We have recently demonstrated that immunization with hepatitis C virus-like particles (HCV-LPs) generated in insect cells can elicit both humoral and cellular immune responses in BALB͞c mice. Here, we evaluate the immunogenicity of HCV-LPs in HLA2.1 transgenic (AAD) mice in comparison to DNA immunization. HCV-LP immunization elicited a significantly stronger humoral immune response than DNA immunization. HCV-LP-immunized mice also developed stronger HCV-specific cellular immune responses than DNA-immunized mice as determined by using quantitative enzyme-linked immunospot (ELISpot) assay and intracellular cytokine staining. In BALB͞c mice, immunization with HCV-LPs resulted in a >5 log10 reduction in vaccinia titer when challenged with a recombinant vaccinia expressing the HCV structural proteins (vvHCV.S), as compared to 1 log 10 decrease in DNA immunization. In HLA2.1 transgenic mice, a 1-2 log10 reduction resulted from HCV-LP immunization, whereas no reduction was seen from DNA immunization. Adoptive transfer of lymphocytes from HCV-LP-immunized mice to naive mice provided protection against vvHCV.S challenge, and this transferred immunity can be abrogated by either CD4 or CD8 depletion. Our results suggest that HCV-LPs can induce humoral and cellular immune responses that are protective in a surrogate HCV challenge model and that a strong cellular immunity provided by both CD4 and CD8 effector lymphocytes may be important for protection from HCV infection.

Recombinant hepatitis C virus (HCV)-like particles (HCV-LPs) containing HCV structural proteins (core, E1, and E2) produced in insect cells resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice and baboons. Here, we present evidence on the immunogenicity and induction of protective immunity by HCV-LPs in chimpanzees. Chimpanzees (two in each group), were immunized with HCV-LPs or HCV-LPs plus AS01B adjuvant. After immunizations, all animals developed an HCV-specific immune response including IFN-␥ ؉ , IL-2 ؉ , CD4 ؉ , and CD8 ؉ T cell and proliferative lymphocyte responses against core, E1, and E2. Upon challenge with an infectious HCV inoculum, one chimpanzee developed transient viremia with low HCV RNA titers (10 3 to 10 4 copies per ml) in the third and fourth weeks after the challenge. The three other chimpanzees became infected with higher levels of viremia (10 4 to 10 5 copies per ml), but their viral levels became unquantifiable (<10 3 copies per ml) 10 weeks after the challenge. After the HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral and intrahepatic T cell and proliferative responses against the HCV structural proteins. These T cell responses coincided with the fall in HCV RNA levels. Four naïve chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with higher viremia in the range of 10 5 to 10 6 copies per ml. Our study suggests that HCV-LP immunization induces HCV-specific cellular immune responses that can control HCV challenge in the chimpanzee model. envelope proteins ͉ prevention ͉ protective immunity ͉ vaccine ͉ viral clearance H epatitis C virus (HCV) is a major public health problem.Approximately 170 million people are infected by the virus worldwide (1, 2). HCV causes a high rate of chronic infection, which can lead to complications of chronic liver disease such as liver cirrhosis and hepatocellular carcinoma. The efficacy of therapy for chronically infected patients is less than satisfactory. Development of an effective vaccine may hold the key to controlling HCV infection. HCV displays high genetic and antigenic diversities, with at least six different genotypes and diverse quasispecies within infected individuals (1, 2). In addition to this inherent problem, the lack of convenient and robust tissue culture systems and small animal models further hampers the effort to develop an effective HCV vaccine (3). The use of recombinant HCV envelope proteins as a vaccine candidate has been met with variable success. High titers of anti-E1/E2 antibodies could be induced and possibly resulted in lower propensity to chronicity upon HCV challenge (3-5). Recently, a combined modality of plasmid DNA and adenoviral vector to deliver HCV structural and nonstructural antigens induced a strong HCV-specific cell-mediated immunity in chimpanzees and resulted in attenuated HCV infection after challenge (6). However, in this study, no obvious difference was observe...

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We have previously reported the production of hepatitis C virus-like particles (HCV-LP) using a recombinant baculovirus containing the cDNA of the HCV structural proteins (core, E1, and E2). Hepatitis C virus (HCV) is a major public health problem; approximately 3% of the world population, about 170 million people, are infected by the virus (19,22). HCV causes high rate of chronic infection, which can lead to severe complications of chronic liver disease such as liver cirrhosis and hepatocellular carcinoma. The efficacy of therapy for chronically infected patients is less than satisfactory. Development of an effective vaccine may hold the key in the control of HCV infection.HCV not only causes chronic infection in the majority of infected people but also displays high genetic and antigenic diversities with at least six different genotypes and diverse quasispecies within the infected individuals (19,22). In addition to this inherent difficulty, the lack of tissue culture systems and small animal models further hampers the development of a successful vaccine for HCV (15).Virus-like particles are attractive as a recombinant protein vaccine, because they mimic closely the properties of native virions. The synthesis of hepatitis C virus-like particle (HCV-LP) using a recombinant baculovirus containing the cDNA of HCV structural proteins, i.e., core, E1, and E2, has been reported (3). HCV-LP induced virus-specific humoral and cellular immune responses in BALB/c mice (20) and HLA-A 2.1 transgenic (AAD) mice (25, 30). These HCV-LP-induced immune responses protected mice from challenge with a recombinant HCV vaccinia expressing HCV structural proteins (vvHCV.S) in a surrogate HCV vaccinia challenge model (25). Furthermore, adoptive transfer of splenocytes from immunized to naïve mice conferred protection against vvHCV.S challenge and the selective depletion of the CD4 ϩ or CD8 ϩ population abolished the protective immunity (25), suggesting that CD4 ϩ and CD8 ϩ may be important for this immunity. Adjuvants have been used with conventional vaccines to elicit an early, robust, and durable immune response, and they can modulate the immune response toward different T-helper response (Th1 versus Th2) (1,5,8,14,17,23,38,40). Vaccination of HCV-LP combined with adjuvant(s), ASO1B (monophosphoryl lipid A and QS21), and/or CpG 10105 (oligonucleotides containing the immunostimulatory CpG motif) enhanced HCV-specific antibody production and promoted cellular immune responses with a Th1 bias in AAD mice (30).In order to optimize the vaccine effect of HCV-LP for use in humans, we evaluated in this paper the safety and immunogenicity of HCV-LP in a nonhuman primate model, the baboon. In addition, we evaluated the effects of vaccine adjuvant ASO1B and the combination of ASO1B and CpG 10105 on the immunogenicity of HCV-LP in these animals. Although chimpanzees are the only animals susceptible to HCV infection (18) and have a Ͼ98% genomic sequence homology with human, they are an endangered species and difficult to work with because of hi...

Treatment of ducks congenitally infected with the duck hepatitis B virus (DHBV) using the guanosine analogue ganciclovir resulted in prompt and profound inhibition of viral DNA replication in serum and liver. By the end of the treatment period all the replicative intermediates, except the supercoiled DNA form, could not be detected. Within 2 weeks of cessation of treatment viral replication returned and, in some cases, rebound occurred. Sequential treatment with prednisolone followed by ganciclovir also resulted in inhibition of viral replication and, even though relapse was observed after therapy was discontinued, the rebound phenomenon was reduced. Ganciclovir significantly and selectively inhibited DHBV DNA replication but may be more efficacious if used in combination with compounds targeted to the viral supercoiled DNA form.

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The structural details of hepatitis C virus (HCV) have been elusive because of the lack of a robust tissue culture system for producing an adequate amount of virions from infectious sources for in-depth three-dimensional (3D) structural analysis. Using both negative-stain and cryo-electron microscopy (cryoEM), we show that HCV virions isolated from cell culture have a rather uniform size of 500 A in diameter and that recombinantly expressed HCV-like particles (HCV-LPs) have similar morphologic, biophysical and antigenic features in spite of the varying sizes of the particles. 3D reconstructions were obtained from HCV-LPs with the same size as the HCV virions in the presence and absence of monoclonal antibodies bound to the E1 glycoprotein. The 3D reconstruction of HCV-LP reveals a multilayered architecture, with smooth outer-layer densities arranged in a 'fishbone' configuration. Reconstruction of the particles in complex with anti-E1 antibodies shows that sites of the E1 epitope are exposed and surround the 5-, 3- and 2-fold axes. The binding pattern of the anti-E1 antibody and the fitting of the structure of the dengue virus E glycoprotein into our 3D reconstructions further suggest that the HCV-LP E1 and E2 proteins form a tetramer (or dimer of heterodimers) that corresponds morphologically and functionally to the flavivirus E homodimer. This first 3D structural analysis of HCV particles offers important insights into the elusive mechanisms of HCV assembly and maturation.

Abstract. A dengue vaccine effective against all four serotypes is urgently needed. However, safety and immunogenicity could be affected by prior exposure to flaviviruses. This open, controlled, phase IIa study was conducted in 35 healthy adults who had received monovalent, live attenuated Vero cell-derived dengue vaccine against dengue virus 1 (VDV1) or 2 (VDV2) or yellow fever (YF) vaccine 1 year before or who were flavivirus-naïve. All participants received one subcutaneous injection of tetravalent dengue vaccine (TDV) and were followed for 180 days. Previous vaccination did not increase reactogenicity, laboratory abnormalities, or incidence of vaccine viremia, but it did increase the neutralizing antibody response to dengue virus that persisted at day 180. There was no increase in YF antibodies in participants previously immunized with YF vaccine. Prior exposure to YF or monovalent dengue vaccines had no adverse effects on the safety or incidence of viremia associated with this TDV, but it increased immunogenicity.