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Neutralizing Antibodies Induced by Cell Culture–Derived Hepatitis C Virus Protect Against Infection in Mice
Accepted Manuscript Neutralizing Antibodies Induced by Cell Culture-Derived Hepatitis C Virus Protect Against Infection in Mice Daisuke Akazawa, Masaki Moriyama, Hiroshi Yokokawa, Noriaki Omi, Noriyuki Watanabe, Tomoko Date, Kenichi Morikawa, Hideki Aizaki, Koji Ishii, Takanobu Kato, Hidenori Mochizuki, Noriko Nakamura, Takaji Wakita PII: DOI: Reference:
S0016-5085(13)00718-X 10.1053/j.gastro.2013.05.007 YGAST 58434
To appear in: Gastroenterology Accepted Date: 5 May 2013 Please cite this article as: Akazawa D, Moriyama M, Yokokawa H, Omi N, Watanabe N, Date T, Morikawa K, Aizaki H, Ishii K, Kato T, Mochizuki H, Nakamura N, Wakita T, Neutralizing Antibodies Induced by Cell Culture-Derived Hepatitis C Virus Protect Against Infection in Mice, Gastroenterology (2013), doi: 10.1053/j.gastro.2013.05.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
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Title Neutralizing Antibodies Induced by Cell Culture-Derived Hepatitis C Virus Protect
Short title Neutralizing effect of HCV-particle vaccine.
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Authors
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Against Infection in Mice
Daisuke Akazawa1,2, Masaki Moriyama1,2, Hiroshi Yokokawa1,2, Noriaki Omi1, Noriyuki
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Watanabe2, Tomoko Date2, Kenichi Morikawa2,3, Hideki Aizaki2, Koji Ishii2, Takanobu Kato2, Hidenori Mochizuki1, Noriko Nakamura1, Takaji Wakita2*
1
Pharmaceutical Research Laboratories, Toray Industries, Inc., Kanagawa, Japan
2
3
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Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Division of Gastroenterology, Department of Medicine, Showa University School of
Grant support
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Medicine, Tokyo, Japan
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This work was partially supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, from the Ministry of Health, Labour and Welfare of Japan, from the Ministry of Education, Culture, Sports, Science and Technology of Japan, from the National Institute of Biomedical Innovation, and from the 1
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Research on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation.
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Abbreviations
CBB, Coomassie brilliant blue; CTL, cytotoxic T lymphocyte; ELISA, enzyme-linked immunosorbent assay; HCV, hepatitis C virus; HCVcc, cell-cultured HCV; HCVpp, HCV
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pseudoparticle; HRP, horseradish peroxidase; MLV, murine leukemia virus; MOI, multiplicity of infection; MPL, monophosphoryl lipid A; MWCO, molecular weight cut-off;
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PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; RTD-PCR, real-time detection reverse transcription-polymerase chain reaction; SCID, severe combined immuno-deficiency; SDS, sodium dodecyl sulfate; TDM, trehalose-6,6– dimycolate; uPA, albumin enhancer/promoter urokinase plasminogen activator; VLDL,
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very low-density lipoprotein
*Correspondence: Takaji Wakita, M.D., Ph.D.
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Department of Virology II, National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
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Phone: +81-3-5285-1111; Fax: +81-3-5285-1161; E-mail: [email protected]
Disclosures
The authors declare that they have nothing to disclose. 2
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Author Contributions
Study concept and design: DA, NO, NN and TW; Acquisition, analysis and interpretation of data: DA, MM, HY, NO, NW, TD, KM, NN and TW; Drafting of the manuscript: DA
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and TW; Study supervision: HA, KI, TK and HM.
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Abstract Background & Aims: Hepatitis C virus (HCV) infection is a major cause of liver
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cancer, so strategies to prevent infection are needed. A system for cell culture of infectious HCV particles (HCVcc) has recently been established; the inactivated HCVcc particles might be used as antigens in vaccine development. We aimed to
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confirm the potential of HCVcc as an HCV particle vaccine.
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Methods: HCVcc derived from the J6/JFH-1 chimeric genome was purified from cultured cells by ultrafiltration and ultracentrifugation purification steps. Purified HCV particles were inactivated and injected into female BALB/c mice with adjuvant. Sera from immunized mice were collected and their ability to neutralize HCV was examined in naïve Huh7.5.1 cells and urokinase-type plasminogen
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activator-severe combined immunodeficiency mice (uPA+/+-SCID mice) given
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transplants of human hepatocytes (humanized livers).
Results: Antibodies against HCV envelope proteins were detected in the sera of
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immunized mice; these sera inhibited infection of cultured cells with HCV genotypes 1a, 1b, and 2a. Immunoglobulin G purified from the sera of immunized mice (iHCV-IgG) inhibited HCV infection of cultured cells. Injection of iHCV-IgG
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into uPA+/+-SCID mice with humanized livers prevented infection with the minimum infectious dose of HCV.
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Conclusions: Inactivated HCV particles derived from cultured cells protect chimeric liver uPA+/+-SCID mice against HCV infection, and might be used in
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development of a prophylactic vaccine.
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Keywords: HCV particle; HCV vaccine; immunization; virology
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Introduction Hepatitis C virus (HCV), an enveloped virus that belongs to the
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Hepacivirus genus of the Flaviviridae family, is a human pathogen that is a major cause of chronic hepatitis, liver cirrhosis and hepatic carcinoma. HCV therapy mainly involves treatment with pegylated interferon and ribavirin. However, these
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agents are not very effective for patients with high titer HCV-RNA and genotype 1. Thus, it is necessary to develop new, more effective therapies and preventive
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care treatments for HCV infection.
A cell culture-derived HCV (HCVcc) was developed using the genome of the genotype 2a JFH-1 strain. HCVcc was shown to be infectious in vitro and in vivo1-3, and its analysis has made it possible to understand the HCV lifecycle. Thus, HCVcc can be considered as a useful model for the screening of new antiHowever, the fact that only humans and chimpanzees are
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HCV drugs.
susceptible to HCV has greatly slowed its in vivo study.
As a tool for
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circumventing this problem, a human liver chimeric albumin enhancer/promoter urokinase plasminogen activator-severe combined immuno-deficiency (uPAThus, the HCV cell culture system and the
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SCID) mouse was established4.
human liver chimeric mouse are potent tools for pharmaceutical research concerning HCV.
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Prevention of HCV infection is important for control of the pathogenesis of Hepatitis C.
However, there is no commercial vaccine for HCV at present,
although vaccines are now being developed using HCV components, peptides,
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DNA and viral vectors5. Since HCVcc is a structural mimic of the native HCV particle, it has the potential to function as an immunogen for the development of an anti-HCV vaccine. In this study, in order to use HCVcc as an HCV-particle
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particle vaccine against HCV infection in mice.
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vaccine, we purified HCVcc and determined the neutralizing effects of the HCV-
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Materials & Methods Cell culture Huh7 and Huh7.5.1 cells2 (a generous gift from Dr. Francis V. Chisari)
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were cultured in 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium
Plasmids
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(DMEM) containing 10% fetal bovine serum (DMEM-10) as previously reported1.
pJ6/JFH1 was generated by replacing the JFH-1 structural region with that
HCV particle purification J6/JFH-1
chimeric
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of the J6CF strain as previously reported6.
HCVcc
was
purified
by
ultrafiltration
and
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ultracentrifugation. The methods for HCVcc production and purification were as described in the Supplementary Materials and Methods.
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Infectivity titration
Huh7.5.1 cells were employed to determine the viral infectivity titer using
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end-point dilution and immunofluorescence methods as described previously7. The titer was expressed as focus-forming units per mL (FFU/mL).
HCV pseudo-particle (HCVpp) production 8
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Murine leukemia virus (MLV) pseudotypes were generated according to methods described previously8. HCVpp harboring the envelope of the genotype 1a (H779), 1b (TH10) or 2a (J6CF11) strain were generated as described in the
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Supplementary Materials and Methods.
Preparation of recombinant proteins
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Supplementary Materials and Methods.
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HCV-E1 and E2 recombinant proteins were generated as described in the
Immunization of mice with the HCV vaccine
Mice were immunized as described in the Supplementary Materials and
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Methods.
Enzyme immunoassay
Anti-E1 and anti-E2 antibodies were detected by enzyme immunoassay
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for stabilized recombinant J6E1/FLAG and J6E2/FLAG protein, respectively, as
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described in the Supplementary Materials and Methods.
HCV inhibition assay
Inhibition of HCV infection in cultured cells was assayed using HCVpp and
HCVcc for infection as described in the Supplementary Materials and Methods. 9
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IgG purification Mouse IgG was purified using KAPTIV-GY resin (Technogen, Tehran, Iran)
Anti-HCV envelope IgG adsorption
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as described in the Supplementary Materials and Methods.
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Anti-HCV envelope antibodies were absorbed using recombinant FLAGtagged HCV-E1 and -E2 proteins. Briefly, 1 mg/mL iHCV-IgG was pre-cleared
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with anti-FLAG M2 agarose, and then mixed with 50 µg each of J6E1/FLAG and J6E2/FLAG protein. The IgG recombinant protein mixture was incubated at 4°C overnight, and anti-FLAG M2 agarose was then added into the mixture, which was centrifuged at 3,000 × g for 2 min and the supernatant collected as anti-
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envelope IgG-adsorbed IgG.
Inhibition of HCV infection in chimeric mice in which the liver was repopulated
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with human hepatocytes
SCID mice that were transgenic for the urokinase-type plasminogen
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activator gene and in which the liver was repopulated with human hepatocytes (chimeric mice) were purchased from Phoenix Bio Co., Ltd. (Hiroshima, Japan). Only mice showing levels of human albumin over 7 mg/mL were used in the experiments. The mice were injected intraperitoneally with 100 µg of Cont-IgG or 10
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iHCV-IgG at 1 week and at day -1 prior to virus challenge. J6/JFH-1 HCVcc particles (103, 104 and 105 RNA copies/mouse) were mixed with 50 µg/mL of Cont-IgG or iHCV-IgG, and used to challenge the mice. Blood samples were
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collected within 1 week, and HCV RNA was determined using real-time detection reverse transcription-polymerase chain reaction (RTD-PCR).
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Western blot analysis
Purified HCV particle samples were resolved on 12% SDS-polyacrylamide
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gels and transferred to an Immobilon-P membrane (Millipore, Billerica, MA). Western blot was performed using an anti-E1 or an anti-E2 antibody as described in the Supplementary Materials and Methods.
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Statistical analysis
Dunnett’s test was used to compare the statistical significance of differences between groups of data. A p value <0.05 was considered significant.
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Data are reported as means ± standard error of the mean (S.E.M.) as indicated.
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Results
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HCV particle purification HCV particles for mouse immunization were obtained using the cell culture system described in the Methods section. J6/JFH-1 chimeric HCV was secreted into culture medium supplemented with 2% fetal bovine serum, which was
using a 500-kDa cut-off membrane.
The concentrated culture fluid was
These purification procedures removed approximately
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diafiltered using PBS.
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collected and centrifuged; the supernatant was concentrated by ultrafiltration
90% of the contaminating proteins in the solution of virus particles. Sucrose cushion or gradient centrifugation was then performed to further purify the virus particles. Following each procedure, the fractionated viruses were concentrated
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by ultrafiltration and the amount of HCV Core and RNA, the concentration of total protein and viral infectivity were determined (Supplementary Table I). As shown in Figure 1, HCV particles, which had two distinct characteristics, were obtained
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in different fractions following sucrose gradient fractionation.
One fraction
(Fraction (Frac) 6) had low infectivity and a relatively high level of HCV Core and
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RNA, and the other fraction (Frac 8) had high infectivity and a lower level of HCV Core and RNA. Thus, the yield of HCV particles was highest in sucrose gradient Frac 6 (Supplementary Table I), whereas Frac 8 displayed approximately 7-fold higher infectivity than Frac 6 (Table I). Following sucrose cushion purification, 12
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the total protein level of the solution of purified virus particles was reduced to less
Immunization of mice with HCV particles
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than 0.03% of that of the initial cell culture supernatant (Supplementary Table I).
We next immunized BALB/c mice with 2 or 5 pmol of HCV particles that were purified through a sucrose-cushion (termed cushion-2 and cushion-5,
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respectively) or with the HCV particles (2 pmol HCV Core) from the abovedescribed sucrose gradient fractions 6 or 8, and confirmed the immunogenicity of Prior to immunization, the purified HCV particles were
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these particles.
inactivated by UV irradiation and conjugated with monophosphoryl lipid A (MPL) plus trehalose-6,6–dimycolate (TDM).
The HCV particles were then injected
intraperitoneally into female BALB/c mice, and blood samples were collected.
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Immunization was repeated four times at 2-week intervals, and the presence of anti-E1 and E2 antibodies in immunized mouse serum was analyzed using an enzyme immunoassay. Both anti-E1 and E2 antibodies were induced in mice
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immunized with the HCV particles and the efficiency of antibody induction was the same regardless of the nature of the antigen (Figure 2A, B). To confirm that
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the sera from these immunized mice could inhibit HCV infection, an HCV pseudotype particle (HCVpp) inhibition assay was performed. In this assay, sera from HCV-particle-immunized mice inhibited HCVpp infection of naïve Huh7.5.1 cells by HCV strains H77 (1a), TH (1b) and J6CF (2a) (Figure 2C-E). 13
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Furthermore, the inhibitory effects of different amounts (2 or 5 pmol) of HCV Core purified by the sucrose cushion method or of HCV particles from different fractions obtained by sucrose gradient purification were not significantly different.
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Inhibitory effects of the serum from immunized mice were detected against TH/JFH-1 (genotype 1b) and J6/JFH-1 (genotype 2a) HCVcc infection (Supplementary Figure S1). The 50% inhibitory dilution titer of Frac 6-vaccine-
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immunized mouse serum was 1/181 and 1/614, and that of Frac 8-vaccineimmunized mouse serum was 1/432 and 1/766 for TH/JFH-1 and J6/JFH-1
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HCVcc, respectively. These results indicated that the neutralizing effects of the serum from immunized mice were inter-genotypic, and that 2 pmol of the HCV Core was a sufficient dose for HCV-particle immunization.
In contrast,
immunization with saline or BSA and adjuvant did not induce any neutralizing
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effects in mice (Figure 2C-E). We used BSA as a control since albumin was assumed to be the main contaminating protein in the purified HCV particles
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(Supplementary Figure S2).
The HCV-particle vaccine induced anti-HCV antibody more efficiently than an
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HCV-component vaccine
For comparative study of HCV-component and HCV-particle vaccines,
recombinant E1 and E2 proteins were produced and used to immunize mice using the same adjuvant as that used above for the HCV-particle vaccine. These 14
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recombinant
FLAG-tagged
J6E1
and
J6E2
proteins
(J6E1/FLAG
and
J6E2/FLAG) were expressed and secreted into the culture supernatant of COS-1 cells, and were purified by affinity chromatography using M2-agarose. Prior to
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injection into mice, the purified recombinant proteins, as well as the inactivated HCV particles obtained from the sucrose gradient fractionation described above, were analyzed by Western blot using anti-E1 and E2 antibodies, and the amount
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of E1 or E2 protein present in the HCV particles was determined. Densitometric analysis of the immunoblot shown in Figure 3 indicated that an aliquot of HCV
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particles that contained 1 pmol of HCV Core protein included approximately 10 ng each of the E1 and E2 proteins. Thus, HCV particles that contained 2 pmol of HCV Core contained approximately 20 ng of each envelope protein. Recombinant J6E2/FLAG was inoculated into mice at a concentration of
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20, 200 or 2000 ng per mouse or was inoculated together with the same dose of J6E1/FLAG (20 or 200 ng each per mouse). Inactivated HCV particles from Frac 6 or Frac 8 were injected into mice using 2 pmol (20 ng each of E1 or E2) of HCV
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Core protein per mouse. Sera from immunized mice were collected and the presence of anti-E2 antibodies was analyzed using an enzyme immunoassay.
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Anti-E2 antibodies were induced in all immunized mice, and the highest level of anti-E2 antibodies was induced in the group of mice that were immunized with 200 or 2000 ng of the J6E2 protein per mouse. On the other hand, the level of anti-E2 antibodies that was induced by immunization with 20 ng of the inactivated 15
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HCV particles was higher than that induced by the same amount of recombinant envelope proteins (Figure 4A). To analyze the inhibitory effects of the sera from immunized mice against HCV infection, HCVpp was incubated with heat-
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inactivated mouse sera, and was then used to inoculate Huh7.5.1 cells. Sera from HCV-particle-immunized mice were able to inhibit HCVpp infection; however, sera from mice immunized with 20 or 200 ng of the recombinant E2 or E2 plus E1
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proteins did not show significant neutralization activity. Although sera from mice immunized with 2 mg of the recombinant E2 protein could neutralize HCVpp
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infection, the sera from mice immunized with only 20 ng of the HCV particles from sucrose gradient Frac 6 or Frac 8 were also able to induce similar levels of anti-envelope antibody and HCV neutralization (Figure 4B).
These results
suggested that HCV particles have greater potential as vaccine candidates than
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recombinant envelope proteins in terms of their potency for antibody induction.
Vaccination with cell-cultured HCV induces anti-HCV IgG
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As described above, inactivated HCV particles can induce an anti-HCV effect in the sera of immunized mice. To confirm that this anti-HCV effect was
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dependent on HCV-specific antibodies, IgG was purified from the sera of control (Cont-IgG) and vaccine-immunized (iHCV-IgG) mice and was assayed in an HCV inhibition assay. The purity of the purified IgG was confirmed by SDS-PAGE analysis (Figure 5A). Purified IgGs were pre-incubated with J6/JFH-1 HCVcc 16
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and this mixture was subsequently used to inoculate Huh7.5.1 cells. iHCV-IgG inhibited HCV infection in an iHCV-IgG dose-dependent manner, whereas ContIgG did not show any neutralizing effects on this in vitro assay (Figure 5B). The
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50% inhibitory concentration (IC50) of iHCV-IgG against HCVcc infection was 16.1 µg/mL. The iHCV-IgG from Frac 6- and Frac 8-immunized mice showed a neutralizing effect on TH (genotype 1b) and J6CF (genotype 2a) HCVpp
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infections (Supplementary Figure S3); the IC50 values were 18.0 (TH) and 30.4 (J6CF) µg/mL for Frac 6-IgG, and 15.1 (TH) and 33.1 (J6CF) µg/mL for Frac 8Similarly, IgG from Frac 6- and Frac 8-immunized mice showed a
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IgG.
neutralizing effect on HCVcc infection (Supplementary Figure S4). Thus, both fractions of the HCV particles appear to have similar immunogenicity.
As a
control, mice were immunized with naive Huh7-cultured media that was
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processed similar to the method used for the purification of HCV particles or saline together with adjuvant. IgG was purified from the mice sera, and HCVcc inhibition assay was performed. The purified IgG did not exhibited significant
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inhibitory effect for HCV infection compared to saline-immunized mice (Supplementary Figure S5). However, iHCV-IgG significantly inhibited the HCV
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infection at the concentration of 30 µg/mL. The effect of adsorption of the anti-E1 and E2 antibodies in iHCV-IgG by incubation with recombinant J6E1 and J6E2/FLAG proteins on the inhibitory effect of iHCV-IgG was then examined. The HCV neutralizing effect of iHCV-IgG was reduced by adsorption of the anti17
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envelope antibodies, and the IC50 value was increased to 52.9 µg/mL (Figure 5C). These data indicated that anti-HCV envelope neutralizing antibodies were
HCV vaccine induced anti-HCV infection in vivo
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induced in mice by injection of the HCV-particle vaccine.
As shown above, anti-HCV envelope neutralizing antibodies were induced To confirm this effect of the HCV-particle
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in HCV-particle-immunized mice.
vaccine in vivo, we next examined its ability to neutralize an HCV challenge to
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human liver chimeric uPA-SCID mice. Purified IgG (100 µg) from HCV-particleimmunized mice was intraperitoneally injected into these chimeric mice 1 week before HCV infection. The mice were then challenged with multiple doses of 103, 104 and 105 RNA copies of J6/JFH-1 HCVcc at 2-week intervals. Blood samples
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were collected and HCV RNA was measured using RTD-PCR. HCV infection was inhibited (no infection) in all 6 mice in the vaccine-treated mouse group (iHCV-IgG) when 103 copies of HCV were inoculated (minimal infectious dose),
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whereas infection was observed in 4 out of the 6 mice in the control mouse group (Cont-IgG). However, the neutralizing effect of iHCV-IgG vaccination was not
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detected when 104 or 105 copies of HCV were used for infection (Table II). These data indicated that an inactivated HCV-particle vaccine could potentially be used as a prophylactic vaccine for HCV.
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Discussion There is no vaccine available for clinical use against HCV infection.
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Development of an HCV cell culture system has facilitated an understanding of the HCV lifecycle, and the production of HCV prophylactic and therapeutic drugs. HCVcc mimics the native HCV particle, and thus has potential for use as an
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HCV-particle vaccine. In the present study, we established an efficient method for HCV particle production and purification, which allowed evaluation of the HCV
HCV of genotype 2a J6/JFH-1.
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particle as a vaccine candidate. HCV particles were purified from cell-cultured The HCVcc in the culture supernatant was
concentrated and partially purified by ultrafiltration using a 500-kDa cut-off membrane.
Subsequently,
HCVcc
was
purified
by
sucrose
gradient
ultracentrifugation. As reported previously12,13, the virus particles present in Frac
characteristics.
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6 and 8 following this sucrose gradient centrifugation displayed different The virus in Frac 6 displayed low infectivity, but had a high
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concentration of viral components, whereas that in Frac 8 displayed high infectivity, but a low concentration of viral components. It is thought that the
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content of lipids or lipoproteins in these HCV particle fractions is different12,13. Finally, each viral fraction was purified and concentrated by ultrafiltration. When these HCV particles were inactivated, conjugated with an MPL plus TDM adjuvant, and injected into mice, anti-E1 and E2 antibodies were induced in the 19
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immunized mice, and the sera from these mice had a neutralizing effect against HCV infection in vitro.
Since these sera displayed cross-reactivity for HCV
strains 1a (H77), 1b (TH) and 2a (J6CF), it was considered that the HCV-particle
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vaccine had a broad neutralizing effect against HCV infection.
Western blot analysis of the envelope proteins in these HCV particles (Figure 3) indicated that the Frac 6 HCV particles contained approximately 10 ng
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each of the E1 and E2 proteins per 1 pmol HCV Core protein.
Since the
molecular weight of the Core, E1 and E2 proteins is approximately 20, 35 and 70
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kDa, respectively14, the molar ratio of the structural proteins in the purified HCV particles (Core:E1:E2) was calculated to be 5:1:1 from Western blot analysis. Western blotting analysis also suggested that the amount of the E1 and E2 proteins in the Frac 8 HCV particle was likely to be higher than that in the Frac 6
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HCV particle. Indeed, densitometric analysis of the blot indicated that the level of both E1 and E2 proteins in Frac 8 was 1.5-fold higher than that in Frac 6. This higher level of E1 and E2 proteins in Frac 8 may be the reason why the Frac 8
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HCV particles had higher infectivity. Interestingly, the expression level of the Core protein relative to that of the envelope proteins was different in infected
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cells and in purified HCV particles (Figure 3). These data suggest that only a limited amount of the envelope protein was incorporated into HCV particles. We compared the potential of the purified HCV particles to function as a
vaccine with that of HCV-envelope components. For this purpose, recombinant 20
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HCV E1 and E2 proteins were prepared and different doses of recombinant E2 (rE2), or recombinant E1 (rE1) plus rE2, were injected into mice with adjuvant. Other groups of mice were immunized with the HCV-particle vaccine (Frac 6 or
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Frac 8) using a dose of 2 pmol of HCV Core per mouse. Since it was calculated that HCV particles that included 2 pmol of HCV Core protein contained approximately 20 ng of recombinant E1 and E2, we used 20 to 2,000 ng of rE2 or
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20 or 200 ng each of rE1 plus rE2 proteins for comparative purposes. Although the sera from all immunized mice contained anti-E2 antibodies, and immunization
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with 200 or 2,000 ng of recombinant E2 protein induced antibody production with the highest efficiency, HCV-particle immunization displayed the highest virusneutralizing effect. Based on these results, it was concluded that purified HCV particles may be a more potent prophylactic HCV vaccine candidate than a
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vaccine that is composed of recombinant viral components. There are several possible explanations for the observed differences between these two types of vaccines. First, HCV envelope proteins that are a part of HCV particles are
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properly folded, which would maintain their function, whereas the recombinant envelope proteins may not be properly folded, and may thus lose their
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antigenicity. Second, other factors associated with the HCV particles may be important for inducing neutralizing antibodies. To address these possibilities, we adsorbed the anti-envelope antibodies in iHCV-IgG with recombinant envelope proteins. This procedure reduced, but did not completely abolish, the neutralizing 21
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effect of iHCV-IgG towards HCVcc infection.
It is possible that, if the
recombinant envelope proteins were not folded properly as mentioned above, then not all of the anti-envelope antibodies were removed in this adsorption
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process. However, this possibility is considered unlikely since virus-neutralizing antibodies were induced by both immunization with the HCV particles and with the recombinant envelope proteins. This result suggests that HCV particles may
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include other factors, such as lipoproteins, that are important for HCV infection1517
. It is also necessary to compare the immunogenic activity of the inactivated
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virus vaccine with the potential immunogenic activities of recombinant HCV-like particles and E1-E2 heterodimers. Furthermore, attention needs to be paid as to whether the inactivated purified vaccine may induce autoantibodies against human proteins associated with the viral particles.
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To confirm the in vivo efficacy of HCV particle-induced neutralizing antibodies, the ability of iHCV-IgG to protect infected animals against HCV challenge was examined using human liver chimeric uPA-SCID mice.
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Interestingly, iHCV-IgG could protect against HCV challenge, at least at the lower virus challenge dose, although the mice challenged with the higher virus doses
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became infected with HCV. This result indicated that the HCV-particle vaccine could exert a prophylactic anti-HCV effect in vivo by induction of virusneutralizing antibodies. However, the therapeutic effect of this vaccine is not yet clear and an understanding of its effect will require detailed study of its effect on 22
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cellular immune responses such as the cytotoxic T lymphocyte (CTL) response. In addition, the prophylactic effect was only partially observed in the in vivo experiment.
The reason for this insufficient protection is unclear; however,
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induction with a higher titer of neutralizing antibody may be needed using adjuvants or more effective forms of the vaccine such as recombinant HCV-like particles.
It may also be important to induce protective cellular immune
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responses in addition to humoral immune responses; effective CTL responses may be necessary for a prophylactic effect against infection at higher virus doses.
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Prophylactic and therapeutic HCV vaccines have been previously developed and some of these vaccines have been tested in clinical trials5,18,19. An HCV vaccine should be able to induce humoral and cellular immune response with specific anti-HCV effects.
Previous vaccine candidates that have been
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tested have included recombinant proteins20-24, peptides25-29, DNA30 and viral vectors31,32 with or without appropriate adjuvants.
Recombinant E1 and E2
proteins with an MF59 adjuvant were developed by Chiron/Novartis as a
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prophylactic vaccine, and the immunogenicity of this vaccine was confirmed in mice and guinea pigs33. The recombinant protein vaccine was well tolerated in a
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Phase I clinical study, and induction of anti-envelope antibodies and an envelope-specific T-cell response was observed24.
In another study, Fab
fragments of anti-E2 antibodies that were obtained using a phage display library generated from an HCV-infected donor showed a broad HCV-neutralizing effect 23
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in human liver chimeric uPA-SCID mice34.
A separate study revealed that
retroviral pseudotype-derived HCVLPs also induced broad neutralizing antibodies in mice and macaques35. This result indicated that a prophylactic vaccine may
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be achievable by activation of the humoral immune response. Moreover, it was reported that insect cell-derived virus-like particles containing HCV structural proteins induced a humoral immune response36 and stimulated dendritic cells37
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or a CTL response38. Thus, it is possible that an HCVcc-derived HCV-particle vaccine also has the capacity to function as a therapeutic vaccine.
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The HCV vaccine described in this study could be used for protection against HCV infection in high-risk patient groups. Given that the HCV particles are used as the vaccine, it will be necessary to improve the production and purification of these particles before they can be used. We recently established a
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serum-free HCV culture system for efficient production and purification of virus particles that may be useful for this purpose39.
It will also be necessary to
examine other cell types for the production of HCV particles, because the Huh7
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cells used in this study are cells of a hepatoma cell line. Once HCV particle production using an industrial method is established and its safety is evaluated
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and confirmed, then it is expected that a purified HCV-particle vaccine will be of prophylactic use. Alternatively, a novel vaccine composed of HCV components should be developed using recombinant E1 and E2 proteins that can induce viral neutralizing antibodies with similar or even higher efficiency as HCV particles. 24
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Acknowledgements The Huh7.5.1 cell line was a kind gift from Dr. Francis V. Chisari. AP33 was a generous gift from Genentech, Inc. HCV cDNA of the H77 and J6CF
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strains were kindly provided by Dr. Robert Purcell and Dr. Jens Bukh, respectively. The authors thank Dr. Junichi Tanabe for his much appreciated
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contribution to this work.
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Figure Legends
Figure 1 HCV-particle purification from cell cultures. Huh7 cells were inoculated
were passaged into CellSTACK (Corning).
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with J6/JFH-1 chimeric HCVcc (multiplicity of infection (MOI) 0.2) and infected cells The cell supernatant was collected and
concentrated by ultrafiltration using a Hollow Fiber UFP-500-C-3MA (GE Healthcare).
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Concentrated virus was then diafiltered using PBS, and the viral fluid was layered on top of a preformed continuous 10% – 60% sucrose gradient in 10 mM Tris - 150 mM
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NaCl - 0.1 mM EDTA (TNE buffer). The gradients were centrifuged using an SW28 rotor (Beckman Coulter) at 28,000 rpm for 4 h at 4°C, and 1-mL fractions were collected from the bottom of the tube. The level of the HCV Core protein, HCV RNA, infectivity towards naïve Huh7.5.1 cells, total protein and buoyant density were determined for
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each fraction. Infectivity is indicated as ‘specific infectivity’, which was calculated as the infectivity titer (FFU: focus-forming unit) divided by the HCV Core protein (fmol).
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Figure 2 Cross-genotypic neutralizing effect of the sera from HCV-particleimmunized mice. HCV-particles that were purified using a sucrose cushion (2 or 5
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pmol HCV Core) or sucrose gradient fractionation (Frac 6 and Frac 8, 2 pmol HCV Core each) were inactivated by UV irradiation.
The inactivated HCV-particles were
conjugated with SIGMA Adjuvant System, and were intraperitoneally injected into BALB/c mice (5 weeks old, female, n = 3). Saline or BSA (150 µg) with adjuvant was 26
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injected into mice as a control.
Immunization was repeated four times at 2-week
intervals (at weeks 0, 2, 4 and 6). Blood was collected at weeks 1, 3, 5 and 7 after immunization. Pre-immune sera and immunized mouse sera were prepared from these
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blood samples and were heat inactivated at 56°C for 1 h. The heat-inactivated sera were diluted (1,000-fold) and their reactivity with J6E1/FLAG (A) and J6E2/FLAG (B) recombinant proteins was analyzed using an enzyme immunoassay.
Assays were
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performed in triplicate. The y-axis indicates absorbance at 450 nm. Mean values ± S.E.M. are shown. Mouse serum-mediated inhibition of HCV infection was assayed
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using HCVpp genotypes 1a (H77, panel C), 1b (TH, panel D) and 2a (J6CF, panel E). The sera were diluted (100-fold) and HCVpp infection in naïve Huh7.5.1 cells was examined. Assays were performed in triplicate and infection rates are expressed as
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mean ± S.E.M.
Figure 3 Western blot analysis of envelope proteins in purified HCV particles. The HCV Core (0.5 pmol) of purified HCV-particles (Frac 6 and Frac 8), as well as 1 to 30 ng
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of recombinant envelope proteins (J6E1/FLAG or J6E2/FLAG) were resolved on 12% SDS-polyacrylamide gels and transferred to a PVDF membrane. Naïve Huh7 cells and
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J6/JFH-1-infected Huh7 cells were lysed using Passive Lysis Buffer (Promega), and were similarly assayed as negative (n) and positive (i) controls respectively.
The
membrane was blocked and probed with primary antibodies against the virus Core (2H9; 3 µg/mL), E1 (B7567; 10 µg/mL) and E2 (AP33; 3 µg/mL) proteins.
The 27
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membrane was then probed with HRP-conjugated secondary antibody, and bound antibody was detected using ECL-plus.
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Figure 4 Neutralizing effects of sera from HCV-component and HCV-particle vaccine-immunized mice. BALB/c mice (n = 5) were immunized with recombinant proteins (20, 200 or 2,000 ng of FLAG-tagged E2, 20 or 200 ng each of FLAG-tagged
Frac 8) together with adjuvant.
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E1 and FLAG-tagged E2) or with purified HCV-particles (2 pmol HCV Core of Frac 6 or The presence of anti-E2 antibodies in sera from
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immunized mice was analyzed using an EIA (A) and the ability of these sera to inhibit J6CF HCVpp infection of cultured Huh7.5.1 cells was also assayed (B). Each assay was performed in triplicate, and the data are presented as box plots. The statistical significance of differences between groups was analyzed using Dunnett’s test (*p < 0.05,
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**p < 0.005, ***p < 0.001 vs. saline).
Figure 5 Immunoglobulin G purified from the sera of HCV-particle-immunized
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mice had a neutralizing effect against HCV infection in vitro. (A) IgG from the sera of mice at 7 weeks after immunization (iHCV-IgG) was purified using a KAPTIV-GY
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resin. Control mouse IgG was similarly prepared from saline-injected mice (Cont-IgG). Each IgG was analyzed for purity by SDS-PAGE and CBB staining. As a control, a purchased mouse IgG was used. (B) Serially diluted Cont-IgG or iHCV-IgG was mixed with J6/JFH-1 HCVcc (MOI 0.1) and this mixture was inoculated into naïve Huh7.5.1 28
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cells. HCV infection was determined by analysis of the intracellular concentration of the HCV Core protein. Infectivity is expressed as the percentage of cells that were infected. The IC50 value of iHCV-IgG was 16.1 µg/mL. (C) Anti-E1 and E2 antibodies in iHCV-
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IgG were adsorbed by recombinant J6E1/FLAG and J6E2/FLAG protein, and the neutralizing effect of the E1E2-adsorbed IgG on HCV infection of Huh7 cells was assessed using the HCVcc inhibition assay as described for (B). The IC50 of the E1E2-
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adsorbed IgG shifted to 52.9 µg/mL.
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Table I Composition of purified HCV particles following ultracentrifugation.
Infectivity/core
Infectivity/RNA
Core/protein
Frac 6
6.54× × 10-7
129.8
8.50× × 10-5
113.8
Frac 8
5.38× ×
987.9
5.32× ×
19.3
Ratio (Frac8/Frac6)
10-7
0.82
7.61 FFU/fmol
6.26
0.17
FFU/copy
fmol/µ µg
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fmol/copy
10-4
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Core/RNA
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Table II Neutralizing effect of immunoglobulin G purified from the sera of HCV-particle-immunized mice against HCV infection in human liver chimeric uPA-SCID mice.
Infected mice Cont-IgG
103
4/6
104
5/6
105
6/6
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Virus challenge (HCV RNA copies)
iHCV-IgG 0/6
6/6
6/6
Figure1 Click here to download high resolution image
Figure2 Click here to download high resolution image
Figure3 Click here to download high resolution image
Figure4 Click here to download high resolution image
Figure5 Click here to download high resolution image
S0016-5085(13)00718-X 10.1053/j.gastro.2013.05.007 YGAST 58434
To appear in: Gastroenterology Accepted Date: 5 May 2013 Please cite this article as: Akazawa D, Moriyama M, Yokokawa H, Omi N, Watanabe N, Date T, Morikawa K, Aizaki H, Ishii K, Kato T, Mochizuki H, Nakamura N, Wakita T, Neutralizing Antibodies Induced by Cell Culture-Derived Hepatitis C Virus Protect Against Infection in Mice, Gastroenterology (2013), doi: 10.1053/j.gastro.2013.05.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
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Title Neutralizing Antibodies Induced by Cell Culture-Derived Hepatitis C Virus Protect
Short title Neutralizing effect of HCV-particle vaccine.
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Authors
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Against Infection in Mice
Daisuke Akazawa1,2, Masaki Moriyama1,2, Hiroshi Yokokawa1,2, Noriaki Omi1, Noriyuki
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Watanabe2, Tomoko Date2, Kenichi Morikawa2,3, Hideki Aizaki2, Koji Ishii2, Takanobu Kato2, Hidenori Mochizuki1, Noriko Nakamura1, Takaji Wakita2*
1
Pharmaceutical Research Laboratories, Toray Industries, Inc., Kanagawa, Japan
2
3
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Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Division of Gastroenterology, Department of Medicine, Showa University School of
Grant support
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Medicine, Tokyo, Japan
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This work was partially supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, from the Ministry of Health, Labour and Welfare of Japan, from the Ministry of Education, Culture, Sports, Science and Technology of Japan, from the National Institute of Biomedical Innovation, and from the 1
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Research on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation.
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Abbreviations
CBB, Coomassie brilliant blue; CTL, cytotoxic T lymphocyte; ELISA, enzyme-linked immunosorbent assay; HCV, hepatitis C virus; HCVcc, cell-cultured HCV; HCVpp, HCV
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pseudoparticle; HRP, horseradish peroxidase; MLV, murine leukemia virus; MOI, multiplicity of infection; MPL, monophosphoryl lipid A; MWCO, molecular weight cut-off;
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PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; RTD-PCR, real-time detection reverse transcription-polymerase chain reaction; SCID, severe combined immuno-deficiency; SDS, sodium dodecyl sulfate; TDM, trehalose-6,6– dimycolate; uPA, albumin enhancer/promoter urokinase plasminogen activator; VLDL,
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very low-density lipoprotein
*Correspondence: Takaji Wakita, M.D., Ph.D.
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Department of Virology II, National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
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Phone: +81-3-5285-1111; Fax: +81-3-5285-1161; E-mail: [email protected]
Disclosures
The authors declare that they have nothing to disclose. 2
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Author Contributions
Study concept and design: DA, NO, NN and TW; Acquisition, analysis and interpretation of data: DA, MM, HY, NO, NW, TD, KM, NN and TW; Drafting of the manuscript: DA
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and TW; Study supervision: HA, KI, TK and HM.
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Abstract Background & Aims: Hepatitis C virus (HCV) infection is a major cause of liver
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cancer, so strategies to prevent infection are needed. A system for cell culture of infectious HCV particles (HCVcc) has recently been established; the inactivated HCVcc particles might be used as antigens in vaccine development. We aimed to
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confirm the potential of HCVcc as an HCV particle vaccine.
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Methods: HCVcc derived from the J6/JFH-1 chimeric genome was purified from cultured cells by ultrafiltration and ultracentrifugation purification steps. Purified HCV particles were inactivated and injected into female BALB/c mice with adjuvant. Sera from immunized mice were collected and their ability to neutralize HCV was examined in naïve Huh7.5.1 cells and urokinase-type plasminogen
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activator-severe combined immunodeficiency mice (uPA+/+-SCID mice) given
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transplants of human hepatocytes (humanized livers).
Results: Antibodies against HCV envelope proteins were detected in the sera of
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immunized mice; these sera inhibited infection of cultured cells with HCV genotypes 1a, 1b, and 2a. Immunoglobulin G purified from the sera of immunized mice (iHCV-IgG) inhibited HCV infection of cultured cells. Injection of iHCV-IgG
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into uPA+/+-SCID mice with humanized livers prevented infection with the minimum infectious dose of HCV.
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Conclusions: Inactivated HCV particles derived from cultured cells protect chimeric liver uPA+/+-SCID mice against HCV infection, and might be used in
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development of a prophylactic vaccine.
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Keywords: HCV particle; HCV vaccine; immunization; virology
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Introduction Hepatitis C virus (HCV), an enveloped virus that belongs to the
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Hepacivirus genus of the Flaviviridae family, is a human pathogen that is a major cause of chronic hepatitis, liver cirrhosis and hepatic carcinoma. HCV therapy mainly involves treatment with pegylated interferon and ribavirin. However, these
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agents are not very effective for patients with high titer HCV-RNA and genotype 1. Thus, it is necessary to develop new, more effective therapies and preventive
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care treatments for HCV infection.
A cell culture-derived HCV (HCVcc) was developed using the genome of the genotype 2a JFH-1 strain. HCVcc was shown to be infectious in vitro and in vivo1-3, and its analysis has made it possible to understand the HCV lifecycle. Thus, HCVcc can be considered as a useful model for the screening of new antiHowever, the fact that only humans and chimpanzees are
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HCV drugs.
susceptible to HCV has greatly slowed its in vivo study.
As a tool for
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circumventing this problem, a human liver chimeric albumin enhancer/promoter urokinase plasminogen activator-severe combined immuno-deficiency (uPAThus, the HCV cell culture system and the
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SCID) mouse was established4.
human liver chimeric mouse are potent tools for pharmaceutical research concerning HCV.
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Prevention of HCV infection is important for control of the pathogenesis of Hepatitis C.
However, there is no commercial vaccine for HCV at present,
although vaccines are now being developed using HCV components, peptides,
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DNA and viral vectors5. Since HCVcc is a structural mimic of the native HCV particle, it has the potential to function as an immunogen for the development of an anti-HCV vaccine. In this study, in order to use HCVcc as an HCV-particle
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particle vaccine against HCV infection in mice.
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vaccine, we purified HCVcc and determined the neutralizing effects of the HCV-
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Materials & Methods Cell culture Huh7 and Huh7.5.1 cells2 (a generous gift from Dr. Francis V. Chisari)
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were cultured in 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium
Plasmids
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(DMEM) containing 10% fetal bovine serum (DMEM-10) as previously reported1.
pJ6/JFH1 was generated by replacing the JFH-1 structural region with that
HCV particle purification J6/JFH-1
chimeric
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of the J6CF strain as previously reported6.
HCVcc
was
purified
by
ultrafiltration
and
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ultracentrifugation. The methods for HCVcc production and purification were as described in the Supplementary Materials and Methods.
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Infectivity titration
Huh7.5.1 cells were employed to determine the viral infectivity titer using
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end-point dilution and immunofluorescence methods as described previously7. The titer was expressed as focus-forming units per mL (FFU/mL).
HCV pseudo-particle (HCVpp) production 8
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Murine leukemia virus (MLV) pseudotypes were generated according to methods described previously8. HCVpp harboring the envelope of the genotype 1a (H779), 1b (TH10) or 2a (J6CF11) strain were generated as described in the
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Supplementary Materials and Methods.
Preparation of recombinant proteins
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Supplementary Materials and Methods.
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HCV-E1 and E2 recombinant proteins were generated as described in the
Immunization of mice with the HCV vaccine
Mice were immunized as described in the Supplementary Materials and
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Methods.
Enzyme immunoassay
Anti-E1 and anti-E2 antibodies were detected by enzyme immunoassay
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for stabilized recombinant J6E1/FLAG and J6E2/FLAG protein, respectively, as
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described in the Supplementary Materials and Methods.
HCV inhibition assay
Inhibition of HCV infection in cultured cells was assayed using HCVpp and
HCVcc for infection as described in the Supplementary Materials and Methods. 9
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IgG purification Mouse IgG was purified using KAPTIV-GY resin (Technogen, Tehran, Iran)
Anti-HCV envelope IgG adsorption
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as described in the Supplementary Materials and Methods.
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Anti-HCV envelope antibodies were absorbed using recombinant FLAGtagged HCV-E1 and -E2 proteins. Briefly, 1 mg/mL iHCV-IgG was pre-cleared
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with anti-FLAG M2 agarose, and then mixed with 50 µg each of J6E1/FLAG and J6E2/FLAG protein. The IgG recombinant protein mixture was incubated at 4°C overnight, and anti-FLAG M2 agarose was then added into the mixture, which was centrifuged at 3,000 × g for 2 min and the supernatant collected as anti-
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envelope IgG-adsorbed IgG.
Inhibition of HCV infection in chimeric mice in which the liver was repopulated
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with human hepatocytes
SCID mice that were transgenic for the urokinase-type plasminogen
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activator gene and in which the liver was repopulated with human hepatocytes (chimeric mice) were purchased from Phoenix Bio Co., Ltd. (Hiroshima, Japan). Only mice showing levels of human albumin over 7 mg/mL were used in the experiments. The mice were injected intraperitoneally with 100 µg of Cont-IgG or 10
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iHCV-IgG at 1 week and at day -1 prior to virus challenge. J6/JFH-1 HCVcc particles (103, 104 and 105 RNA copies/mouse) were mixed with 50 µg/mL of Cont-IgG or iHCV-IgG, and used to challenge the mice. Blood samples were
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collected within 1 week, and HCV RNA was determined using real-time detection reverse transcription-polymerase chain reaction (RTD-PCR).
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Western blot analysis
Purified HCV particle samples were resolved on 12% SDS-polyacrylamide
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gels and transferred to an Immobilon-P membrane (Millipore, Billerica, MA). Western blot was performed using an anti-E1 or an anti-E2 antibody as described in the Supplementary Materials and Methods.
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Statistical analysis
Dunnett’s test was used to compare the statistical significance of differences between groups of data. A p value <0.05 was considered significant.
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Data are reported as means ± standard error of the mean (S.E.M.) as indicated.
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Results
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HCV particle purification HCV particles for mouse immunization were obtained using the cell culture system described in the Methods section. J6/JFH-1 chimeric HCV was secreted into culture medium supplemented with 2% fetal bovine serum, which was
using a 500-kDa cut-off membrane.
The concentrated culture fluid was
These purification procedures removed approximately
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diafiltered using PBS.
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collected and centrifuged; the supernatant was concentrated by ultrafiltration
90% of the contaminating proteins in the solution of virus particles. Sucrose cushion or gradient centrifugation was then performed to further purify the virus particles. Following each procedure, the fractionated viruses were concentrated
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by ultrafiltration and the amount of HCV Core and RNA, the concentration of total protein and viral infectivity were determined (Supplementary Table I). As shown in Figure 1, HCV particles, which had two distinct characteristics, were obtained
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in different fractions following sucrose gradient fractionation.
One fraction
(Fraction (Frac) 6) had low infectivity and a relatively high level of HCV Core and
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RNA, and the other fraction (Frac 8) had high infectivity and a lower level of HCV Core and RNA. Thus, the yield of HCV particles was highest in sucrose gradient Frac 6 (Supplementary Table I), whereas Frac 8 displayed approximately 7-fold higher infectivity than Frac 6 (Table I). Following sucrose cushion purification, 12
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the total protein level of the solution of purified virus particles was reduced to less
Immunization of mice with HCV particles
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than 0.03% of that of the initial cell culture supernatant (Supplementary Table I).
We next immunized BALB/c mice with 2 or 5 pmol of HCV particles that were purified through a sucrose-cushion (termed cushion-2 and cushion-5,
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respectively) or with the HCV particles (2 pmol HCV Core) from the abovedescribed sucrose gradient fractions 6 or 8, and confirmed the immunogenicity of Prior to immunization, the purified HCV particles were
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these particles.
inactivated by UV irradiation and conjugated with monophosphoryl lipid A (MPL) plus trehalose-6,6–dimycolate (TDM).
The HCV particles were then injected
intraperitoneally into female BALB/c mice, and blood samples were collected.
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Immunization was repeated four times at 2-week intervals, and the presence of anti-E1 and E2 antibodies in immunized mouse serum was analyzed using an enzyme immunoassay. Both anti-E1 and E2 antibodies were induced in mice
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immunized with the HCV particles and the efficiency of antibody induction was the same regardless of the nature of the antigen (Figure 2A, B). To confirm that
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the sera from these immunized mice could inhibit HCV infection, an HCV pseudotype particle (HCVpp) inhibition assay was performed. In this assay, sera from HCV-particle-immunized mice inhibited HCVpp infection of naïve Huh7.5.1 cells by HCV strains H77 (1a), TH (1b) and J6CF (2a) (Figure 2C-E). 13
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Furthermore, the inhibitory effects of different amounts (2 or 5 pmol) of HCV Core purified by the sucrose cushion method or of HCV particles from different fractions obtained by sucrose gradient purification were not significantly different.
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Inhibitory effects of the serum from immunized mice were detected against TH/JFH-1 (genotype 1b) and J6/JFH-1 (genotype 2a) HCVcc infection (Supplementary Figure S1). The 50% inhibitory dilution titer of Frac 6-vaccine-
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immunized mouse serum was 1/181 and 1/614, and that of Frac 8-vaccineimmunized mouse serum was 1/432 and 1/766 for TH/JFH-1 and J6/JFH-1
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HCVcc, respectively. These results indicated that the neutralizing effects of the serum from immunized mice were inter-genotypic, and that 2 pmol of the HCV Core was a sufficient dose for HCV-particle immunization.
In contrast,
immunization with saline or BSA and adjuvant did not induce any neutralizing
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effects in mice (Figure 2C-E). We used BSA as a control since albumin was assumed to be the main contaminating protein in the purified HCV particles
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(Supplementary Figure S2).
The HCV-particle vaccine induced anti-HCV antibody more efficiently than an
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HCV-component vaccine
For comparative study of HCV-component and HCV-particle vaccines,
recombinant E1 and E2 proteins were produced and used to immunize mice using the same adjuvant as that used above for the HCV-particle vaccine. These 14
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recombinant
FLAG-tagged
J6E1
and
J6E2
proteins
(J6E1/FLAG
and
J6E2/FLAG) were expressed and secreted into the culture supernatant of COS-1 cells, and were purified by affinity chromatography using M2-agarose. Prior to
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injection into mice, the purified recombinant proteins, as well as the inactivated HCV particles obtained from the sucrose gradient fractionation described above, were analyzed by Western blot using anti-E1 and E2 antibodies, and the amount
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of E1 or E2 protein present in the HCV particles was determined. Densitometric analysis of the immunoblot shown in Figure 3 indicated that an aliquot of HCV
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particles that contained 1 pmol of HCV Core protein included approximately 10 ng each of the E1 and E2 proteins. Thus, HCV particles that contained 2 pmol of HCV Core contained approximately 20 ng of each envelope protein. Recombinant J6E2/FLAG was inoculated into mice at a concentration of
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20, 200 or 2000 ng per mouse or was inoculated together with the same dose of J6E1/FLAG (20 or 200 ng each per mouse). Inactivated HCV particles from Frac 6 or Frac 8 were injected into mice using 2 pmol (20 ng each of E1 or E2) of HCV
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Core protein per mouse. Sera from immunized mice were collected and the presence of anti-E2 antibodies was analyzed using an enzyme immunoassay.
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Anti-E2 antibodies were induced in all immunized mice, and the highest level of anti-E2 antibodies was induced in the group of mice that were immunized with 200 or 2000 ng of the J6E2 protein per mouse. On the other hand, the level of anti-E2 antibodies that was induced by immunization with 20 ng of the inactivated 15
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HCV particles was higher than that induced by the same amount of recombinant envelope proteins (Figure 4A). To analyze the inhibitory effects of the sera from immunized mice against HCV infection, HCVpp was incubated with heat-
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inactivated mouse sera, and was then used to inoculate Huh7.5.1 cells. Sera from HCV-particle-immunized mice were able to inhibit HCVpp infection; however, sera from mice immunized with 20 or 200 ng of the recombinant E2 or E2 plus E1
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proteins did not show significant neutralization activity. Although sera from mice immunized with 2 mg of the recombinant E2 protein could neutralize HCVpp
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infection, the sera from mice immunized with only 20 ng of the HCV particles from sucrose gradient Frac 6 or Frac 8 were also able to induce similar levels of anti-envelope antibody and HCV neutralization (Figure 4B).
These results
suggested that HCV particles have greater potential as vaccine candidates than
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recombinant envelope proteins in terms of their potency for antibody induction.
Vaccination with cell-cultured HCV induces anti-HCV IgG
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As described above, inactivated HCV particles can induce an anti-HCV effect in the sera of immunized mice. To confirm that this anti-HCV effect was
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dependent on HCV-specific antibodies, IgG was purified from the sera of control (Cont-IgG) and vaccine-immunized (iHCV-IgG) mice and was assayed in an HCV inhibition assay. The purity of the purified IgG was confirmed by SDS-PAGE analysis (Figure 5A). Purified IgGs were pre-incubated with J6/JFH-1 HCVcc 16
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and this mixture was subsequently used to inoculate Huh7.5.1 cells. iHCV-IgG inhibited HCV infection in an iHCV-IgG dose-dependent manner, whereas ContIgG did not show any neutralizing effects on this in vitro assay (Figure 5B). The
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50% inhibitory concentration (IC50) of iHCV-IgG against HCVcc infection was 16.1 µg/mL. The iHCV-IgG from Frac 6- and Frac 8-immunized mice showed a neutralizing effect on TH (genotype 1b) and J6CF (genotype 2a) HCVpp
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infections (Supplementary Figure S3); the IC50 values were 18.0 (TH) and 30.4 (J6CF) µg/mL for Frac 6-IgG, and 15.1 (TH) and 33.1 (J6CF) µg/mL for Frac 8Similarly, IgG from Frac 6- and Frac 8-immunized mice showed a
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IgG.
neutralizing effect on HCVcc infection (Supplementary Figure S4). Thus, both fractions of the HCV particles appear to have similar immunogenicity.
As a
control, mice were immunized with naive Huh7-cultured media that was
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processed similar to the method used for the purification of HCV particles or saline together with adjuvant. IgG was purified from the mice sera, and HCVcc inhibition assay was performed. The purified IgG did not exhibited significant
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inhibitory effect for HCV infection compared to saline-immunized mice (Supplementary Figure S5). However, iHCV-IgG significantly inhibited the HCV
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infection at the concentration of 30 µg/mL. The effect of adsorption of the anti-E1 and E2 antibodies in iHCV-IgG by incubation with recombinant J6E1 and J6E2/FLAG proteins on the inhibitory effect of iHCV-IgG was then examined. The HCV neutralizing effect of iHCV-IgG was reduced by adsorption of the anti17
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envelope antibodies, and the IC50 value was increased to 52.9 µg/mL (Figure 5C). These data indicated that anti-HCV envelope neutralizing antibodies were
HCV vaccine induced anti-HCV infection in vivo
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induced in mice by injection of the HCV-particle vaccine.
As shown above, anti-HCV envelope neutralizing antibodies were induced To confirm this effect of the HCV-particle
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in HCV-particle-immunized mice.
vaccine in vivo, we next examined its ability to neutralize an HCV challenge to
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human liver chimeric uPA-SCID mice. Purified IgG (100 µg) from HCV-particleimmunized mice was intraperitoneally injected into these chimeric mice 1 week before HCV infection. The mice were then challenged with multiple doses of 103, 104 and 105 RNA copies of J6/JFH-1 HCVcc at 2-week intervals. Blood samples
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were collected and HCV RNA was measured using RTD-PCR. HCV infection was inhibited (no infection) in all 6 mice in the vaccine-treated mouse group (iHCV-IgG) when 103 copies of HCV were inoculated (minimal infectious dose),
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whereas infection was observed in 4 out of the 6 mice in the control mouse group (Cont-IgG). However, the neutralizing effect of iHCV-IgG vaccination was not
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detected when 104 or 105 copies of HCV were used for infection (Table II). These data indicated that an inactivated HCV-particle vaccine could potentially be used as a prophylactic vaccine for HCV.
18
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Discussion There is no vaccine available for clinical use against HCV infection.
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Development of an HCV cell culture system has facilitated an understanding of the HCV lifecycle, and the production of HCV prophylactic and therapeutic drugs. HCVcc mimics the native HCV particle, and thus has potential for use as an
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HCV-particle vaccine. In the present study, we established an efficient method for HCV particle production and purification, which allowed evaluation of the HCV
HCV of genotype 2a J6/JFH-1.
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particle as a vaccine candidate. HCV particles were purified from cell-cultured The HCVcc in the culture supernatant was
concentrated and partially purified by ultrafiltration using a 500-kDa cut-off membrane.
Subsequently,
HCVcc
was
purified
by
sucrose
gradient
ultracentrifugation. As reported previously12,13, the virus particles present in Frac
characteristics.
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6 and 8 following this sucrose gradient centrifugation displayed different The virus in Frac 6 displayed low infectivity, but had a high
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concentration of viral components, whereas that in Frac 8 displayed high infectivity, but a low concentration of viral components. It is thought that the
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content of lipids or lipoproteins in these HCV particle fractions is different12,13. Finally, each viral fraction was purified and concentrated by ultrafiltration. When these HCV particles were inactivated, conjugated with an MPL plus TDM adjuvant, and injected into mice, anti-E1 and E2 antibodies were induced in the 19
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immunized mice, and the sera from these mice had a neutralizing effect against HCV infection in vitro.
Since these sera displayed cross-reactivity for HCV
strains 1a (H77), 1b (TH) and 2a (J6CF), it was considered that the HCV-particle
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vaccine had a broad neutralizing effect against HCV infection.
Western blot analysis of the envelope proteins in these HCV particles (Figure 3) indicated that the Frac 6 HCV particles contained approximately 10 ng
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each of the E1 and E2 proteins per 1 pmol HCV Core protein.
Since the
molecular weight of the Core, E1 and E2 proteins is approximately 20, 35 and 70
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kDa, respectively14, the molar ratio of the structural proteins in the purified HCV particles (Core:E1:E2) was calculated to be 5:1:1 from Western blot analysis. Western blotting analysis also suggested that the amount of the E1 and E2 proteins in the Frac 8 HCV particle was likely to be higher than that in the Frac 6
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HCV particle. Indeed, densitometric analysis of the blot indicated that the level of both E1 and E2 proteins in Frac 8 was 1.5-fold higher than that in Frac 6. This higher level of E1 and E2 proteins in Frac 8 may be the reason why the Frac 8
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HCV particles had higher infectivity. Interestingly, the expression level of the Core protein relative to that of the envelope proteins was different in infected
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cells and in purified HCV particles (Figure 3). These data suggest that only a limited amount of the envelope protein was incorporated into HCV particles. We compared the potential of the purified HCV particles to function as a
vaccine with that of HCV-envelope components. For this purpose, recombinant 20
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HCV E1 and E2 proteins were prepared and different doses of recombinant E2 (rE2), or recombinant E1 (rE1) plus rE2, were injected into mice with adjuvant. Other groups of mice were immunized with the HCV-particle vaccine (Frac 6 or
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Frac 8) using a dose of 2 pmol of HCV Core per mouse. Since it was calculated that HCV particles that included 2 pmol of HCV Core protein contained approximately 20 ng of recombinant E1 and E2, we used 20 to 2,000 ng of rE2 or
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20 or 200 ng each of rE1 plus rE2 proteins for comparative purposes. Although the sera from all immunized mice contained anti-E2 antibodies, and immunization
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with 200 or 2,000 ng of recombinant E2 protein induced antibody production with the highest efficiency, HCV-particle immunization displayed the highest virusneutralizing effect. Based on these results, it was concluded that purified HCV particles may be a more potent prophylactic HCV vaccine candidate than a
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vaccine that is composed of recombinant viral components. There are several possible explanations for the observed differences between these two types of vaccines. First, HCV envelope proteins that are a part of HCV particles are
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properly folded, which would maintain their function, whereas the recombinant envelope proteins may not be properly folded, and may thus lose their
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antigenicity. Second, other factors associated with the HCV particles may be important for inducing neutralizing antibodies. To address these possibilities, we adsorbed the anti-envelope antibodies in iHCV-IgG with recombinant envelope proteins. This procedure reduced, but did not completely abolish, the neutralizing 21
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effect of iHCV-IgG towards HCVcc infection.
It is possible that, if the
recombinant envelope proteins were not folded properly as mentioned above, then not all of the anti-envelope antibodies were removed in this adsorption
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process. However, this possibility is considered unlikely since virus-neutralizing antibodies were induced by both immunization with the HCV particles and with the recombinant envelope proteins. This result suggests that HCV particles may
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include other factors, such as lipoproteins, that are important for HCV infection1517
. It is also necessary to compare the immunogenic activity of the inactivated
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virus vaccine with the potential immunogenic activities of recombinant HCV-like particles and E1-E2 heterodimers. Furthermore, attention needs to be paid as to whether the inactivated purified vaccine may induce autoantibodies against human proteins associated with the viral particles.
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To confirm the in vivo efficacy of HCV particle-induced neutralizing antibodies, the ability of iHCV-IgG to protect infected animals against HCV challenge was examined using human liver chimeric uPA-SCID mice.
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Interestingly, iHCV-IgG could protect against HCV challenge, at least at the lower virus challenge dose, although the mice challenged with the higher virus doses
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became infected with HCV. This result indicated that the HCV-particle vaccine could exert a prophylactic anti-HCV effect in vivo by induction of virusneutralizing antibodies. However, the therapeutic effect of this vaccine is not yet clear and an understanding of its effect will require detailed study of its effect on 22
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cellular immune responses such as the cytotoxic T lymphocyte (CTL) response. In addition, the prophylactic effect was only partially observed in the in vivo experiment.
The reason for this insufficient protection is unclear; however,
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induction with a higher titer of neutralizing antibody may be needed using adjuvants or more effective forms of the vaccine such as recombinant HCV-like particles.
It may also be important to induce protective cellular immune
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responses in addition to humoral immune responses; effective CTL responses may be necessary for a prophylactic effect against infection at higher virus doses.
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Prophylactic and therapeutic HCV vaccines have been previously developed and some of these vaccines have been tested in clinical trials5,18,19. An HCV vaccine should be able to induce humoral and cellular immune response with specific anti-HCV effects.
Previous vaccine candidates that have been
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tested have included recombinant proteins20-24, peptides25-29, DNA30 and viral vectors31,32 with or without appropriate adjuvants.
Recombinant E1 and E2
proteins with an MF59 adjuvant were developed by Chiron/Novartis as a
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prophylactic vaccine, and the immunogenicity of this vaccine was confirmed in mice and guinea pigs33. The recombinant protein vaccine was well tolerated in a
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Phase I clinical study, and induction of anti-envelope antibodies and an envelope-specific T-cell response was observed24.
In another study, Fab
fragments of anti-E2 antibodies that were obtained using a phage display library generated from an HCV-infected donor showed a broad HCV-neutralizing effect 23
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in human liver chimeric uPA-SCID mice34.
A separate study revealed that
retroviral pseudotype-derived HCVLPs also induced broad neutralizing antibodies in mice and macaques35. This result indicated that a prophylactic vaccine may
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be achievable by activation of the humoral immune response. Moreover, it was reported that insect cell-derived virus-like particles containing HCV structural proteins induced a humoral immune response36 and stimulated dendritic cells37
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or a CTL response38. Thus, it is possible that an HCVcc-derived HCV-particle vaccine also has the capacity to function as a therapeutic vaccine.
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The HCV vaccine described in this study could be used for protection against HCV infection in high-risk patient groups. Given that the HCV particles are used as the vaccine, it will be necessary to improve the production and purification of these particles before they can be used. We recently established a
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serum-free HCV culture system for efficient production and purification of virus particles that may be useful for this purpose39.
It will also be necessary to
examine other cell types for the production of HCV particles, because the Huh7
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cells used in this study are cells of a hepatoma cell line. Once HCV particle production using an industrial method is established and its safety is evaluated
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and confirmed, then it is expected that a purified HCV-particle vaccine will be of prophylactic use. Alternatively, a novel vaccine composed of HCV components should be developed using recombinant E1 and E2 proteins that can induce viral neutralizing antibodies with similar or even higher efficiency as HCV particles. 24
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Acknowledgements The Huh7.5.1 cell line was a kind gift from Dr. Francis V. Chisari. AP33 was a generous gift from Genentech, Inc. HCV cDNA of the H77 and J6CF
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strains were kindly provided by Dr. Robert Purcell and Dr. Jens Bukh, respectively. The authors thank Dr. Junichi Tanabe for his much appreciated
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contribution to this work.
25
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Figure Legends
Figure 1 HCV-particle purification from cell cultures. Huh7 cells were inoculated
were passaged into CellSTACK (Corning).
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with J6/JFH-1 chimeric HCVcc (multiplicity of infection (MOI) 0.2) and infected cells The cell supernatant was collected and
concentrated by ultrafiltration using a Hollow Fiber UFP-500-C-3MA (GE Healthcare).
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Concentrated virus was then diafiltered using PBS, and the viral fluid was layered on top of a preformed continuous 10% – 60% sucrose gradient in 10 mM Tris - 150 mM
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NaCl - 0.1 mM EDTA (TNE buffer). The gradients were centrifuged using an SW28 rotor (Beckman Coulter) at 28,000 rpm for 4 h at 4°C, and 1-mL fractions were collected from the bottom of the tube. The level of the HCV Core protein, HCV RNA, infectivity towards naïve Huh7.5.1 cells, total protein and buoyant density were determined for
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each fraction. Infectivity is indicated as ‘specific infectivity’, which was calculated as the infectivity titer (FFU: focus-forming unit) divided by the HCV Core protein (fmol).
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Figure 2 Cross-genotypic neutralizing effect of the sera from HCV-particleimmunized mice. HCV-particles that were purified using a sucrose cushion (2 or 5
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pmol HCV Core) or sucrose gradient fractionation (Frac 6 and Frac 8, 2 pmol HCV Core each) were inactivated by UV irradiation.
The inactivated HCV-particles were
conjugated with SIGMA Adjuvant System, and were intraperitoneally injected into BALB/c mice (5 weeks old, female, n = 3). Saline or BSA (150 µg) with adjuvant was 26
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injected into mice as a control.
Immunization was repeated four times at 2-week
intervals (at weeks 0, 2, 4 and 6). Blood was collected at weeks 1, 3, 5 and 7 after immunization. Pre-immune sera and immunized mouse sera were prepared from these
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blood samples and were heat inactivated at 56°C for 1 h. The heat-inactivated sera were diluted (1,000-fold) and their reactivity with J6E1/FLAG (A) and J6E2/FLAG (B) recombinant proteins was analyzed using an enzyme immunoassay.
Assays were
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performed in triplicate. The y-axis indicates absorbance at 450 nm. Mean values ± S.E.M. are shown. Mouse serum-mediated inhibition of HCV infection was assayed
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using HCVpp genotypes 1a (H77, panel C), 1b (TH, panel D) and 2a (J6CF, panel E). The sera were diluted (100-fold) and HCVpp infection in naïve Huh7.5.1 cells was examined. Assays were performed in triplicate and infection rates are expressed as
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mean ± S.E.M.
Figure 3 Western blot analysis of envelope proteins in purified HCV particles. The HCV Core (0.5 pmol) of purified HCV-particles (Frac 6 and Frac 8), as well as 1 to 30 ng
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of recombinant envelope proteins (J6E1/FLAG or J6E2/FLAG) were resolved on 12% SDS-polyacrylamide gels and transferred to a PVDF membrane. Naïve Huh7 cells and
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J6/JFH-1-infected Huh7 cells were lysed using Passive Lysis Buffer (Promega), and were similarly assayed as negative (n) and positive (i) controls respectively.
The
membrane was blocked and probed with primary antibodies against the virus Core (2H9; 3 µg/mL), E1 (B7567; 10 µg/mL) and E2 (AP33; 3 µg/mL) proteins.
The 27
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membrane was then probed with HRP-conjugated secondary antibody, and bound antibody was detected using ECL-plus.
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Figure 4 Neutralizing effects of sera from HCV-component and HCV-particle vaccine-immunized mice. BALB/c mice (n = 5) were immunized with recombinant proteins (20, 200 or 2,000 ng of FLAG-tagged E2, 20 or 200 ng each of FLAG-tagged
Frac 8) together with adjuvant.
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E1 and FLAG-tagged E2) or with purified HCV-particles (2 pmol HCV Core of Frac 6 or The presence of anti-E2 antibodies in sera from
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immunized mice was analyzed using an EIA (A) and the ability of these sera to inhibit J6CF HCVpp infection of cultured Huh7.5.1 cells was also assayed (B). Each assay was performed in triplicate, and the data are presented as box plots. The statistical significance of differences between groups was analyzed using Dunnett’s test (*p < 0.05,
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**p < 0.005, ***p < 0.001 vs. saline).
Figure 5 Immunoglobulin G purified from the sera of HCV-particle-immunized
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mice had a neutralizing effect against HCV infection in vitro. (A) IgG from the sera of mice at 7 weeks after immunization (iHCV-IgG) was purified using a KAPTIV-GY
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resin. Control mouse IgG was similarly prepared from saline-injected mice (Cont-IgG). Each IgG was analyzed for purity by SDS-PAGE and CBB staining. As a control, a purchased mouse IgG was used. (B) Serially diluted Cont-IgG or iHCV-IgG was mixed with J6/JFH-1 HCVcc (MOI 0.1) and this mixture was inoculated into naïve Huh7.5.1 28
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cells. HCV infection was determined by analysis of the intracellular concentration of the HCV Core protein. Infectivity is expressed as the percentage of cells that were infected. The IC50 value of iHCV-IgG was 16.1 µg/mL. (C) Anti-E1 and E2 antibodies in iHCV-
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IgG were adsorbed by recombinant J6E1/FLAG and J6E2/FLAG protein, and the neutralizing effect of the E1E2-adsorbed IgG on HCV infection of Huh7 cells was assessed using the HCVcc inhibition assay as described for (B). The IC50 of the E1E2-
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adsorbed IgG shifted to 52.9 µg/mL.
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glycoprotein immunization of rodents elicits cross-reactive neutralizing antibodies.
Law M, Maruyama T, Lewis J, et al. Broadly neutralizing antibodies protect
against hepatitis C virus quasispecies challenge. Nat Med 2008;14:25-27. [35]
Garrone P, Fluckiger AC, Mangeot PE, et al. A prime-boost strategy using
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virus-like particles pseudotyped for HCV proteins triggers broadly neutralizing antibodies in macaques. Sci Transl Med 2011;3:94ra71. [36]
Steinmann D, Barth H, Gissler B, et al. Inhibition of hepatitis C virus-like
EP
particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C. J Virol 2004;78:9030-9040. Barth H, Ulsenheimer A, Pape GR, et al. Uptake and presentation of
AC C
[37]
hepatitis C virus-like particles by human dendritic cells. Blood 2005;105:36053614.
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[38]
Elmowalid GA, Qiao M, Jeong SH, et al. Immunization with hepatitis C
virus-like particles results in control of hepatitis C virus infection in chimpanzees. Proc Natl Acad Sci U S A 2007;104:8427-8432. Akazawa D, Morikawa K, Omi N, et al. Production and characterization of
RI PT
[39]
AC C
EP
TE D
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SC
HCV particles from serum-free culture. Vaccine 2011;29:4821-4828.
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Table I Composition of purified HCV particles following ultracentrifugation.
Infectivity/core
Infectivity/RNA
Core/protein
Frac 6
6.54× × 10-7
129.8
8.50× × 10-5
113.8
Frac 8
5.38× ×
987.9
5.32× ×
19.3
Ratio (Frac8/Frac6)
10-7
0.82
7.61 FFU/fmol
6.26
0.17
FFU/copy
fmol/µ µg
SC
fmol/copy
10-4
RI PT
Core/RNA
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Table II Neutralizing effect of immunoglobulin G purified from the sera of HCV-particle-immunized mice against HCV infection in human liver chimeric uPA-SCID mice.
Infected mice Cont-IgG
103
4/6
104
5/6
105
6/6
AC C
EP
TE D
Virus challenge (HCV RNA copies)
iHCV-IgG 0/6
6/6
6/6
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