Vaccine Development for Hepatitis C: Lessons from the Past Turn into Promise for the Future

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1 Vaccine development for hepatitis C REVIEW ARTICLE Vaccine Development for Hepatitis C: Lessons from the Past Turn into Promise for the Future Chi-Tan Hu Division of Gastroenterology, Department of Internal Medicine, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan ABSTRACT Most natural hepatitis C virus (HCV) infection elicits poor immune responses and 75% to 85% of HCV infections become chronic; therefore, the development of an effective vaccine is of paramount importance. HCV was discovered in 1988 and there is still no vaccine for it. Impediments in the development of a globally effective hepatitis C vaccine include the following: (1) the lack of a susceptible small animal model; (2) the genetic and serological heterogeneity of hepatitis C virus, especially the hypervariable region of the envelope glycoprotein E2, which contains a principal neutralization epitope; (3) modest titers of short-lived neutralizing antibody responses to the envelope proteins emerge too late to prevent chronic infection; (4) failure to produce high quantities of HCV in tissue culture. Lessons from the past indicate that a globally effective HCV vaccine should elicit high-titer, long-lasting, cross-reactive anti-envelope antibodies and induce a vigorous, multispecific cellular immune response that includes both helper and cytotoxic T lymphocytes, particularly of the Th1 type. The successful vaccine may require multiple components that target various aspects of protective immunity, possibly in parallel when more of the various HCV epitopes are found. A polyepitope-based strategy combining HCV-like particles (containing core, E1, and E2 proteins) and conserved T-cell epitopes in HCV non-structural proteins could fulfill these criteria. This review summarizes the present status and highlights novel promising strategies in HCV vaccine development. (Tzu Chi Med J 2005; 17:61-74) Key words: hepatitis C, epitopes, vaccination, cytotoxic T lymphocytes, helper T lymphocytes INTRODUCTION Approximately 200 million people are chronically infected with HCV worldwide. Although there have been innovations in the various methodologies of vaccine development, (Fig. 1), there have also been many obstacles facing the development of a globally effective hepatitis C vaccine. First, substantial sequence diversity exists among HCV strains isolated within and between geographic areas and there are at least 6 HCV genotypes associated with more than 50 subtypes. This makes the development of a global HCV vaccine rather difficult. Second, HCV exists as a quasispecies. Even within an infected person, HCV isolates with rather divergent sequences in certain regions of the viral genome 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 [1,2]. 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-reactive epitopes are still unclear. These problems are further complicated by the Received: March 15, 2004, Revised: April 7, 2004, Accepted: April 29, 2004 Address reprint requests and correspondence to: Dr. Chi-Tan Hu, Division of Gastroenterology, Department of Internal Medicine, Buddhist Tzu Chi General Hospital, 707, Section 3, Chung Yang Road, Hualien, Taiwan SN

2 C. T. Hu lack of a reliable infectious tissue culture system for testing neutralizing antibodies or passage and propagating of the virus, which is in part resolved by the development of a novel HCV replicon system. In the past, the only reliable animal model for HCV infection was the chimpanzee, which as an endangered species is not only costly but also difficult to study. Similarly, the development of HLA-A2.1 transgenic mice can in part solve the problem. Furthermore, the course of HCV infection in chimpanzees or HLA-A2.1 transgenic mice 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 re-infection suggest an absence of protective immunity from natural infection. 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. Because the availability of small animal models would greatly facilitate 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 [3] and has been evaluated as a small animal model for HCV [4]. Alternatively, mouse models for HCV have 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 pre-exposure prophylaxis active vaccine development for HCV. In this article, the problems associated with developing a vaccine against HCV infection are discussed and recent progress made towards overcoming these problems in the development of a global HCV vaccine are described. ACUTE AND PASSIVE IMMUNIZATION Active and passive immunizations are available for hepatitis A and B but so far not for hepatitis C. Postexposure hepatitis C immune globulin treatment markedly prolonged the incubation period of acute hepatitis C but did not prevent or delay HCV infection. Intravenous immune globulin had no effect on the course of HCV infection [5]. The efficacy of anti-hcv immunoglobulin (neutralizing antibodies to HCV envelope glycoproteins) to protect from hepatitis C infection in chimpanzees has been evaluated in some studies. 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 immunoglobulin-treated animal and the period of acute hepatitis C was prolonged [5]. In another study, a significant decrease of HCV RNA levels in serum could be detected during treatment [6]. 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. The combination of both passive and active immunizations has the advantage of immediate protection due to the immunoglobulin which lasts until the active immunization induces an endogenous antibody production [7]. CELL-MEDIATED AND HUMORAL RESPONSES Patients with self-limited hepatitis C have evidence of a polyclonal, multi-specific CD8 + T-cell response along with a coordinated CD4 + T-cell response that is associated with eradication of HCV infection. Thus, HCV clearance may result from an appropriate balance between cellular-mediated (CMI) and humoral-mediated immune responses (Fig. 2). Previous studies showed a reduced effectiveness of cytotoxic T lymphocytes (CTL) [8], CD4 + helper T cells [9], or antibody-producing B cells [10,11] in the acute phase of infection is associated with long-term viral persistence (Fig. 3). Importantly, a virus-specific T helper type 1 (CD4 + /Th1) T- cell response is important in viral clearance during the acute phase of disease, which has to be maintained permanently to achieve long-term control of the virus [12], indicating a vaccine candidate should direct a predominantly Th1 response. Besides, patients without viraemia after HCV infection frequently have strong Th1 lymphocyte responses to multiple HCV antigens many years after the onset of infection, whereas antibody responses are less marked [13]. Most HCV proteins have been shown to be targets of helper T-cell responses and CTL activities. Besides, multi-specific and vigorous T-cell proliferative responses against structural and nonstructural (NS) proteins are important in controlling HCV infection. Several highly conserved CD4 + T-cell immunodominant epitopes within the NS3 protein have been particularly linked to viral clearance in acute hepatitis C [14]. Strong T-cell proliferative responses against HCV core [15-19], E2 [17], NS3 [17,19, 20], NS4 [16-19] and NS5 [16-18] proteins have been found to be associated with self-limited infection. CTL escape mutants, including CTL antagonists, may contribute to the manifestation of chronic in- SO

3 Vaccine development for hepatitis C Fig. 1. Traditional and recent approaches in vaccine development. (A) shows the traditional methods of manufacturing vaccines containing live attenuated or killed whole bacteria or viruses. (B) demonstrates the genetic or molecular engineering strategies by which researchers have pursued an HCV vaccine. See text for detailed description of vaccine development. fection [21,22]. This observation is confirmed by studies in chimpanzees showing that during acute infection, CD8 + CTL activities correlated better with protection than the antibodies [23]. The envelope protein E2 of HCV contains highly variable sequences within the N-terminal region (HVR1) that encodes neutralizing B-cell epitopes [24-26]. Escape mutants arise in the hypervariable region, which leads to loss of immune control. The hypervariability of this region has been suggested as a possible mechanism through which the virus evades the immune response [24,27,28]. Besides, antibody responses to the envelope proteins develop slowly and achieve only modest titers during primary infection [29]. Therefore, neutralizing antibodies may emerge too late to prevent chronic infection. In addition, anti-envelope antibodies tend to be short-lived and disappear gradually after viral clearance [29]. Recently it was found that CpG immunostimulatory motifs enhanced humoral immune but not cellular responses against hepatitis C virus core protein after DNA-based vaccine immunization [30]. Fig. 2. Balanced components of humoral and cellular immune responses in viral infection. Cell-mediated immune responses (CMI) are regulated by cytokines that are produced by CD4 + T-helper cells. The major cytokines associated with the development of antiviral CMI are IL-2 and interferon-gamma, whereas cytokines such as IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 are thought to inhibit the development of CMI. IL: interleukin; TCR: T cell receptor; Th: T helper; MHC: major histocompatibility complex; TNF: tumor necrosis factor; Th1/Th2: helper T cells with a type 1/type2 cytokine profile. Fig. 3. Influence of interactions between cellular and humoral immune responses on viral escape. The left arm shows adequate viral control by early cellular immune responses after viral infection. The right arm demonstrates two subdivisions in which either the CD4 + T cells or CD8 + T cells (or both) have functional defects. Other mechanisms that have been shown to influence the generation or maintenance of early cellular immune responses after viral infection include rapid replication kinetics of the virus, high antigen load, poor or improper activation of Th1 and Th2 helper cells, antigenic sin, interference of the virus with the function of the antigen presenting cells, generation of altered peptide ligands, and genetic factors in the host such as low interferon levels. SP

4 C. T. Hu DNA VACCINES DNA-based immunization is the most recent approach in vaccine development (Fig. 4) and there have been many DNA immunization approaches and strategies targeted for HCV (Table 1). It could be of prophylactic and therapeutic value for HCV infection and induce immunity against multiple viral epitopes. DNAbased vaccines mimic the effect of live attenuated viral vaccines, eliciting cell mediated immunity in addition to inducing humoral responses. Efficacy may further be improved by addition of DNA encoding immunomodulatory cytokines and more recently, direct genetic modulation of antigen-presenting cells, such as dendritic cells, has been shown to increase antigen-specific immune responses [31]. A DNA-based vaccine usually consists of purified plasmid DNA carrying sequences encoding for an antigen of interest under the control of a eukaryotic 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 ease in generating and manipulating DNA and its potency in priming different arms of the immune response such as CTL, T helper cell, and antibody responses. However, one of the limiting factors in DNA vaccine immunogenicity is the low level of gene expression from the injected plasmid. THE POLYEPITOPE APPROACH TO DNA VACCINATION Fig. 4. Principles of DNA vaccination. Production of a DNA vaccine involves the isolation of genes from a selected pathogen followed by their insertion into a mammalian expression plasmid. Different arms of the immune responses including CTL (MHC class I restricted), T helper cells (MHC class II restricted) and antibody responses are elicited by this approach. The identification of the epitope as the smallest immunogenic subunit derived from antigenic proteins has led to the development of polyepitope-based vaccines. An effective, global HCV vaccine can be developed using DNA plasmid encoding a polyepitope protein, which contains multiple contiguous minimal CTL epitopes (Fig. 5). To select useful epitopes among those identified in the HCV structural and nonstructural proteins, one has to consider the following points: (1) HLA-A2 binding; (2) CD8 + recognition; (3) induction of CTL response; (4) interferon-gamma production; and (5) conservancy among different HCV genotypes. The Table 1. HVR1 Mimotopes with the Highest Frequency of Reactivity Amino acid sequences Highest crossreactivity (%) R9 QTTVVGGSQSHTVRGLTSLFSPGASQN 60 F78 QTHTTGGQAGHQAHSLTGLFSPGAKQN 70 M122 QTTTTGGSASHAVSSLTGLFSPGSKON 44 G31 TTHTVGGSVARQVHSLTGLFSPGPQQK 77 H1 QTHTTGGVVGHATSGLTSLFSPGPSQK 42 D6 QTTTTGGQVSHATHGLTGLFSLGPQQK 60 SQ

5 Vaccine development for hepatitis C Fig. 5. Principles of the polyepitope-based vaccine. Several identified HLA-A2-restricted CTL epitopes from various HCV proteins are linked together and cloned into an expression vector. After the polyepitope protein is synthesized, it will be delivered to the proteosome to undergo proteolytic processing to generate different peptides carrying different CTL epitopes. In this example, the NS2B epitope peptide is degraded and the NS4B peptide is truncated, rendering loss of immune responses from both of these epitopes. C: core protein; E: envelop protein, NS: nonstructural proteins; ampr: ampicillin resistance gene; Ori: origin of replication of the plasmid. multiple CTL responses induced by this multiepitopebased strategy can target to numerous antigens to avoid escape from immune detection by antigen loss variants. ANTIBODY RESPONSES The interaction between the virus and the host immune system determines the outcome of HCV infection. Apparently, the persistence of infection in most HCVinfected individuals, despite the presence of HCV-directed antibodies, suggests that most of the antibody responses described below fail to induce viral clearance. Antibody responses to the core protein Most DNA-based vaccines targeted the HCV core protein because it is highly conserved among various genotypes. However, the anti-core antibody response is frequently weak or transient. Studies in which mice were immunized with HCV core DNA alone showed no or only weak antibody responses [32-34]. To enhance the humoral immune responses against this non-secreted viral protein, DNA encoding interleukin 4 (IL-4), IL-2, or granulocyte/macrophage colony-stimulating factor (GM-CSF) was co-administered along with the HCV core DNA [32]. Co-immunization 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 anti-core antibodies, whereas core gene immunization alone could not generate an IgG response in mice [35]. 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 [34,36]. In two independent studies, the chimeric expression plasmids induced anti-core antibodies in all immunized mice as compared with 0% [34] and 40% [36] 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 [34]. Antibody responses to the envelope protein The HCV E2 protein has been a major focus for developing a DNA-based HCV vaccine because HCV is an enveloped virus and neutralizing epitopes likely reside on the surface of the envelope. However, 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 [37]. 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 fulllength E2 [38]. Based on these results, two chimpanzees were immunized three times with this construct. Only one animal developed anti-e2 antibodies but preliminary data from challenge studies showed no protection against viral challenge [39]. The co-delivery of cytokines (i.e. GM-CSF) was also explored to enhance the immune responses against the HCV envelope proteins [40]. A combined vaccine regimen, consistent of priming with E2 DNA and boosting with recombinant E2 protein, could also enhance antibody (IgG2a) responses [41]. The mode of DNA delivery has also been shown 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 intramuscular injections in mice [42]. The combination of both delivery routes may be more efficient in inducing broad antibody responses [43]. Antibody responses to NS proteins Two studies on the DNA immunization using plasmids encoding NS3, NS4, and NS5 proteins individu- SR

6 C. T. Hu ally or together demonstrated the successful induction of HCV-specific antibodies against NS3, NS4, and NS5 in mice and buffalo rats [44,45]. However, the co-delivery of GM-CSF did not enhance the antibody titers to HCV NS3, NS4, and NS5 [45]. LYMPROPROLIFERATIVE RESPONSES The most commonly used method to assess specific CMI in vitro has been to test the ability of peripheral blood mononuclear cells (PBMC) derived from patients to proliferate in response to viral antigens. It has been assumed that these assays largely measure CD4 + T-cell responses; however, CD8 + T cells or natural killer cells can also contribute to the response. DNA immunization can induce lymphoproliferative responses against the structural proteins including core [32,35,36,46], E1 [47], E2 [47], and the NS3, NS4, and NS5 in mice [44] and buffalo rats [45]. 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 co-delivery of GM-CSF, IL-2, or IL-4 genes can increase the lymphoproliferative responses significantly [32,45]. Additionally, boosting with a recombinant HCV core protein after HCV core DNA immunization appears to enhance the T helper cell proliferation [35]. 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 [36]. CYTOKINE RESPONSES It has been shown that patients with persistent viremia and chronic hepatitis C have less PBMC showing type 1 cytokine (IL-2, IFN-gamma) responses to HCV core protein than patients with self-limited HCV infection. The mechanism is still unknown and a Th1 to Th2 shift has been rejected as the mechanism in chronic hepatitis C [48]. Intramuscular DNA immunization induced predominantly interferon-gamma but not IL-4 production, suggesting a Th1 response [32,44,49]. Analysis of the IgG subtypes showed an almost exclusive IgG 2a and 2b antibody production [42,43,47], which is also consistent with a Th1-like response. CYTOTOXIC T-CELL RESPONSES CTL responses to the core protein The CTL activities in mice immunized with HCV core plasmids were generally demonstrated [35,36,46, 50-52]. Specific CTL responses were detected only in mice injected with plasmid constructs encoding the core alone or together with E1 and E2 [52], implicating that the core region might have the strongest CTL epitope in the structural region for BALB/c mice. Core-specific CTL activities were highest in mice co-immunized with an IL-2 expressing plasmid, whereas GM-CSF did not significantly augment CTL activities [32]. In addition, immunization with HBV and HCV chimeric proteins [36] or combined DNA-protein immunization [35] did not alter the generation of core-specific CTL responses. On the contrary, co-administration with an IL-4 construct suppressed HCV core-specific CTL activity [32]. Furthermore, mice injected with an HCV core construct truncated [39], which removes a known CTL epitope, showed almost no induction of CTL activities in BALB/ c mice [36]. CTL responses to the envelope protein The CTL responses of DNA immunization against the envelope proteins E1 and E2 have been addressed in some studies [41,52-54]. 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 was closely associated with the protection of mice against challenge with an E2 expressing tumor cell line [41]. CTL responses to the non-structural proteins CD8 + CTL activities have also been demonstrated for NS3 and NS5 after three intramuscular injections with NS3 and NS5-encoding plasmids [44]. In addition, a tumor model in which syngeneic cells stably transfected with an NS5 expression plasmid was used to assess CTL activity in vivo [44]. About 60% of immunized mice were protected against tumor formation, and in those which developed tumors, the tumor weight was significantly reduced compared with unimmunized mice. CTL responses in the HLA-A2.1 expressing transgenic mice Since most of the above studies used BALB/c mice (their MHC is called H-2), a transgenic mouse model expressing human HLA-A2.1 was developed to better approximate HCV infection in humans [51,55]. This transgenic mouse model was used to study the generation of humanlike HLA-A2.1 restricted CTL responses. In this study [51], the HCV core DNA vaccine generated a long-lasting protection against infection with re- SS

7 Vaccine development for hepatitis C combinant vaccinia virus expressing HCV core in vivo, despite a weak anti-core CTL response requiring at least three stimulations with peptides to detect the HCV corespecific CTL activities in the standard 51 Cr release assay. The authors also showed that the protection was mediated by CD8 + T cells. CTL responses to different promoter-driven expression In one study, plasmids encoding core, E1 and E2 individually or together, under the control of a cytomegalovirus promoter were constructed [53]. The authors compared the efficacy of these constructs with a plasmid containing coding sequences for core, E1 and E2 together under the control of the human elongation factor l alpha (EF-la) promoter. BALB/c mice were immunized only once with these constructs. Spleen cells obtained from the DNA-immunized mice were assessed for their ability to lyse MHC-matched target cells. Only mice inoculated with the plasmid expressing core, E1, and E2 under the EF-la promoter generated HCV-specific responses against all three proteins after a single immunization. RECOMBINANT VIRAL VACCINE VECTORS Most of the DNA-based vaccine studies against various HCV proteins 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 NS proteins. Recombinant viruses are an efficient vehicle for DNA delivery that can result in a high level of recombinant protein expression in host cells. Several recombinant viral vectors described below are being evaluated for HCV vaccine development. In addition, a highly attenuated, recombinant rabies virus vaccine strain-based vector was utilized as a new immunization strategy to induce humoral and cellular responses against HCV glycoprotein E2 [56]. Recombinant adenovirus 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. 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 [57]. Besides, strong CTL responses against core and E1 could be detected in splenocytes from mice immunized with an adenovirus carrying core and E1 genes [58]. Co-administration of a recombinant adenovirus expressing IL-12 led to a marked increase in cellular immune responses when administered at a dose of 107 plaque-forming units [59]. Vaccination with an adenoviral vector encoding HCV NS3 protein protects against infection with HCV-recombinant vaccinia virus [60]. Recombinant vaccinia virus Spleen cells from mice immunized with a recombinant vaccinia virus expressing the HCV core gene exhibited strong core-specific CTL activities [61]. In addition, studies 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 persistence of HCV infection [62]. Recombinant canarypox virus A non-replicating canarypox virus encoding polycistronic core/e1/e2/ns2/ns3 genes was used to potentiate the immune response to HCV DNA immunization. Preliminary data suggest that a booster injection with this recombinant canarypox virus enhanced the HCV-specific immune response and generated broader T-cell activity [55]. RECOMBINANT BACTERIAL VACCINE VECTORS Listeria monocytogenes Recombinant Listeria monocytogenes (LM) have been exploited as live vaccine vectors for the induction of protective cell mediated immunity. LM is a gram positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell-to-cell without encountering the extracellular milieu. Infection of mice induces an LM specific CD8 + CTL response which contributes to long lasting CMI. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the MHC class I antigen processing and presentation pathway. A plasmid DNA vaccine encoding a ubiquitin-hcv NS3 fusion protein was generated, and its efficacy was confirmed by in vivo induction of NS3-specific, gamma ST

8 C. T. Hu interferon-secreting T cells following vaccination of BALB/c mice. These immunized mice also exhibited specific in vivo protection against subsequent challenge with a recombinant LM (TC-LNS3) expressing the NS3 protein. Notably, sublethal infection of naive mice with the strain TC-LNS3 induced similar NS3-specific T-cell responses. These findings suggest that recombinant strains of L. monocytogenes expressing HCV antigens should prove useful for evaluating, or even inducing, protective immune responses against HCV antigens [63]. Recombinant attenuated Salmonella typhimurium The promoter of phop-activated gene C (P(pagC)) of Salmonella typhimurium was cloned and used as transcriptionally regulating element for a plasmid that expresses hepatitis C virus core antigen [64]. The resultant plasmid was transformed into attenuated Salmonella typhimurium SL7207. This recombinant strain and another bacterial strain that constitutively expresses HCV core antigen were orally inoculated in BALB/c mice. The results showed that the in vivo activated P(pagC) promoter could stabilize the plasmid in the bacteria and enhance the humoral and cellular immune responses greatly, showing a novel way to produce an effective, cheap oral vaccine against hepatitis C. PEPTIDE VACCINES Major histocompatibility complex The class I MHC molecules found on almost all cell types present only peptide fragments generated intracellularly to CD8 + T cytotoxic cells whereas class II MHC molecules on antigen presenting cells present antigenic peptides to T helper cells. Therefore, peptide vaccines under this principle use 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 candidates for HCV. CMI responses to peptides A peptide vaccine can be designed using amino acid motifs to predict the binding of peptides to MHC class I or II molecules. Several CTL and T helper epitopes on the HCV polyprotein have been found to be important for viral clearance. Peptides containing epitopes from the core [65-67], NS4 [67], and NS5 [65] 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 [65-67]. Surprisingly, enhancement of the immunogenicity of a core-specific CTL epitope has been achieved by substitution of only one amino acid on the native peptide [68]. Furthermore, covalently linked T helper and CTL epitopes were more potent immunogens when delivered as lipidated peptides [67]. Antibody responses to peptides Peptide vaccines can induce antibodies against linear epitopes. The HVR1 is an attractive target for a peptide-based vaccine because it contains a neutralizing epitope. 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 [69]. 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 [70] produced high titers of broadly cross-reactive antibodies to HCV that could block the binding of antibodycaptured HCV to MOLT-4 cells. HVR1 mimotopes The most difficult problem of choosing the HVR1 as the target for a HCV vaccine is the existence of quasispecies in this region of the 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 antibodies that cross-react with a large panel of HVR1 were identified [71]. 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 (Table 2) could generate antibodies that recognized 95% of the same panel of natural HVR1 variants. The major obstacles for a peptide-based approach lie in the observation that a 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 co-administration of potent adjuvants or the use of a polyepitope vaccine that contains a mixture of peptides. SU

9 Vaccine development for hepatitis C Table 2. DNA Immunization Approaches and Strategies Approach HCV Immunogens Antibody Th Cells CTL References DNA Structural proteins Core + IL Core, E1, E2 Low Low Low 32-34, 38, 40 Core + IL-4 32 Core + IL-2 32 Core + GM-CSF 32 tcore-hbv 34, 36 E2 + CpG motifs nd 87 te2surf* nd nd 38 te2s -GM-CSF + tels -GM-CSF nd 40 Core-E1-E2 under EF-1 α promoter nd nd 53 DNA + protein boost Core + recombinant core 35 DNA + protein boost te2s + recombinant E2-HSV 1gpD nd 41 Nonsturctural proteins NS3 (liposome-mediated) nd nd 88 NS3, NS4, NS NS3, NS4, NS5 + GM-CSF 45 Mixed Core, E1, E2, NS2, NS3 + recombinant canarypox virus boost nd 55 *: C-terminus of E2 replaced by platelet-derived growth factor receptor transmembrane domain targeting to the cell surface (surf); Th: T helper; CTL: cytotoxic T lymphocyte; IL: interleukin; t: truncated; : signal sequence of E1 and E2 proteins replaced by the signal sequence of HSV 1 protein D; +: positive response; : enhanced response; : decreased response; : no change in response; nd: not done RECOMBINANT PROTEIN SUBUNIT VACCINES Immunoprophylaxis function A subunit vaccine composed of recombinant HCV proteins may protect from infection or chronic infection by different HCV genotypes. The initial attempt to develop an HCV vaccine was directed toward generating a recombinant protein subunit vaccine. Because it has been shown for several flaviviruses that antibodies to the envelope protein can provide protection, recombinant HCV E1 and E2 proteins were used in early vaccination studies from Chiron [72]. Although no sterilizing immunity was achieved in chimpanzee experiments using recombinant HCV E1 and E2 proteins, chronic infection might have been prevented. Immunotherapeutic function Recombinant HCV proteins have been shown to have an immunotherapeutic function. Recombinant E1 protein of genotype 1b was purified as homodimers that associated into particles of about 9 nm in diameter [73]. 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 decreases in alanine aminotransferase (ALT) levels in both animals. Although HCV RNA levels in the serum remained steady 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 Given the structural proteins of HCV-like particles (HCV-LPs) are presented in a native, virion-like conformation, the HCV-LP is superior in eliciting a protective immune response compared with the recombinant subunit-based vaccine. In addition, HCV-LP, as a particular antigen, may elicit a CTL response [74-76], which plays a critical role in viral clearance. The HCV- LP, which was synthesized in insect cells using a recombinant baculovirus containing the cdna of the HCV structural core, E1, and E2 proteins [77], exhibits morphologic, biophysical, and antigenic properties simi- SV

10 C. T. Hu lar to the putative virions isolated from HCV infected humans. These noninfectious nm HCV-LPs consist of a lipid envelope containing E1 and E2. Mice immunized with HCV-LP generated a strong humoral immune response against the structural proteins core and E2 [78]. HCV-LPs are also capable of inducing strong cellular immune responses in BALB/c mice [79]. 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-γ but not IL-4 was produced by the HCV-specific activated T cells, suggesting a type I-like response. PRIME-BOOST MODALITY Only a few studies have explored the DNA-protein prime-boost regimen for an HCV vaccine [35,41]. However, the development of an HCV vaccine may follow some promising studies from the HIV field. The efficacy of prime-boost approaches by priming with DNA and boosting with recombinant virus vectors or viral proteins 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 an FPV boost [80,81]. Priming with DNA followed by boosting with gp 120 also generated protective responses that were far superior to either DNA or recombinant protein immunization alone [81]. CONCLUSION A successful HCV vaccine may require a multi-component approach that stimulates various aspects of the immune response including broad humoral, T helper, and CTL responses. The promising strategy might be a combination of different approaches, such as a combination of DNA and recombinant subunit protein vaccines. The combination of multiple epitopes residing on the HCV-LP and HCV nonstructural proteins, which can stimulate CTL and interferon responses, may be a focus to be investigated. To induce a broad cellular immune response in the general population, it is also necessary that the vaccine candidate contains epitopes that are restricted by diverse HLA 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 crossprotection. Mimotope sequences derived from the HVR1 that induce cross-reactivity are encouraging and may be valuable for the development of a broadly active vaccine. Another spotlight is the codelivery of cytokines to enhance the immune responses against DNA-based HCV vaccines. Thus, the advantages of other costimulatory or immunomodulating molecules such as B7 [82], CD40 ligand [83,84], or CTLA4 [85] that have been shown to enhance nucleic acid immunization should be explored for HCV DNA vaccination. The delivery of DNA is also a crucial step that should be studied in more detail; factors such as the routes and methods of delivery and the delivery systems should be optimized. Furthermore, the prime-boost combination of DNA and protein vaccines should be carefully evaluated to establish an immunization protocol that maximizes the potency of both approaches. The progress in recombinant DNA technique, molecular cloning and eukaryotic protein expression has a great impact on molecular and cellular immunology. Issues such as HCV epitope mutations which cause the failure of strong binding to different alleles of the HLA- A2 superfamily, the loss of motif recognition by CD8 + T-cells, molecular interactions between HCV and host cells, and the emergence of HCV variants with altered peptide ligands as TCR antagonists accompanied by a limited TCR repertoire remain to be explored [86]. In the development of a globally effective HCV vaccine, one of the most challenging tasks is to look for epitopes which have a high conservancy rate for the 6 different genotypes and which can induce interferon production and CTL response. The use of short synthetic peptides (i.e. 15 to 20 amino acids), covering an entire HCV protein region (i.e. the core protein) can help to identify immunodominant T cell epitopes. The constrained peptides mimic at least a part of a conformational epitope and are thus called mimotopes. The sequential information contained in those epitopes found so far tends to be minimized and effective in eliciting neutralizing antibodies, or cell-mediated responses. Besides, the importance of molecular and topologic determinants in the elicitation of immune response can be further illustrated by the virion-like conformation of the HCV-LP by which stronger humoral and cellular immune responses than DNA immunization per se were induced. However, the molecular basis for viral induced B cell proliferation is still unknown. One possibility is that HCV stimulates the proliferation of monoclonal B cells via their HCV-specific B cell receptor on the cell surface. Binding of the HCV envelope proteins to a cel- TM

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