV proteins
      were injected with an oil/water microemulsified adjuvants im 18
      times into two chimpanzees and three times in additional five
      animals. Two to 3 weeks after the final boost, chimpanzees were
      challenged with a low dose of homologous HCV-1 virus. The
      five animals with the highest anti-E1/E2 titers did not show any
      signs of viral infection. The two remaining animals became
      infected but resolved their infection. In comparison, four similarly
      challenged naive chimpanzees developed chronic infection.
      However, rechallenge of the five protected animals with
      heterologous. HCV-H isolate after additional boosts with
      recombinant E1 and E2 proteins derived from a constitutively
      expressing Chinese hamster ovary cell line led to infection in all
      animals, although none developed persistent infection.[18] In
      general, the Chinese hamster ovary cell-derived vaccine
      exhibited lower immuno-genicity than the recombinant envelope
      proteins derived from vaccinia virus-infected HeLa cells and did
      not protect any of the vaccinated chimpanzees after challenge
      with the homologous virus. However, self-limited infection
      occurred more frequently than in nonvaccinated animals.
      Nonetheless, these results are encouraging in the sense that
      although no sterilizing immunity was achieved, chronic infection
      might be prevented.

      Recombinant HCV proteins have also been explored for an
      immunotherapeutic approach. Recombinant E1 protein of
      genotype 1b was purified as homodimers that associated into
      particles of about 9 nm in diameter.[111] Two chimpanzees with
      chronic HCV infection received a total of nine doses of 50 µg
      recombinant E1 protein. One of the chimpanzees was infected
      with HCV genotype 1a and the other one with 1b. Vaccination
      resulted in improved liver histology, disappearance of viral
      antigens from the liver as detected by immunostaining, and
      decrease in alanine aminotransferase (ALT) levels in both
      animals. Although HCV RNA levels in the serum did not change
      during treatment, liver inflammation and HCV antigens
      reappeared and ALT levels rose after the end of treatment. An
      association between high levels of anti-E1 antibodies and the
      improvement of hepatitis C was observed.
      HCV-Like Particles

      Viruslike particles are attractive as a recombinant protein
      vaccine, because they mimic more closely the properties of
      native viruses than recombinant protein subunit vaccines. Indeed,
      studies in various animal models have demonstrated that
      papillomavirus- and rotarvirus-like particles synthesized in insect
      cells can induce protective immunity.[112-114] In addition, several
      studies have shown the efficacy of viruslike particles to induce
      not only a strong antibody response but also a CTL response in
      immunized animals.[115-118] Our laboratory recently reported the
      synthesis of HCV-like particles (HCV-LPs) in insect cells using
      a recombinant baculovirus containing the cDNA of the HCV
      structural proteins (core, E1, and E2).[119] The HCV-LP exhibit
      similar morphologic, biophysical, and antigenic properties as the
      putative virions isolated from HCV infected humans. These
      noninfectious 40-50 nm HCV-LPs consist of a lipid envelope
      containing E1 and E2 (Fig. 2). Mice immunized with HCV-LP
      generated a strong humoral immune response against the
      structural proteins core and E2 (Table 4).[120] The antienvelope
      titers were detectable after the third immunization and were
      highest after the seventh immunization with titers ranging between
      12,800 and 204,800. Antienvelope antibodies recognize E2
      proteins from different genotypes and were broadly directed
      against various regions of the E2 protein. HCV-LPs are also
      capable of inducing a strong cellular immune responses in
      BALB/c mice.[121] Splenocytes from HCV-LP immunized mice
      showed T-cell proliferative responses against core and E1/E2
      proteins. In addition, HCV-specific CTL activities predominantly
      directed against the E2 protein could be detected in HCV-LP
      immunized mice. Furthermore, interferon-gamma but not IL-4
      production produced by the HCV-specific activated T cells,
      suggesting a type 1-like response. Studies in which HCV-LPs
      were denatured before injection revealed that the immunogenicity
      is strongly dependent on particle formation. These studies
      suggest that HCV-LP may be promising as a potential vaccine
      candidate.


      Figure 2. (click image to zoom) Electron
      microscopy of HCV-like particles. The sucrose
      gradient purified viruslike particles were fixed and
      visualized by electron microscopy (arrow; bar, 40
      nm). The inserts below show labeling of partially
      purified particles with monoclonal anticore,
      anti-E1, anti-E2 antibodies, and serum from an
      HCV-infected human (HUM) (bar, 50 nm).
      (Reproduced with permission from Ref. 120.)
      Passive Immunization

      Because passive immunization has been successfully used for the
      prevention of hepatitis A and HBV infection, the efficacy of
      anti-HCV immunoglobulin to protect from hepatitis C infection
      has been evaluated in several studies. In one study, three
      chimpanzees were inoculated with HCV.[122] One hour after
      inoculation, one chimpanzee was treated once intravenously with
      hepatitis C immunoglobulin, the second with anti-HCV negative
      immunoglobulin, and the third animal received no treatment.
      Anti-HCV immunoglobulin did not prevent infection as shown by
      the presence of HCV RNA in serum and HCV antigen in the
      liver. However, liver enzyme activity was delayed in the
      anti-HCV immuno-globulin- treated animal and the period of
      acute hepatitis C was prolonged. In a more recent study, four
      chimpanzees received multiple infusions of anti-HCV
      immunoglobulins over a period of 13 weeks after inoculation
      with HCV.[123] In this study, HCV viremia lasted only 73 to 105
      days, and there was no evidence of acute hepatitis in the treated
      animals. HCV RNA reoccurred in serum samples of one animal
      after passively transferred anti-HCV immunoglobulins
      disappeared. In addition, three chronically infected chimpanzees
      were also treated with repeated infusions of anti-HCV immuno-
      globulins twice a week over a period of 15 weeks. In two
      chimpanzees, a significant decrease of HCV RNA levels in
      serum could be detected during treatment. After the end of
      treatment, HCV RNA levels decreased further. Interestingly, a
      substantial decrease in HCV antigen was detected in the liver.
      These studies suggest that antibodies alone can prevent acute
      HCV infection and are even beneficial when administered in the
      chronic phase of HCV infection. Further studies are needed to
      clarify which antibodies are essential for preventing acute HCV
      infection. The knowledge of the mechanism for
      antibody-mediated clearance of HCV antigens in the liver of
      chronically infected chimpanzees may provide further insights in
      preventing persistent HCV infection.
      Combination Modality

      Given the lack of clearly defined correlates for a protective
      immunity and the relatively weak immunogenicity of recombinant
      subunit vaccines, a successful HCV vaccine may require a
      multicomponent approach that stimulates various aspects of the
      immune response. Promising new studies on the prime-boost
      approaches in the HIV field could be adapted for the
      development of a HCV vaccine. Most encouraging approaches
      are priming with DNA and boosting with recombinant virus
      vectors or viral proteins. The efficacy of this strategy has been
      shown by the protection of macaque monkeys from a pathogenic
      challenge with simian HIV chimera (SHIV) after a prime boost
      regimen with DNA followed by FPV boost.[124,125] Priming with
      DNA followed by boosting with gp120 also generated
      protective responses that was far superior as either DNA or
      recombinant protein immunization alone.[125] Only a few studies
      have explored the DNA-protein prime-boost regimen for an
      HCV vaccine.[65,71] These studies demonstrated the potency of
      this strategy. Future studies, especially protection studies in
      chimpanzees, are needed to determine if the combination
      modality indeed holds the promise for the induction of broadly
      directed humoral and cellular immune response that is sufficient
      for a protective immunity against HCV.
      The development of an effective vaccine against HCV faces a
      variety of obstacles. The lack of a convenient experimental
      model system makes it very difficult to study the correlates of
      protective immunity and viral clearance. Studies in humans and in
      chimpanzees indicate thus far that an ideal vaccine should induce
      broad humoral, T helper, and cytotoxic T-cell responses.
      Therefore, the final product might be a combination of different
      approaches, such as a combination of DNA and recombinant
      subunit protein vaccines. To induce a broad cellular immune
      response in the general population, it is also necessary that the
      vaccine candidate contain epitopes that are restricted by diverse
      MHC alleles. In addition, given the high degree of genetic
      heterogenicity of HCV, this vaccine should also be able to exert
      cross-protective immunity against various HCV genotypes. This
      could be achieved by either including antigens from different
      genotypes or by using antigens that induce cross-protection.
      Mimotope sequences derived from the HVR1 that induce
      cross-reactivity are encouraging and may be valuable for the
      development of a broadly active vaccine.

      Finally, because HCV-associated morbidity and mortality result
      from long-term sequelae of chronic infection, sterilizing immunity
      may not be necessary as long as vaccine-induced immunity is
      effective in preventing chronic infection. Following this strategy,
      there are encouraging reports that immunization with anti-HCV
      immunoglobulins or recombinant envelope proteins can alter the
      outcome of infection and prevent chronic infection in
      chimpanzees. Other promising strategies for the development of
      an HCV vaccine also have been evaluated in small animal
      models. However, their prophylactic potential would have to be
      determined in primate models. As we move from bench to
      bedside, clinical trials to test the efficacy of any vaccine
      candidate would have to be carefully conducted in large high-risk
      populations and possibly in countries where the incidence of new
      HCV infection remains high. Finally, the question remains as to
      who should receive the vaccine. High-risk populations such as
      health care workers, IV drug users, hemophiliacs, renal dialysis
      patients, infants born to infected mothers, partners of infected
      individuals, and highly sexually active persons should be the
      primary target of a vaccine program. However, universal
      vaccination, like the case for HBV, may be necessary to achieve
      the ultimate goal of global control of HCV infection.

      Abbreviations

      CTL cytotoxic T lymphocyte
      E envelop
      HBV hepatitis B virus
      GM-CSF granulocyte-macrophage colony-stimulating factor
      HCV hepatitis C virus
      HCV-LP HCV-like particles
      HLA human histocompatibility leukocyte
      HVR hypervariable region
      IL interleukin
      MHC major histocompatibility complex
      NS nonstructural.


      Section 12 of 12



      CONTENTS
      Abstract &
      Introduction

      Humoral Immunity

      Cell-Mediated
      Immunity

      Obstacles In
      Developing An
      Hcv Vaccine

      DNA Vaccine

      Recombinant Virus

      Peptide Vaccine

      Recombinant Protein
      Subunit
      Vaccine

      HCV-Like Particles

      Passive Immunization

      Combination Modality

      Concluding Remarks

      References


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