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Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006.

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Chapter 15Recombinant Vesicular Stomatitis Virus (VSV) and Other Strategies in HCV Vaccine Designs and Immunotherapy

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Abstract

Several vaccine strategies have been attempted in chimpanzee and smaller animal models to generate immune responses to hepatitis C virus (HCV). While neutralizing antibody may play a role in preventing HCV infection, studies in chimpanzees and humans during rare cases of acute resolving HCV infection indicate that, HCV immunity appears to be associated with vigorous, sustained and multi-specific Th1 intra hepatic CD8+ and CD4+ T cell responses. Several new promising technologies utilizing viral based vaccine approaches that appear to generate both antibody and cell mediated immune responses have recently been reported. These include viral vectors that express HCV products and non-replicating viral like particles (VLPs) that appear to induce T-helper type 1 (Th1) immune responses considered important in resolving HCV infection. In addition, viral vectors based on recombinant vesicular stomatitis virus (rVSV) may offer safe yet potent stimulation of both innate and adaptive immune responses. Here, we review the successful application of viral based vaccines, including VSV in generating viral immunity in animal models and describe the potential usefulness of this technology as a strategy for HCV vaccine design and immunotherapy.

Introduction

The inhibition of virus replication by the immune system is of paramount importance to limit the spread of infection and moderate the course of the disease. Essentially, control of viral infections consists of non-specific innate immune responses and adaptive responses to viral specific proteins (Parkin and Cohen, 2001). Vaccine intervention aims to stimulate B cell antibody production (humoral) and cell mediated (CD4+ and CD8+) immunity (Begue, 2001b). However, there have been several major obstacles that have hampered the development of an effective HCV vaccine. Firstly, apart from humans, the only infectious HCV animal model is the chimpanzee, a protected species that is costly and hence limited in its availability (Bukh, 2004). Secondly, although extensive studies from chimpanzees and patients have provided insights into immune responses, it remains unclear as to why the immune system, in many cases, is inefficient in eliminating HCV infection (Gremion and Cerny, 2005). Despite detectable humoral and cell mediated immunity, HCV induces 50–80% chronicity of infected individuals leading potentially to cirrhosis and hepatocellular carcinoma (Harris et al., 2002). HCV is also highly variable, divided into at least 6 major genotypes and more than 50 subtypes based on nucleotide diversity within core, E1 and NS5 genes (Zein, 2000). In addition, as observed with other RNA viruses, HCV exists as groups of related but distinct viral populations termed quasispecies variants that differ in sequence diversity within distinct hypervariable regions along the genome. The N-terminus of the E2 glycoprotein (a prime vaccine target) contains a major hypervariable region 1 (HVR-1) and sequence variation in the HVR1 has been associated with immunologically driven HVR1-antibody escape mutants (Farci et al., 1997; Majid et al., 1999). The first neutralizing antibody epitopes were located within the HVR-1 region, and hyperimmune serum obtained from immunization of a rabbit with HVR-1 peptide has been found to protect against homologous HCV in chimpanzees, but did not protect against 'escape' mutants that persisted during chronic infection (Farci et al., 1996). Nevertheless, studies associated with intravenous drug users who resolved previous HCV infection and who are less likely to be re-infected suggests that immunity against HCV can be successfully generated in some individuals (Mehta et al., 2002). Furthermore, recent work illustrates that targeting multiple epitopes in the HCV envelope proteins (E1 and E2) may facilitate a more broad neutralization capacity (Bartosch et al., 2003a). Immunological studies of acute phase HCV infection suggest that this period is critical for determining the outcome of infection (Thimme et al., 2001). In humans and chimpanzees, the clearance of acute infection is accompanied by a strong and multi-specific CD4+ and CD8+ T cell response (Haefelin-Neumann et al., 2005; Rehermann and Nascimbeni, 2005). Immune mediated control of HCV infection is also evident from intrahepatic compartmentalization of HCV specific T cells, aggressive disease progression in HCV/HIV co-infected individuals and increased viral loads with immunosuppression of patients (Gremion and Cerny, 2005).

Here, we examine the problems encountered in the development of HCV vaccines and evaluate current as well as future vaccine strategies to generate effective immune responses to HCV. Several approaches designed to elicit immune responses to HCV structural proteins (Core, E1 and E2 glycoproteins) and the bi-functional viral serine protease/helicase (NS3) that contains conserved 'immunodominant' regions, have been tested in non-human primates, monkeys and murine models. Although a comprehensive review of these technologies is beyond the scope of this chapter, we briefly describe the need for more optimal approaches to HCV vaccine design. In this regard, we discuss the recombinant Vesicular Stomatitis Virus (VSV) as a vector that could be useful in the fight against HCV infection.

Classical Strategies in Viral Vaccines

The principle of vaccination is to induce a 'primed' state in the host so that infection with a pathogen will result in a rapid secondary immune response. The goal to eliminate or inhibit replication of the organism and protect from clinical disease is dependent upon memory T and B cells as well as neutralizing antibody in the serum. Virus infection and replication in host cells is known to elicit long-lived antibody and cell-mediated immunity. These features make viral vectors attractive as vaccine candidates after safety issues are addressed by attenuation or inactivation, since they can often induce long-term immunity following a single dose.

Non-virulent viral vaccine strategies have been successfully developed for a number of viral pathogens that can be grown in cell culture to facilitate their attenuation or inactivation (Begue, 2001b, Gershon, 1990; Hinman and Orenstein, 1990; Matter, 1997). Live attenuated viruses typically have reduced virulence caused by repetitive passage during in vitro cell culture growth conditions. Selected mutants replicate poorly in the host and do not cause disease but efficiently induce long-lived antibody and cell-mediated immunity. Indeed, Measles, Mumps and Rubella (MMR vaccine) are controlled in many developed countries through this live attenuated vaccine approach (Burgess, 1994; Wharton et al., 1990; Zimmerman and Burns, 1994). The worldwide eradication of smallpox is another example of a live attenuated heterologous vaccine. In this case, the cross reacting immunity of vaccinia (less virulent) is protective against variola virus, the causative agent of small pox (Begue, 2001a, Enders et al., 2002). Selectively targeted live attenuated vaccines also include single doses of yellow fever for travellers and varicellazoster virus for the elderly (Hill, 1992; Marfin et al., 2005; Senterre, 2004; Takahashi, 2004). The main drawback of live attenuated vaccines however, is the danger of reversion to virulence and the possibility of causing extensive disease in immunocompromised individuals.

When live attenuated vaccines are unavailable, inactivated preparations of the virulent organism using beta-propiolactone or formaldeheyde are an option (Bachmann et al., 1993; Jiang et al., 1986; King, 1991). However, these inactivated vaccines generally only stimulate humoral responses, are expensive to prepare and in addition, the chemical inactivation can directly impair certain immune responses such as T cell activation (Bachmann et al., 1993). However, the Salk poliovirus vaccine containing all 3 polio-virus strains is a successful example of this approach and is particularly useful in protecting immuno-suppressed children (Pearce, 2004). The live, less expensive Sabin polio-vaccine has been adopted in many parts of the world due to lower costs and elicits effective induction of mucosal immunity (Pearce, 2004). Seasonal Influenza vaccines similarly comprise inactivated Influenza A and B strains (Hill, 1992; Marfin et al., 2005; Schwartz and Gellin, 2005; Takahashi, 2004). For Rabies virus, a human diploid cell culture-derived inactivated vaccine is administered either for post exposure prophylaxis following a rabid animal bite or pre-exposure prophylaxis to protect animal workers at risk of infection from occupatioal exposure (Lodmell and Ewalt, 2004).

In regard to other major etiological agents of liver disease, effective hepatitis A virus formalin inactivated cell culture vaccines are available (Provost et al., 1986; Strader and Seeff, 1996). Two doses administered one month apart appear to induce high levels of neutralizing antibodies. For hepatitis B virus (HBV), HBV surface protein purified from viral carriers, or a recombinant viral approach have been successfully utilized to protect against HBV infection (Coursaget et al., 1990; Goilav and Piot, 1989; Magnani et al., 1989; Prince et al., 1984; Szmuness, 1979). In the first strategy, a trial in homosexual men in the USA showed that 3 intramuscular injections at 0, 1 and 6 months appeared to protect 95% of vaccinees (Goilav and Piot, 1989). However, the latter HBV vaccine is now more widely used in the universal childhood immunization scheme and is given at 6, 10, and 14 weeks of age, and likely requires a booster later in life (Milne et al., 1992; Miskovsky et al., 1991; Murata et al., 1989). Importantly, HAV and HBV vaccination demonstrates the plausibility of protection against hepatopathogens that replicate primarily in immune-compromised environment of the liver (Willberg et al., 2003). However, it has been more difficult to develop HCV vaccine along similar lines since HCV does not replicate efficiently in cell culture. Recently, three independent reports (see chapter 16) describe complete HCV replication of a chimeric genotype 2a replicon in cell culture, which may facilitate vaccine strategies (Lindenbach et al., 2005). Thus, large scale purification of chemically inactivated or attenuated HCV strains using this technology remains an exciting prospect for HCV vaccine studies. Meanwhile, alternative recombinant approaches to engineer immune responses to HCV have currently been used in HCV vaccine studies.

Studies of Recombinant HCV Protein Sub-Units in Chimpanzees

In the early 1990s, chimpanzees were immunized with purified recombinant HCV E1 and E2 glycoproteins as these were presumed targets for virus neutralization. The source vectors for these subunit vaccines were recombinant vaccinia (rVV) since rVV expressed products were known to elicit potent antibody responses in alternate vaccine regimens (Choo et al., 1994; Ralston et al., 1993). These rVV vectors demonstrated high-level protein expression from prototype HCV-1 structural (core-E1-E2 1-906) cDNA (Ralston et al., 1993). Purified E1 and E2 products mixed with the MF59 adjuvant also produced neutralizing antibodies since protection was observed following challenge with low doses of homologous HCV but not against heterologous HCV infection (Choo et al., 1994; Houghton et al., 1997; Ott et al., 1995). Subsequently, since E1/E2 antigens appeared susceptible to genetic variation amongst HCV types, the HCV core protein that is highly conserved amongst HCV genotypes was also evaluated. However, since subunit vaccines alone appear inefficient at inducing cytotoxic T lymphocyte (CTL) responses, the core protein was combined with a 40nm matrix composed of saponins, cholesterol and phospholipids (ISCOM) (Polakos et al., 2001). The classical ISCOM method entraps the antigen inside the adjuvant platform to facilitate priming of CD4+ and CD8+ mediated responses (Takahashi et al., 1990). For HCV vaccine studies, a non-classical ISCOM approach was used where E. coli purified core protein was adsorbed onto ISCOMATRIX. The resulting particulates stimulated strong long-lived, CD4+ and CD8+ responses and induced Th0-type (Th1 and Th2-type cytokines) as well as anti-core antibodies in Rhesus Macaques (Polakos et al., 2001). The prospects of HCV ISCOM-vaccines to produce sterilizing immunity awaits to be reported but a core vaccine itself may have therapeutic value since core-specific CTLs in HLA-B44+ patients co-incided with lower viral titers, and core-CD4+ T cell responses correlated with milder courses of liver disease (Bottarelli et al., 1993; Hiroishi et al., 2004)

To faciliate broader responses to HCV types, other studies have utilized truncated forms of E1aa192-330 and E2aa 390-683 (HCV-N2) purified from baculovirus-infected cells combined with HVR-1 peptides from different isolates (HCV-6) (Esumi et al., 1999). However, despite high antibody responses to E1/E2 in chimpanzees, the low level immune responses to HVR-1 peptides resulted in lack of sterilizing immunity to HCV-6 challenge that was achieved only by boosting HVR-1 (HCV-6) antibody responses. It seemed that antibody responses alone were incapable of neutralizing HCV infection and these observations pointed to the requirement for technologies that may facilitate broader immune responses to control HCV infection. In this regard, the introduction of DNA vaccination technologies offered alternative or complementary approaches to E1/E2 subunit vaccines.

Efficacy of Recombinant DNA Approaches in Generating HCV Immunity

Naked DNA or plasmid vaccines encode viral genes that following inoculation of a host are expressed to stimulate immune responses. DNA vaccination was reported to prime good antibody responses and offer the advantage of increasing CTL responses (Donnelly et al., 1997). In fact, DNA-derived immunogens encoding HCV core and E2 sequences alone or fused with hepatitis B surface antigen induced antibody and CTL responses to HCV or HBV in BALB/c mice (Inchauspe et al., 1997). However, the recombinant DNA-vaccinated animals lacked neutralizing antibodies that could block binding of purified HCV E2 to the putative cellular receptor, CD81 (Heile et al., 2000; Pileri et al., 1998). Furthermore, the same study highlighted that endoplasmic retained recombinant E2 protein expressed in mammalian cells was superior in eliciting neutralizing antibodies. The latter finding stresses the importance of generating antigens that are correctly proteolytically processed and therefore authentically folded (discussed below).

Combining DNA vaccination with boosts of purified protein or recombinant viruses expressing the same antigens appear to enhance immune responses (Pancholi et al., 2000; Pancholi et al., 2003; Song et al., 2000). However, in order to assess the effectiveness of immune responses generated using these and other HCV vaccine approaches, surrogate HCV challenge technologies have been created to replace the need for expensive chimpanzee models in preliminary studies. Two examples include: vaccinia-expressing HCV structural proteins, or Listeria monocytogenes-expressing HCV NS3, both of which were developed to monitor the efficacy of elicited immune responses in murine and potentially higher animal models (Pancholi et al., 2003; Simon et al., 2003). The HLA-A2.1 transgenic mouse model has been especially useful for refining cell-mediated HCV immunity (Pascolo et al., 1997). These mice are devoid of murine MHC-I molecules and are transgenic for human HLA-A2.1 (A0201) monochain major histocompatability class I molecule. Studies have shown that these mice recognized the same HCV-derived peptides found in human HLA-A2.1-restricted CTLs (Arichi et al., 2000; Brinster et al., 2001). DNA vaccines can be positively modulated as shown in murine studies with adjuvants such as CpG motifs and Quil A that appear to induce Th1-(IL2 and IFN γ cytokine profile) biased immune responses in addition to strong antibody responses as demonstrated in DNA vaccination to HCV NS3 (Hong et al., 2004). Furthermore, efficient presentation and priming of cell-mediated responses can be optimized using cationic microparticles that carry DNA-based vaccines (Hagan et al., 2004). Alternatively, recombinant semliki forest virus (rSFV) particles expressing NS3, in combination with DNA vaccination or alone, have been found to stimulate strong immune responses in mice (Brinster et al., 2002). The rSFV particles infect host cells but do not replicate. However they express high levels of NS3, which appear to induce (as with DNA vaccine) NS3-specific CTLs targeted to dominant HLA-A2 epitopes described in patients (Urbani et al., 2001). However, only rSFV-NS3/DNA combination experiments elicited anti-NS3 antibodies in non-transgenic BALB/c mice (Brinster et al., 2002). Unfortunately, in chimpanzees, DNA immunization-encoding HCV E2 appeared incapable of generating sufficient antibody and cellular immune responses to clear HCV infection (Xavier et al., 2000). A good HCV vaccine should elicit strong antibody and cellular immune responses., and current studies in HCV-DNA vaccine approaches suggest combinatory vaccine strategies are likely required to enhance HCV antigen responses.

Advancements in recombinant viral technologies have led to additional strategies that may generate safer approaches to viral based vaccine regimens. These include production of virus-like particles (VLPs) that closely resemble properties of the native virion. These VLPs have been shown to elicit potent humoral and importantly cellular (CTL) activity as demonstrated with human papillomavirus-VLPs, recombinant HIV and HBV VLPs in animal studies (Kahn et al., 2001; Roberts et al., 1999; Roberts et al., 1998; Roberts et al., 2004; Rose et al., 2001). Recently, HCV-like particles based on C, E1 and E2 that are capable of stimulating CTL and humoral activity have also been reported (see discussion below).

Hepatitis C Virus-like Particles

Recombinant baculovirus expression of the HCV structural proteins (core/E1/E2) has been reported to generate HCV-like particles (HCV-VLPs) that have biophysical, ultrastructural, and antigenic properties similar to those of the putative virions (Baumert et al., 1998; Baumert et al., 1999). Immunization of BALB/c mouse strains or human HLA-A2 transgenic (AAD) mice with these 40–50nm non-infectious HCV-VLPs suggests that they are efficient at generating broad and vigorous humoral and cellular immune responses compared to immunization with DNA (Murata et al., 2003). Mice vaccinated with HCV-VLPs developed antibodies to HCV E1/E2. These anti-HCV E1/E2 antibody responses were enhanced by HCV-VLP plus monophosphoryl lipid A [MPL] and QS21 adjuvant (AS01B) or CpG 10105 and especially with combination of AS01B plus CpG 10105 (Qiao et al., 2003). Furthermore, isotype analysis of the induced anti-HCV envelope proteins demonstrated that HCV-VLP alone induced immunoglobulin (Ig) G1 response while the use of adjuvants ASO1B and CpG 10105 combined facilitated predominately IgG2a response that is indicative of a Th1 response proposed to be important for HCV clearance. The neutralization capacity of these anti-E1/E2 antibodies induced by HCV-VLP cannot be tested easily due to the lack of a suitable small-animal model system. However, by challenging mice with recombinant vaccinia expressing HCV structural antigens (vv.HCV.S-genotype 1b), investigators were able to examine the induction of immune protection in murine and higher animal models (Jeong et al., 2004; Murata et al., 2003). Although vvHCV.S infection is not representative of natural HCV infection, studies analyzing immunization strategies with baculovirus generated HCV-VLPs showed that HCV-VLP vaccinated mice were better protected against vvHCV.S than DNA immunization (Murata et al., 2003). This protection is due in part to the fact that, as compared with animals immunized with DNA methods, HCV-VLPs elicit strong CTL responses and vigorous CD8+ T cell responses against HCV core and E2 proteins. It appears that unlike recombinant protein subunit vaccines, these virion like structures can possibly be processed efficiently through the major histocompatibility complex 1 pathway and subsequently effectively prime CD8+ responses. HCV-VLPs therefore offer promise for further study in chimpanzee or human trials.

Recently, infectious particle systems have also been described where pseudo-particles are assembled that display unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles (Bartosch et al., 2003b, Flint et al., 2004). These particles were primarily designed to understand HCV cell entry but could provide useful information for vaccine development especially with regard to the potential neutralization of HCV glycoproteins to their target receptors using anti-sera of animals treated with different vaccine regimens. In fact, the presence of a green fluoresent protein marker packaged within these HCV-pseudotype allowed determination of infectivity mediated by the HCV glycoproteins in primary hepatocytes and hepato-carcinoma cells (Bartosch et al., 2003b). This infectivity was neutralized using patient sera and by some anti-E2 monoclonal antibodies, indicating a role for neutralizing antibodies against HCV glycoproteins. The potential modification of these particles to render them non-infectious may allow for in vivo vaccine studies in animal models.

In addition to the above studies, our laboratory and others have focused on using vesicular stomatitis virus (VSV) as a candidate for evaluation as a virus-based strategy for HCV vaccination and/or immuntherapeutic studies (Ezelle et al., 2002; Majid et al., 2005). The advantages of using VSV-based approaches to generate immune responses against HCV are discussed below.

VSV as a Safe and Potent Vector for Generating Immunity against Viral Pathogens

VSV is a member of the Rhabdoviridae family and is a negative-stranded cytopathic virus. Rhabdoviruses are classed into at least 5 genera. Vesiculoviruses, Lyssaviruses and Ephemeroviruses infect animals (both vertebrates and invertebrates), whereas Cytorhabdoviruses and Nucleorhabdoviruses infect plants. VSV is composed of an 11-kilobase negative-sense RNA genome and is relatively simple since it encodes for only five viral proteins: the nucleocapsid (N) that encases the virus genome, 2 polymerase proteins (L and P), a single surface glycoprotein (G) and a peripheral matrix protein (M). Rhabdoviruses are enveloped viruses and their glycoproteins are classed as typical type 1 membrane proteins consisting of polypeptide trimers of the viral G protein (Coll, 1995). This G protein is a dominant viral antigen and is responsible for viral infection through unidentified cellular receptor(s) on a variety of mammalian and insect cells, and has been shown to be a target for neutralizing antibody (Beebe and Cooper, 1981; Chan et al., 1982; Hardgrave et al., 1993; Lefrancois and Lyles, 1983).

There are several features that make VSV an excellent candidate as a vaccine vector. This weak human pathogen does not undergo genetic recombination or genomic reassortment and has no known transforming properties. Furthermore, VSV does not integrate any of its genomic material into host cell DNA (Barber, 2004; Lawson et al., 1995; McKenna et al., 2003). As a vaccine strategy, VSV is known to elicit strong humoral and cellular immune responses in vivo and naturally infects at mucosal surfaces (Haglund et al., 2002; Martinez et al., 2004; McKenna et al., 2003). This feature offers an alternative less invasive intranasal route of immunization that has been shown to induce both mucosal and systemic immunity (Kahn et al., 2001; Reuter et al., 2002; Roberts et al., 1999). In addition, recombinant VSVs can be generated to accommodate large foreign gene inserts or multiple genes into their genomes (Ezelle et al., 2002; Fernandez et al., 2002; Obuchi et al., 2003). Other RNA viral vectors, such as those based on alphaviruses and poliovirus, do not typically tolerate incorporation of large foreign genes to form replication-competent RNA viruses (Falkner and Holzer, 2004; Schlesinger, 2001). Furthermore, VSV grows to high titers in vitro, thus facilitating rapid purification of large amounts of virus and viral proteins.

However, natural VSV infection has been reported in cattle, horses and swine causing significant disease, including vesicular lesions around the mouth, hoofs, and teats with loss of beef and milk production (Martinez and Wertz, 2005). In the US, livestock is periodically infected with one of two serotypes of VSV (Indiana, VSVI or New Jersey, VSVNJ) VSV infection is asymptomatic in humans but in rare cases, chills, myalgia and nausea have been reported (Coll, 1995; Fields and Hawkins, 1967; Johnson et al., 1966; Wagner, 1996). As a consequence of rare infectivity in humans, seroprevalence of VSV antibodies within the general population is low except in limited regions in Georgia (VSVNJ) or Central America (VSVI and VSVNJ). Antibodies are also detected in individuals who have high risk of exposure such as laboratory workers, veterinarians and ranchers (Johnson et al., 1966). This low VSV seropositivity in the general population and the lack of serious pathogenicity in humans are advantages in the potential use of live recombinant VSV-vectored vaccines in humans.

VSV-HCV Pseudotypes as a Prelude to Recombinant VSV in HCV Immuno-Intervention

Since VSV was known to infect many cell types and can incorporate foreign viral glycoproteins on its surface, it was used first in HCV research as a tool to generate reagents to understand HCV infection in cell culture. The use of VSV in HCV research initially focused in generating pseudotype viruses expressing HCV envelope proteins on their surface (Matsuura et al., 2001; Meyer et al., 2000). These recombinant VSV pseudotypes were used to understand HCV cell entry in the absence of conventional cell culture systems. Several lines of evidence using VSV-HCV pseudotypes pointed to functional role for both E1 and E2 glycoproteins in pseudotype virus infectivity. First, sera derived from chimpanzees immunized with homologous HCV envelope glycoproteins were found to neutralize virus infectivity. Secondly, as with many pH-dependent enveloped viruses, VSV-HCV pseudotype entry was negatively influenced by low pH pre-treatment in a number of susceptible cell lines (Meyer et al., 2000). The same study also demonstrated that Concavalin A (plant lectin) neutralized both E1 and E2 pseudotype virus infectivity. These findings illustrated that carbohydrate structures on HCV envelope glycoproteins may influence binding of HCV to cells directly or by attachment to carbohydrate binding proteins for attachment to or infection of cells. Furthermore, the use of monoclonal antibodies to block putative HCV cell entry receptors, CD81 or LDL, appeared to reduce pseudotype plaque titers demonstrating specificity of VSV-HCV pseudotype binding to cells (Agnello et al., 1999; Cormier et al., 2004; Matsuura et al., 2001; Meyer et al., 2000; Pileri et al., 1998). However, technical limitations in generating VSV-HCV pseudotypes resulted in expression of either HCV E1 or E2 alone, and not as a functional E1/E2 non-covalently linked glycoprotein complex as found in natural HCV infection (Beek et al., 2004). Furthermore, the chimeric nature of the recombinant HCV E1 or E2 fused to the cytoplasmic tail of VSV glycoprotein may influence interpretation of the results (Meyer et al., 2000). However, others also showed that VSV-HCV pseudotypes could be generated that possesed chimeric E1 or E2 glycoproteins either individually or together (Matsuura et al., 2001). In their report, VSV glycoprotein was replaced with the green fluorescent protein (GFP) and infectivity of pseudotypes was determined by GFP-expressing cells. Importantly, their study illustrated that co-expression of both HCV glycoproteins in the VSV-pseudotypes was required for maximal infectivity. Subsequently, we and others adapted an alternative VSV approach that utilized the genetic manipulation of the full length VSV genome to generate novel reagents for vaccine strategies.

Replication Competent and Defective Recombinant VSV as a Vaccine Strategy to Generate Immune Responses

The availability of recombinant DNA technology has allowed for genetic manipulation of the VSV genome and recovery of infectious VSV entirely from cDNA clones (Lawson et al., 1995). Rabies virus was the first Rhabdovirus to be recovered from a complete cDNA clone (Schnell et al., 1994). An important quality of the approach utilized the initiation of the infectious cycle by expressing the antigenomic RNA rather than the genomic RNA in cells expressing the viral N, P, and L proteins. This strategy avoids potential anti-sense problems effecting viral replication in which mRNAs encoding the N, P, and L proteins would hybridize to the negative-strand genomic RNA. This same approach has been successfully applied to full-length positive-strand cDNA for VSV. The high recovery of VSV using this approach combined with the genetic malleability of the VSV cDNA has useful applications to vaccine development. Gene expression of the non-segmented negative-strand RNA viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter (Wagner, 1996). The rearrangement of these genes appears to affect virus phenotype and although live attenuated viruses can be generated, they appear to have reduced capacity to generate clinical disease in the natural hosts such as the domestic swine (Flanagan et al., 2001). Nevertheless, these attenuated viruses were still effective delivery mechanisms for generating VSV-G immune responses and protection against wild type VSV following challenge of vaccinated animals (Flanagan et al., 2000; Flanagan et al., 2001).

In subsequent studies, it was demonstrated that by introducing an extra foreign transcription unit between G and L proteins, rVSVs could be recovered that highly expressed and efficiently incorporated foreign proteins into their virions (McKenna et al., 2003; Schnell et al., 1996). These include CD4 receptor, measles virus glycoprotein (Schnell et al., 1996), and influenza virus hemagglutinin (HA) and neuraminidase (NA) (Kretzschmar et al., 1997). Furthermore, EM analysis of rVSV-HA/NA influenza particles demonstrated that the recombinants were mosaics carrying both VSV-G and influenza glycoproteins. There appears therefore significant space in the VSV membrane that can accommodate foreign membrane proteins.

From the features described above it is clear that vaccines based on live VSV recombinants have advantages over other live recombinant vaccine vectors. First, compared to large complex genomes of the Poxviridae family that encode numerous proteins that include immunoevasive and immunosuppressive proteins, the VSV genome is relatively simple, well understood and easier to manipulate (Lawson et al., 1995). Second, there is a potential for generating live attenuated viruses with reduced pathogenic phenotypes (Flanagan et al., 2000). Third, compared to segmented genomes of viruses in the Orthomyxoviridae family, the single-stranded genome of VSV does not undergo re-assortment and therefore these attenuated viruses cannot genetically recombine with wild-type viruses in vivo.

Recombinant VSV Vaccine Studies

VSV infection can be neuropathic in mice, following high dose intranasal infection, since olfactory receptors are highly tropic for VSV (Bi et al., 1995). Lethal encephalitis can occur especially in young mice but the virus can also be cleared by innate and adaptive immune responses (Balachandran et al., 2000a, Durbin et al., 1996; Meraz et al., 1996; Muller et al., 1994). Several recombinant VSV studies against human pathogens have been tested in murine models. For example, antibodies to influenza glycoprotein HA appear to be neutralizing in natural infection and a recombinant VSV-HA (rVSV-HA) has been shown to successfully generate protective immunity to lethal doses of influenza A/WSN/33 (H1N1) virus in BALB/c strain of mice (Roberts et al., 1998). Furthermore, the rVSV-HA was administered intranasally to elicit immune responses to the expressed influenza HA protein. As described above, the rVSV cloned from cDNA appears to have reduced pathogenicity compared to wild-type VSV Indiana strain. In related studies, an attenuated live rVSV expressing influenza HA with a truncation of the cytoplasmic domain of the VSV-G protein was intranasally administered to mice and found to completely protect against influenza challenge (Roberts et al., 1999). Similarly, in the same study a non-propagating rVSV devoid of VSV-G was also capable of eliciting protective immunity to influenza HA. This vector was also non-pathogenic and additional advantages over its replication counterpart include a lack of neutralizing antibody stimulation against the vector itself and the replication defective strategy improves potential safety attributes as a viral vaccine vector.

In the case of respiratory syncitial virus (RSV), replication competent or attenuated non-propagating VSV expressing RSV G (attachment) and RSV F (fusion) glycoproteins has been reported (Kahn et al., 2001). The VSV-RSV-F virus appeared to elicit RSV-specific antibodies in serum as well as neutralizing antibodies to RSV that were found to be protective against RSV challenge in BALB/c mice.

Finally, the rVSV approach has also been successfully applied to higher animal models. For example an AIDS vaccine based on attenuated VSV vectors expressing SHIV env and gag genes was tested in rhesus monkeys (Rose et al., 2001). This approach provided significant protection as demonstrated in vaccinated animals challenged with pathogenic AIDS virus. Since VSV was shown to be a potent inducer of cellular and humoral immunity in several viral models, we considered VSV as a tool for delivering HCV immunity.

VSV Expressing HCV Core, E1 and E2 Structural Proteins as a Potential Vaccine and Immunotherapeutic Strategy against HCV Infection

In our laboratory we have generated VSV-based HCV vaccine vectors that express all the HCV structural proteins, to maximize an immune response to multiple HCV epitopes (Ezelle et al., 2002; Majid et al., 2006). In the first strategy we developed a recombinant live or replication competent VSV antigen delivery system (Ezelle et al., 2002). To accomplish this, the contigous HCV core, E1 and E2aa 1-746 (HCV genotype 1b) open reading frame (ORF) was cloned into the Indiana (VSVI) cDNA backbone encoded from the plasmid pVSV-XN2 (Fig. 1). In order to create recombinant virus, BHK cells are infected with vaccinia encoding the T7 polymerase (vTF7-3) that drives expression of the N, P and L proteins of VSV encoded in pBL-N, pBL-P, and PBL-L plasmids respectively. Co-transfection of the attenuated VSV cDNA genome vector (pVSV-XN2-C/E1/E2) in this set up allows for efficient recovery of recombinant VSV (rVSV) expressing foreign genes (as described above).

Fig. 1. Construction of rVSV (VSV-HCV-C/E1/E2) expressing HCV Core, E1 and E2.

Fig. 1

Construction of rVSV (VSV-HCV-C/E1/E2) expressing HCV Core, E1 and E2. HCV NIHJ1 (genotype 1b provided by T. Miyamura) Core, E1 and E2 regions (aa 1-746) were cloned into the pVSV-XN2 vector (provided by J. Rose). Co-transfection of the recombinant pVSV-XN2-C/E1/E2 (more...)

The methodology above allows for large scale preparations of replication competent rVSV for cell and animal studies. Importantly, we observed only slight attenuation in the growth properties of replication competent VSV-HCV-C/E1/E2 as compared to wild type virus counterparts as shown in Fig. 2 (Ezelle et al., 2002). Furthermore, VSV-HCV-C/E1/E2 expressed high levels of HCV antigen illustrated in Fig. 3 (Ezelle et al., 2002). In recent studies, analysis using a panel of conformational sensitive mouse and human antibodies illustrated that rVSV expressed authentically folded non-covalently linked E1/E2 heterodimers (Ezelle et al., 2002; Majid et al., 2006).

Fig. 2. Growth curve analysis of rVSV.

Fig. 2

Growth curve analysis of rVSV. VSV-HCV-C/E1/E2 demonstrates a similar growth rate to VSV-GFP. Infections were undertaken at an MOI of 1 for 30 min. Cell medium was analyzed for viral titers at 6, 12, 18, and 24hr post-infection by standard plaque assay (more...)

Fig. 3. Expression of HCV core, E1, and E2.

Fig. 3

Expression of HCV core, E1, and E2. BHK cells were infected with VSV-HCV-C/E1/E2 (VSV-C/E1/E2) or control wild type VSV (VSV-XN2) at an MOI of 1. Cell lysates were analyzed for HCV protein expression by immunoblot analysis 18 hr post-infection. The results (more...)

The binding of these VSV expressed HCV glycoproteins to patient antibodies from native HCV infection is an important potential recognition of these immunogens in HCV vaccine design. Furthermore, intravenous or intraperitoneal immunization of BALB/c mice with VSV-HCV-C/E1/E2 elicited potent secondary serum antibody responses to HCV E2 as detected by ELISA demonstrated in Fig. 4 (Ezelle et al., 2002). Vaccine regimens involved intravenous injections with VSV-HCV-C/E1/E2 or control VSV-GFP, or PBS followed by a secondary inoculation 2 weeks later. The sera were tested for antibody responses on day 21 post initial injection. Our data indicated that VSV-HCV-C/E1/E2 is an efficient vehicle for generating HCV E2 antibodies and warrant further study to assess their capacity in neutralizing HCV infection. In addition, we have observed that VSV-HCV-C/E1/E2-injected mice produced strong HCV core antibodies indicating that immune responses to all the HCV structural proteins were being successfully generated.

Fig. 4. Humoral immunity generated to HCV structural antigens using VSV.

Fig. 4

Humoral immunity generated to HCV structural antigens using VSV. (A) Sera were collected from BALB/c mice vaccinated by intravenous injection (6 mice per group) with VSV-HCV-C/E1/E2 (VSV-C/E1/E2), VSV-GFP or PBS only. Anti-HCV-E2 was detected only in (more...)

In addition to strong humoral responses, the generation of multispecific CTL responses are proposed to be essential for clearance of HCV during acute infections in humans and chimpanzees (Cooper et al., 1999; Erickson et al., 2001; Neumann-Haefelin et al., 2005; Thimme et al., 2001). Therefore, to determine if VSV-HCV-C/E1/E2 was able to generate CD8+ T cell responses, we investigated CTL responses in splenocytes from vaccinated mice using IFNγ-ELISPOT analysis against HCV genotype 1b peptides known to stimulate CTL activity in BALB/c models (Gordon et al., 2000; Nishimura et al., 1999). The release of IFNγ-cytokine from CD8+ T cells is an indication of Th1 type immunity, and our studies illustrated that VSV-HCV-C/E1/E2 could indeed elicit Th1 type CTL responses against HCV core, E1 and E2 peptides 4 weeks after the initial injection as illustrated in Table 1 (Ezelle et al., 2002).

Table 1. CTL activation by VSV-HCV-C/E1/E2 following intravenous injection.

Table 1

CTL activation by VSV-HCV-C/E1/E2 following intravenous injection. IFNγ ELISPOT analysis was determined by ELISPOT. Splenocytes were harvested from intravenously vaccinated BALB/c mice 4 weeks after initial injection and pulsed against core, E1, (more...)

We have also evaluated whether routes of inoculation using rVSV could affect the strength and type of immunity generated against HCV antigens. Intraperitoneal injections were performed using VSV-HCV-C/E1/E2, VSV-GFP or PBS. Serum collected on day 28 was tested again in an ELISA format and demonstrated significant anti-E2 antibody levels in VSV-HCV-C/E1/E2 injected mice shown in Fig. 4B (Ezelle et al., 2002). Interestingly, splenocytes harvested 7 days after injection demonstrated CTL responses to HCV structural peptides as shown by IFN- ELISPOT and presented in Table 2 (Ezelle et al., 2002). Collectively, our data indicate that rVSV expressing HCV antigens can stimulate potent humoral and cellular immunity in animal models. Studies ongoing in our laboratory also suggest that a non-propagating VSV strategy may also be a feasible option for generating immunotheraputic intervention to combat HCV infection. Given the genetic malleability of VSV, the possibility of generating a number of vectors that are safe and yet very effective at generating immune responses to HCV remain an exciting prospect.

Table 2. CTL activation by VSV-HCV-C/E1/E2 following intraperitoneal route of inoculation.

Table 2

CTL activation by VSV-HCV-C/E1/E2 following intraperitoneal route of inoculation. Splenocytes were harvested and analyzed for IFNγ production by ELISPOT 7 days following the injection. The results indicate again that only VSV-HCV-C/E1/E2 (VSV-C/E1/E2) (more...)

Conclusions

The window against the quest for developing HCV vaccines and immunotherapy has certainly shortened as result of a wealth of knowledge distributed with regard to the immune responses that appear to be hallmarks of acute HCV clearance in chimpanzees and humans. In addition, understanding key targets for virus neutralization, particularly the HCV glycoprotein complex will aid the design of immunogens and generate effective immune responses potentially to epitopes broadly conserved amongst viral types. Furthermore, it appears increasingly likely that viral neutralization by antibody alone may not be sufficient in generating effective immune responses against a prophylactic HCV vaccine approach. Nevertheless, antibody responses alone using subunit vaccines appears to reduce chronicity of HCV infection, suggesting a therapeutic role in preventing HCV related liver disease that may account to decreased morbidity and mortality in patients. Indeed, an HCV glycoprotein-based subunit vaccine trial in humans demonstrated a potential immunotherapeutic role for vaccination (Nevens et al., 2003).

The innovation of new technologies that can elicit potent humoral and cellular responses to multi-specific HCV antigens is likely key to a first line of defense against HCV infection. Several promising approaches have been described in this work. However, many studies are in preliminary phases using small animal models and their findings require confirmation in HCV animal models, such as the chimpanzees. The ability to effectively 'tune' individual immune responses against key HCV antigens; core, E1, E2 (and NS3) may provide an opportunity for the immune system to prevent HCV infection before this virus can outpace the immune system as described in chronically infected individuals (Willberg et al., 2003). In this regard, the VSV system described in this report offers significant promise since this recombinant viral approach appears to be a potent stimulator of HCV immunity in the murine model. Furthermore, several features of this system, for example, the malleability of the VSV genome, ability to recovery high titer replication competent or non-propagating recombinant viruses, high level expression of foreign genes, and lack of pathogenicity in humans makes VSV an exciting tool for further endeavors in development of a vaccine against HCV infection.

Future Trends

Although there are still technical and immunological obstacles to overcome, many exciting technologies being tested may improve vaccine studies and also immunotherapy. Essential antigen presenting cells that initiate the immunological cascade, such as dendritic cells (DCs) have been proposed to be impaired during HCV infection although initial reports have been controversial and recent studies suggest normal functions of circulating plamacytoid or myeloid DCs (Bain et al., 2001; Longman et al., 2005; Sarobe et al., 2003). In any case, it may be possible to overcome this defect by autologous transfusion of HCV antigen-loaded mature DC (Gowans et al., 2004). In addition, self replicating cytopathic and non-cytopathic replicon transfection of DCs ex-vivo, illustrates efficient processing of HCV antigens and stimulates efficient priming of T-cell responses following transfusion into murine models (Racanelli et al., 2004). Furthermore, HCV-VLP uptake and presentation has also been demonstrated by human DC (Barth et al., 2005). Key goals therefore include better antigen delivery platforms and priming of efficient immune responses.

The non-propagating VSV approach offers enhanced safety for a recombinant viral vector. This virus can infect many cells, including DC, and can effectively activate unknown innate responses that will inevitably facilitate efficient transduction of adaptive immunity (Balachandran et al., 2000a, Balachandran et al., 2004). Furthermore, modification of these viruses to express cell specific markers for targeting or cytokines for adjuvant purposes is plausible. Finally, discovery of novel innate intracellular molecules that are involved in anti-viral host defense may also be inserted into VSV-based vectors to further facilitate potency of vaccine against virus infection (Balachandran et al., 2004). These exciting advancements and better understanding of HCV immunology suggest an adventurous future in the quest for prophylactic and or immunotherapy against HCV infection.

References

  1. Agnello V, Gyorgy A, Elfahal M, Knight GB, Zhang Q-X. Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci USA. 1999;96:12766–12771. [PMC free article: PMC23090] [PubMed: 10535997]
  2. Arichi T, Saito T, Major ME, Belyakov IM, Shirai M, Engelhard VH, Feinstone S, Berzofsky JA. Prophylactic DNA vaccine for hepatitis C virus infection: HCV-specific cytotoxic T lymphocyte induction and protection from HCV-recombinant vaccinia infection in a HLA-A2.1 transgenic mouse model. Proc Natl Acad Sci USA. 2000;97:297–302. [PMC free article: PMC26657] [PubMed: 10618412]
  3. Bachmann MF, Kundig TM, Kalberer CP, Hengartner H, Zinkernagel RM. Formalin inactivation of vesicular stomatitis virus impairs T-cell- but not T-help-independent B-cell responses. J Virol. 1993;67:3917–3922. [PMC free article: PMC237758] [PubMed: 8389912]
  4. Bain C, Fatmi A, Zoulim F, Zarski JP, Trepo C, Inchauspe G. Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection. Gastroenterology. 2001;120:512–524. [PubMed: 11159892]
  5. Balachandran S, Roberts PC, Brown LE, Truong H, Pattnaik AK, Archer DR, Barber GN. Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection. Immunity. 2000a;13:129–141. [PubMed: 10933401]
  6. Balachandran S, Roberts PC, Kipperman T, Bhalla KN, Compans RW, Archer DR, Barber GN. Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/Caspase-8 death signaling pathway. J Virol. 2000b;74:1513–1523. [PMC free article: PMC111487] [PubMed: 10627563]
  7. Balachandran S, Thomas E, Barber GN. A FADD-dependent innate mechanism in mammalian cells. Nature. 2004;432:401–405. [PubMed: 15549108]
  8. Barber GN. Vesicular Stomatitis virus as an oncolytic vector. Viral Immunol. 2004;17:516–527. [PubMed: 15671748]
  9. Barth H, Ulsenheimer A, Pape GR, Diepolder HM, Hoffmann M, Neumann-Haefelin C, Thimme R, Henneke P, Klein R, Paranhos-Baccala G, Depla E, Liang TJ, Blum HE, Baumert TF. Uptake and presentation of hepatitis C virus-like particles by human dendritic cells. Blood. 2005;105:3605–3614. [PubMed: 15657184]
  10. Bartosch B, Bukh J, Meunier JC, Granier C, Engle RE, Blackwelder WC, Emerson SU, Cosset FL, Purcell RH. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proc Natl Acad Sci USA. 2003a;100:14199–14204. [PMC free article: PMC283569] [PubMed: 14617769]
  11. Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes. J Exp Med. 2003b;197:633–642. [PMC free article: PMC2193821] [PubMed: 12615904]
  12. Baumert TF, Ito S, Wong DT, Liang TJ. Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J Virol. 1998;72:3827–3836. [PMC free article: PMC109606] [PubMed: 9557666]
  13. Baumert TF, Vergalla J, Satoi J, Thomson M, Lechmann M, Herion D, Greenberg HB, Ito S, Liang TJ. Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate. Gastroenterology. 1999;117:1397–1407. [PubMed: 10579981]
  14. Beebe DP, Cooper NR. Neutralization of vesicular stomatitis virus (VSV) by human complement requires a natural IgM antibody present in human serum. J Immunol. 1981;126:1562–1568. [PubMed: 6259260]
  15. Beek ODA, Voisset C, Bartosch B, Ciczora Y, Cocquerel L, Keck Z, Foung S, Cosset FL, Dubuisson J. Characterization of functional hepatitis C virus glycoproteins. J Virol. 2004;78:2994–3002. [PMC free article: PMC353750] [PubMed: 14990718]
  16. Begue P. [Eradication of infectious diseases and vaccination] Bull Acad Natl Med. 2001a;185:777–784. [PubMed: 11503363]
  17. Begue P. [Impact of vaccinations on the epidemiology of infective diseases] Bull Acad Natl Med . 2001b;185:927–939. discussion 939–941. [PubMed: 11717848]
  18. Bi Z, Barna M, Komatsu T, Reiss CS. Vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity. J Virol. 1995;69:6466–6472. [PMC free article: PMC189547] [PubMed: 7545248]
  19. Bottarelli P, Brunetto MR, Minutello MA, Calvo P, Unutmaz D, Weiner AJ, Choo QL, Schuster JR, Kuo G, Bonino F. T-lymphocyte response to hepatitis C virus in different clinical courses of infection. Gastroenterology. 1993;104:580. [PubMed: 8425701]
  20. Brinster C, Chen M, Boucreux D, Baccala-Paranhos G, Liljestrom P, Lemmonier F, Inchauspe G. Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles. J Gen Virol. 2002;83:369–381. [PubMed: 11807230]
  21. Brinster C, Muguet S, Lone YC, Boucreux D, Renard N, Fournillier A, Lemonnier F, Inchauspe G. Different hepatitis C virus nonstructural protein 3 (Ns3)-DNA- expressing vaccines induce in HLA-A2.1 transgenic mice stable cytotoxic T lymphocytes that target one major epitope. Hepatology. 2001;34:1206–1217. [PubMed: 11732011]
  22. Bukh J. A critical role for the chimpanzee model in the study of Hepatitis C. Hepatology. 2004;39:1469–1475. [PubMed: 15185284]
  23. Burgess MA. Two dose MMR vaccine schedule. J Paediatr Child Health. 1994;30:453. [PubMed: 7833088]
  24. Chan JC, East JL, Bowen JM, Massey R, Schochetman G. Monoclonal and polyclonal antibody studies of VSV(hrMMTV) pseudotypes. Virology. 1982;120:54–64. [PubMed: 6179293]
  25. Choo QL, Kuo G, Ralston R, Weiner A, Chien D, Van Nest G, Han J, Berger K, Thudium K, Kuo C, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci USA. 1994;91:1294–8. [PMC free article: PMC43144] [PubMed: 7509068]
  26. Coll J. The glycoprotein G of rhabdoviruses. Arch Virol. 1995;140:827–851. [PubMed: 7605197]
  27. Cooper S, Erickson AL, Adams EJ, Kansopon J, Weiner AJ, Chien DY, Houghton M, Parham P, Walker CM. Analysis of a successful immune response against hepatitis C virus. Immunity. 1999;10:439–449. [PubMed: 10229187]
  28. Cormier EG, Tsamis F, Kajumo F, Durso RJ, Gardner JP, Dragic T. CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci USA. 2004;101:7270–7274. [PMC free article: PMC409908] [PubMed: 15123813]
  29. Coursaget P, Buisson Y, Bourdil C, Yvonnet B, Molinie C, Diop MT, Chiron JP, Bao O, Diop-Mar I. Antibody response to preS1 in hepatitis-B-virus-induced liver disease and after immunization. Res Virol. 1990;141:563–570. [PubMed: 2148981]
  30. Donnelly JJ, Ulmer JB, Schiver JW, Liu MA. DNA vaccines. Annu Rev Immunol. 1997;15:617–648. [PubMed: 9143702]
  31. Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell. 1996;84:443–450. [PubMed: 8608598]
  32. Enders M, Tewald F, Zoller G, Stemmler M, Meyer H. [Smallpox--a review] Dtsch Med Wochenschr. 2002;127:1195–1198. [PubMed: 12035116]
  33. Erickson AL, Kimura Y, Igarashi S, Eichelberger J, Houghton M, Sidney J, McKinney D, Sette A, Hughes AL, Walker CM. The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes. Immunity. 2001;15:883–895. [PubMed: 11754811]
  34. Esumi M, Rikihisa T, Nishimura S, Goto J, Mizuno K, Zhou YH, Shikata T. Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees. Arch Virol. 1999;144:973–980. [PubMed: 10416378]
  35. Ezelle HJ, Markovic D, Barber GN. Generation of hepatitis C virus-like particles by use of a recombinant vesicular stomatitis virus vector. J Virol. 2002;76:12325–12334. [PMC free article: PMC136870] [PubMed: 12414973]
  36. Falkner F, Holzer G. Vaccinia viral/retroviral chimeric vectors. Curr Gene Ther. 2004;4:417–426. [PubMed: 15578991]
  37. Farci P, Bukh J, Purcell RH. The quasispecies of hepatitis C virus and the host immune response. Springer Seminars In Immunopathology. 1997;19:5–26. [PubMed: 9266628]
  38. Farci P, Shimoda A, Wong D, Cabezon T, De Gioannis D, Strazzera A, Shimizu Y, Shapiro M, Alter HJ, Purcell RH. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Natl Acad Sci USA. 1996;93:15394–15399. [PMC free article: PMC26415] [PubMed: 8986822]
  39. Fernandez M, Porosnicu M, Markovic D, Barber GN. Genetically engineered vesicular stomatitis virus in gene therapy: application for treatment of malignant disease. J Virol. 2002;76:895–904. [PMC free article: PMC136833] [PubMed: 11752178]
  40. Fields BN, Hawkins K. Human infection with the virus of vesicular stomatitis during an epizootic. N Engl J Med. 1967;277:989–994. [PubMed: 4293856]
  41. Flanagan EB, Ball LA, Wertz GW. Moving the glycoprotein gene of vesicular stomatitis virus to promoter-proximal positions accelerates and enhances the protective immune response. J Virol. 2000;74:7895–7902. [PMC free article: PMC112320] [PubMed: 10933697]
  42. Flanagan EB, Zamparo JM, Ball LA, Rodriguez LL, Wertz GW. Rearrangement of the genes of vesicular stomatitis virus eliminates clinical disease in the natural host: new strategy for vaccine development. J Virol. 2001;75:6107–6114. [PMC free article: PMC114326] [PubMed: 11390612]
  43. Flint M, Logvinoff C, Rice CM, McKeating JA. Characterization of infectious retroviral pseudotype particles bearing hepatitis C virus glycoproteins. J Virol. 2004;78:6875–6882. [PMC free article: PMC421632] [PubMed: 15194763]
  44. Gershon AA. Viral vaccines of the future. Pediatr Clin North Am. 1990;37:689–707. [PubMed: 2161508]
  45. Goilav C, Piot P. Vaccination against hepatitis B in homosexual men. A review. Am J Med. 1989;87:21S–25S. [PubMed: 2528293]
  46. Gordon EJ, Bhat R, Liu Q, Wang YF, Tackney C, Prince AM. Immune responses to hepatitis C virus structural and nonstructural proteins induced by plasmid DNA immunizations. J Infect Dis. 2000;181:42–50. [PubMed: 10608749]
  47. Gowans EJ, Jones KL, Bharadwaj M, Jackson DC. Prospects for dendritic cell vaccination in persistent infection with hepatitis C virus. J Clin Virol. 2004;30:283–290. [PMC free article: PMC4526278] [PubMed: 15163415]
  48. Gremion C, Cerny A. Hepatitis C virus and the immune system: a concise review. Rev Med Virol. 2005;15:235–268. [PubMed: 15782389]
  49. Haefelin-Neumann C, Blum HE, Chisari FV, Thimme R. T cell response in hepatitis C virus infection. J Clin Virol. 2005;32:75–85. [PubMed: 15653409]
  50. Hagan DTO, Singh M, Dong C, Ugozolli M, Berger K, Glazer E, Selby M, Wininger M, Ng P, Crawford K, Paliard X, Coates S, Houghton M. Cationic microparticles are a potent delivery system for a HCV DNA vaccine. Vaccine. 2004;23:672–680. [PubMed: 15542189]
  51. Haglund K, Leiner I, Kerksiek K, Buonocore L, Pamer E, Rose JK. Robust recall and long-term memory T-cell responses induced by prime-boost regimens with heterologous live viral vectors expressing human immunodeficiency virus type 1 Gag and Env proteins. J Virol. 2002;76:7506–7017. [PMC free article: PMC136360] [PubMed: 12097563]
  52. Hardgrave KL, Neas BR, Scofield RH, Harley JB. Antibodies to vesicular stomatitis virus proteins in patients with systemic lupus erythematosus and in normal subjects. Arthritis Rheum. 1993;36:962–70. [PubMed: 8391264]
  53. Harris HE, Ramsay ME, Andrews N, Eldridge KP. Clinical course of hepatitis C virus during the first decade of infection: cohort study. Br Med J. 2002;324:450–453. [PMC free article: PMC65664] [PubMed: 11859045]
  54. Heile JM, Fong YL, Rosa D, Berger K, Saletti G, Campagnoli S, Bensi G, Capo S, Coates S, Crawford K, Dong C, Wininger M, Baker G, Cousens L, Chien D, Ng P, Archangel P, Grandi G, Houghton M, Abrignani S. Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates. J Virol. 2000;74:6885–6892. [PMC free article: PMC112206] [PubMed: 10888628]
  55. Hill DR. Immunizations for foreign travel. Yale J Biol Med. 1992;65:293–315. [PMC free article: PMC2589588] [PubMed: 1337807]
  56. Hinman AR, Orenstein WA. Immunisation practice in developed countries. Lancet. 1990;335:707–710. [PubMed: 1969069]
  57. Hiroishi K, Matsumura T, Imawari M. [Role of CTL in liver injury of patients with HCV infection] Nippon Rinsho. 2004;62(Suppl 7):159–163. [PubMed: 15359785]
  58. Hong Y, Lorne A, Littel-van den Hurk SVD, Littel-van den Hurk B. Priming with CpG-enriched plasmid and boosting with potein formulated with CpG oligodeoxynucleotides and Quil A induces strong cellular and humoral immune responses to hepatitis C virus NS3. J Gen Virol. 2004;85:1533–1543. [PubMed: 15166437]
  59. Houghton M, Choo QL, Chien D, Kuo G, Weiner A. Development of an HCV vaccination for the induction of immune responses against hepatitis C virus proteins. Vaccine. 1997;15:853–856. [PubMed: 9234532]
  60. Inchauspe G, Major ME, Nakano I, Vitvitski L, Trepo C. DNA vaccination for the induction of immune responses against hepatitis C virus proteins. Vaccine. 1997;15:853–856. [PubMed: 9234532]
  61. Jeong SH, Qiao M, Nascimbeni M, Hu Z, Rehermann B, Murthy K, Liang TJ. Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates. J Virol. 2004;78:6995–7003. [PMC free article: PMC421664] [PubMed: 15194776]
  62. Jiang SD, Pye D, Cox JC. Inactivation of poliovirus with beta-propiolactone. J Biol Stand. 1986;14:103–109. [PubMed: 3020055]
  63. Johnson KM, Vogel JE, Peralta PH. Clinical and serological response to laboratory-acquired human infection by Indiana type vesicular stomatitis virus (VSV). Am J Trop Med Hyg. 1966;15:244–246. [PubMed: 4286381]
  64. Kahn JS, Roberts A, Weibel C, Buonocore L, Rose JK. Replication-competent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (RSV) antigens protect mice against RSV challenge. J Virol. 2001;75:11079–11087. [PMC free article: PMC114687] [PubMed: 11602747]
  65. King DJ. Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum. Avian Dis. 1991;35:505–14. [PubMed: 1835374]
  66. Kretzschmar E, Buonocore L, Schnell MJ, Rose JK. High-efficiency incorporation of functional influenza virus glycoproteins into recombinant vesicular stomatitis viruses. J Virol. 1997;71:5982–5989. [PMC free article: PMC191854] [PubMed: 9223488]
  67. Lawson ND, Stillman EA, Whitt MA, Rose JK. Recombinant vesicular stomatitis viruses from DNA. Proc Natl Acad Sci USA. 1995;92:4477–81. [PMC free article: PMC41967] [PubMed: 7753828]
  68. Lefrancois L, Lyles DS. Antigenic determinants of vesicular stomatitis virus: analysis with antigenic variants. J Immunol. 1983;130:394–398. [PubMed: 6183358]
  69. Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA, Rice CM. Complete replication of hepatitis C virus in cell culture. Science. 2005;309:623–626. [PubMed: 15947137]
  70. Lodmell DL, Ewalt LC. Rabies cell culture vaccines reconstituted and stored at 4 degrees C for 1 year prior to use protect mice against rabies virus. Vaccine. 2004;22:3237–3239. [PubMed: 15308344]
  71. Longman RS, Talal AH, Jacobson IM, Rice CM, Albert ML. Normal functional capacity in circulating myeloid and plasmacytoid dendritic cells in patients with chronic hepatitis C. J Infect Dis. 2005;192:497–503. [PubMed: 15995965]
  72. Magnani G, Bertoletti A, Calzetti C, Campari M, Pizzaferri P, Schianchi C, Vitali P. [Immune response to plasma-derived hepatitis B vaccine in hospital health personnel of Parma] Acta Biomed Ateneo Parmense. 1989;60:73–79. [PubMed: 2535096]
  73. Majid A, Jackson P, Lawal Z, Pearson GM, Parker H, Alexander GJ, Allain JP, Petrik J. Ontogeny of hepatitis C virus (HCV) hypervariable region 1 (HVR1) heterogeneity and HVR1 antibody responses over a 3 year period in a patient infected with HCV type 2b. J Gen Virol. 1999;80:317–25. [PubMed: 10073690]
  74. Majid AM, Ezelle HJ, Shah S, Barber GN. Evaluating replication-defective vesicular stomatitis sirus (VSV) as a vaccine vehicle. 2006 Submitted for publication. [PMC free article: PMC1489030] [PubMed: 16809305]
  75. Marfin AA, Eidex RS, Kozarsky PE, Cetron MS. Yellow fever and Japanese encephalitis vaccines: indications and complications. Infect Dis Clin North Am. 2005;19:151–68. [PubMed: 15701552]
  76. Martinez I, Barrera JC, Rodriguez LL, Wertz GW. Recombinant vesicular stomatitis (Indiana) virus expressing New Jersey and Indiana glycoproteins induces neutralizing antibodies to each serotype in swine, a natural host. Vaccine. 2004;22:4035–4043. [PubMed: 15364454]
  77. Martinez I, Wertz GW. Biological differences between vesicular stomatitis virus Indiana and New Jersey serotype glycoproteins: identification of amino acid residues modulating pH-dependent infectivity. J Virol. 2005;79:3578–3585. [PMC free article: PMC1075735] [PubMed: 15731252]
  78. Matsuura Y, Tani H, Suzuki K, Kimura-Someya T, Suzuki R, Aizaki H, Ishii K, Moriishi K, Robison CS, Whitt MA, Miyamura T. Characterization of pseudotype VSV possessing HCV envelope proteins. Virology. 2001;286:263–275. [PubMed: 11485395]
  79. Matter L. [Vaccinations: the necessary and the desirable] Schweiz Med Wochenschr. 1997;127:377–381. [PubMed: 9132924]
  80. McKenna PM, McGettigan JP, Pomerantz RJ, Dietzschold B, Schnell MJ. Recombinant rhabdoviruses as potential vaccines for HIV-1 and other diseases. Curr HIV Res. 2003;1:229–237. [PubMed: 15043205]
  81. Mehta HS, Cox A, Hoover DR, Wang H-X, Mao Q, Ray S, Strathdee SA, Vlahov D, Thomas DL. Protection against persistence of hepatitis C. The Lancet. 2002;359:1478–1483. [PubMed: 11988247]
  82. Meraz MA, White JM, Sheehan KC, Bach EA, Rodig SJ, Dighe AS, Kaplan DH, Riley JK, Greenlund AC, Campbell D, Carver-Moore K, DuBois RN, Clark R, Aguet M, Schreiber RD. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell. 1996;84:431–442. [PubMed: 8608597]
  83. Meyer K, Basu A, Ray R. Functional features of hepatitis C virus glycoproteins for pseudotype virus entry into mammalian cells. Virology. 2000;276:214–226. [PubMed: 11022009]
  84. Milne A, Krugman S, Waldon JA, Hadler SC, Lucas CR, Moyes CD, Pearce NE. Hepatitis B vaccination in children: five year booster study. N Z Med J. 1992;105:336–338. [PubMed: 1508451]
  85. Miskovsky E, Gershman K, Clements ML, Cupps T, Calandra G, Hesley T, Ioli V, Ellis R, Kniskern P, Miller W, et al. Comparative safety and immunogenicity of yeast recombinant hepatitis B vaccines containing S and pre-S2 + S antigens. Vaccine. 1991;9:346–350. [PubMed: 1872019]
  86. Muller U, Steinhoff U, Reis LF, Hemmi S, Pavlovic J, Zinkernagel RM, Aguet M. Functional role of type I and type II interferons in antiviral defense. Science. 1994;264:1918–1921. [PubMed: 8009221]
  87. Murata K, Lechmann M, Qiao M, Gunji T, Alter HJ, Liang TJ. Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection. Proc Natl Acad Sci USA. 2003;100:6753–6758. [PMC free article: PMC164519] [PubMed: 12748380]
  88. Murata R, Isshiki G, Yoshioka H, Chiba Y, Tada H, Koike M, Kimura M. Prevention of vertical transmission of hepatitis B virus by yeast recombinant hepatitis B vaccine. Acta Paediatr Jpn. 1989;31:180–185. [PubMed: 2516698]
  89. Neumann-Haefelin C, Blum HE, Chisari FV, Thimme R. T cell response in hepatitis C virus infection. J Clin Virol. 2005;32:75–85. [PubMed: 15653409]
  90. Nevens F, Roskams T, Van Vlierberghe H, Horsmans Y, Sprengers D, Elewaut A, Desmet V, Leroux-Roels G, Quinaux E, Depla E, Dincq S, Vander Stichele C, Maertens G, Hulstaert F. A pilot study of therapeutic vaccination with envelope protein E1 in 35 patients with chronic hepatitis C. Hepatology. 2003;38:1289–1296. [PubMed: 14578869]
  91. Nishimura Y, Kamei A, Uno-Furuta S, Tamaki S, Kim G, Adachi Y, Kuribayashi K, Matsuura Y, Miyamura T, Yasutomi Y. A single immunization with a plasmid encoding hepatitis C virus (HCV) structural proteins under the elongation factor 1-alpha promoter elicits HCV-specific cytotoxic T-lymphocytes (CTL). Vaccine. 1999;18:675–680. [PubMed: 10547427]
  92. Obuchi M, Fernandez M, Barber GN. Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity. J Virol. 2003;77:8843–8856. [PMC free article: PMC167243] [PubMed: 12885903]
  93. Ott G, Barchfeld GL, Chernoff D, Radhakrishnan R, van Hoogevest P, Van Nest G. MF59. Design and evaluation of a safe and potent adjuvant for human vaccines. Pharm Biotechnol. 1995;6:277–296. [PubMed: 7551221]
  94. Pancholi P, Liu Q, Tricoche N, Zhang P, Perkus ME, Prince AM. DNA prime-canarypox boost with polycistronic hepatitis C virus (HCV) genes generates potent immune responses to HCV structural and nonstructural proteins. J Infect Dis. 2000;182:18–27. [PubMed: 10882577]
  95. Pancholi P, Perkus M, Tricoche N, Liu Q, Prince A. DNA Immunization with Hepatitis C Virus (HCV) Polycistronic Genes or Immunization by HCV DNA Priming-Recombinant Canarypox Virus Boosting Induces Immune Responses and Protection from Recombinant HCV-vaccinia Virus Infection in HLA-A2.1-Transgenic Mice. J. Virology. 2003;77:382–390. [PMC free article: PMC140575] [PubMed: 12477843]
  96. Parkin J, Cohen B. An overview of the immune system. Lancet. 2001;357:1777–1789. [PubMed: 11403834]
  97. Pascolo S, Bervas N, Ure JM, Smith AG, Lemmonier FA, Perarnau B. HLA-A2.1-restricted education and cytolytic activity of CD8+ T lymphocytes from β2 microglobulin (β2m) HLA-A2.1 monochain transgenic H-2Db β2m double knockout mice. J. Exp. Med. 1997;185:2043–2051. [PMC free article: PMC2196346] [PubMed: 9182675]
  98. Pearce JM. Salk and Sabin: poliomyelitis immunisation. J Neurol Neurosurg Psychiatry. 2004;75:1552. [PMC free article: PMC1738787] [PubMed: 15489385]
  99. Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, Weiner AJ, Houghton M, Rosa D, Grandi G, Abrignani S. Binding of hepatitis C virus to CD81. Science. 1998;282:938–941. [PubMed: 9794763]
  100. Polakos NK, Drane D, Cox J, Ng P, Selby M, Chien D, O'Hagan DT, Houghton M, Paliard X. Characterization of Hepatitis C Virus Core-Specific Immune Responses Primed in Rhesus Macaques by a Nonclassical ISCOM Vaccine. J Immunol. 2001;166:3589–3598. [PubMed: 11207320]
  101. Prince AM, Vnek J, Brotman B. An affordable multideterminant plasma-derived hepatitis B virus vaccine. IARC Sci Publ. 1984:355–372. [PubMed: 6085626]
  102. Provost PJ, Hughes JV, Miller WJ, Giesa PA, Banker FS, Emini EA. An inactivated hepatitis A viral vaccine of cell culture origin. J Med Virol. 1986;19:23–31. [PubMed: 3009703]
  103. Qiao M, Murata K, Davis AR, Jeong SH, Liang TJ. Hepatitis C virus-like particles combined with novel adjuvant systems enhance virus-specific immune responses. Hepatology. 2003;37:52–59. [PubMed: 12500188]
  104. Racanelli V, Behrens SE, Aliberti J, Rehermann B. Dendritic cells transfected with cytopathic self-replicating RNA induce crosspriming of CD8+ T cells and antiviral immunity. Immunity. 2004;20:47–58. [PubMed: 14738764]
  105. Ralston R, Thudium K, Berger K, Kuo C, Gervase B, Hall J, Selby M, Kuo G, Houghton M, Choo QL. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. J Virol. 1993;67:6753–6761. [PMC free article: PMC238116] [PubMed: 8411378]
  106. Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol. 2005;5:215–229. [PubMed: 15738952]
  107. Reuter JD, Vivas-Gonzalez BE, Gomez D, Wilson JH, Brandsma JL, Greenstone HL, Rose JK, Roberts A. Intranasal vaccination with a recombinant vesicular stomatitis virus expressing cottontail rabbit papillomavirus L1 protein provides complete protection against papillomavirus-induced disease. J Virol. 2002;76:8900–8909. [PMC free article: PMC136419] [PubMed: 12163609]
  108. Roberts A, Buonocore L, Price R, Forman J, Rose JK. Attenuated vesicular stomatitis viruses as vaccine vectors. J Virol. 1999;73:3723–3732. [PMC free article: PMC104148] [PubMed: 10196265]
  109. Roberts A, Kretzschmar E, Perkins AS, Forman J, Price R, Buonocore L, Kawaoka Y, Rose JK. Vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge. J Virol. 1998;72:4704–4711. [PMC free article: PMC109996] [PubMed: 9573234]
  110. Roberts A, Reuter JD, Wilson JH, et al. Complete protection from papillomavirus challenge after a single vaccination with a vesicular stomatitis virus vector expressing high levels of L1 protein. J Virol. 2004;78:3196–3199. [PMC free article: PMC353748] [PubMed: 14990742]
  111. Rose NF, Marx PA, Luckay A, Nixon DF, Moretto WJ, Donahoe SM, Montefiori D, Roberts A, Buonocore L, Rose JK. An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants. Cell. 2001;106:539–549. [PubMed: 11551502]
  112. Sarobe P, Lasarte JJ, Zabaleta A, Arribillaga L, Arina A, Melero I, Borras-Cuesta F, Prieto J. Hepatitis C virus structural proteins impair dendritic cell maturation and inhibit in vivo induction of cellular immune responses. J Virol. 2003;77:10862–10871. [PMC free article: PMC224971] [PubMed: 14512536]
  113. Schlesinger S. Alphavirus vectors: development and potential therapeutic applications. Expert Opin Biol Ther. 2001;1:177091. [PubMed: 11727528]
  114. Schnell MJ, Buonocore L, Kretzschmar E, Johnson E, Rose JK. Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles. Proc Natl Acad Sci USA. 1996;93:11359–11365. [PMC free article: PMC38062] [PubMed: 8876140]
  115. Schnell MJ, Mebatsion T, Conzelmann KK. Infectious rabies virus form cloned cDNA. Embo J. 1994;13:4195–4203. [PMC free article: PMC395346] [PubMed: 7925265]
  116. Schwartz B, Gellin B. Vaccination strategies for an influenza pandemic. J Infect Dis. 2005;191:1207–1209. [PubMed: 15776363]
  117. Senterre J. [Varicella vaccination] Rev Med Brux. 2004;25:A223–6. [PubMed: 15516045]
  118. Simon BE, Cornell KA, Clark TR, Chou S, Rosen HR, Barry RA. DNA Vaccination Protects Mice Against Challenge with Listeria monocytogenes Expressing Hepatitis C Virus NS3 protein. Infect. Immun. 2003;71:6372–6380. [PMC free article: PMC219586] [PubMed: 14573658]
  119. Song MK, Lee SW, Suh YS, Lee KJ, Sung Y. Enhancement of immunoglobulin G2a and cytotoxic T-lymphocyte responses by a booster immunization with recombinant hepatitis C virus E2 protein in E2 DNA-primed mice. J Virol. 2000;74:2920–2925. [PMC free article: PMC111786] [PubMed: 10684312]
  120. Strader DB, Seeff LB. New hepatitis A vaccines and their role in prevention. Drugs. 1996;51:359–366. [PubMed: 8882375]
  121. Szmuness W. Large-scale efficacy trials of hepatitis B vaccines in the USA: baseline data and protocols. J Med Virol. 1979;4:327–340. [PubMed: 541683]
  122. Takahashi H, Takeshita T, Morein B, Putney S, Germain R, Berzofsky J. Induction of CD8+ cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMs. Nature. 1990;344:873. [PubMed: 2184369]
  123. Takahashi M. Effectiveness of live varicella vaccine. Expert Opin Biol Ther. 2004;4:199–216. [PubMed: 14998778]
  124. Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med. 2001;194:1395–406. [PMC free article: PMC2193681] [PubMed: 11714747]
  125. Urbani S, Uggeri J, Matsuura Y, Miyamura T, Penna A, Boni C, Ferrari C. Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens. Hepatology. 2001;33:1533–1543. [PubMed: 11391544]
  126. Wagner RR. Rhabdoviridae: The Viruses and Their Replication. In: Fields, Howley, et al., editors. Fields Virology. 3. Philadelphia: Lipincott-Raven Publishers; 1996. pp. 1121–1135.
  127. Wharton M, Cochi SL, Williams WW. Measles, mumps, and rubella vaccines. Infect Dis Clin North Am. 1990;4:47–73. [PubMed: 2407778]
  128. Willberg C, Barnes E, Klenerman P. HCV immunology-death and the maiden T cell. Cell Death Differ. 2003;10 (Suppl 1):S39–47. [PubMed: 12655345]
  129. Xavier F, Paul JP, Xiaoying M, William S, Gerald E, Isa KM, et al. Vaccination of chimpanzees with plasmid DNA encoding the hepatitis C virus (HCV) envelope E2 protein modified the infection after challenge with homologous monoclonal HCV. Hepatology. 2000;32:618–625. [PubMed: 10960458]
  130. Zein NN. Clinical significance of hepatitis C virus genotypes. Clin. Microbiol. Rev. 2000;13:223–235. [PMC free article: PMC100152] [PubMed: 10755999]
  131. Zimmerman RK, Burns IT. Childhood immunization guidelines: current and future. Prim Care. 1994;21:693–715. [PubMed: 7855158]
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