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Previous Article | Next Article 
Journal of Virology, July 2006, p. 6993-7008, Vol. 80, No. 14
0022-538X/06/$08.00+0 doi:10.1128/JVI.00365-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Microbiology and Immunology, University of Miami School of Medicine and Sylvester Comprehensive Cancer Center, Miami, Florida
Received 21 February 2006/ Accepted 2 May 2006
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ABSTRACT |
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G-C/E1/E2) vesicular stomatitis virus (VSV) contiguously expressing the structural proteins of hepatitis C virus (HCV; core [C] and glycoproteins E1 and E2) and report on their immunogenicity in murine models. VSV-C/E1/E2 and VSVG-C/E1/E2 expressed high levels of HCV C, E1, and E2, which were authentically posttranslationally processed. Both VSV-expressed HCV E1-E2 glycoproteins were found to form noncovalently linked heterodimers and appeared to be correctly folded, as confirmed by coimmunoprecipitation analysis using conformationally sensitive anti-HCV-E2 monoclonal antibodies (MAbs). Intravenous or intraperitoneal immunization of BALB/c mice with VSV-C/E1/E2 or VSVG-C/E1/E2 resulted in significant and surprisingly comparable HCV core or E2 antibody responses compared to those of control mice. In addition, both virus types generated HCV C-, E1-, or E2-specific gamma interferon (IFN-
)-producing CD8+ T cells, as determined by enzyme-linked immunospot (ELISPOT) analysis. Mice immunized with VSVG-C/E1/E2 were also protected against the formation of tumors expressing HCV E2 (CT26-hghE2t) and exhibited CT26-hghE2t-specific IFN--producing and E2-specific CD8+ T-cell activity. Finally, recombinant vaccinia virus (vvHCV.S) expressing the HCV structural proteins replicated at significantly lower levels when inoculated into mice immunized with VSV-C/E1/E2 or VSVG-C/E1/E2, but not with control viruses. Our data therefore illustrate that potentially safer replication-defective VSV can be successfully engineered to express high levels of antigenically authentic HCV glycoproteins. In addition, this strategy may therefore serve in effective vaccine and immunotherapy-based approaches to the treatment of HCV-related disease. |
INTRODUCTION |
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HCV comprises a 9.5-kb single-stranded positive RNA genome that encodes a single large open reading frame of 3,000 amino acids (aa) translated by an internal ribosome entry site located at the 5' untranslated region. The polyprotein is cleaved into 10 HCV gene products: three structural proteins, namely the capsid (core [C]) and the envelope glycoproteins (E1 and E2); a small hydrophobic polypeptide P7 ion channel; and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) that are essential for viral replication (8). The cleavage of structural proteins from the polyprotein is mediated by host signal peptidase(s), and the remainder of the polypeptide is cleaved by cointeraction of NS2 and NS3 zinc-dependent proteinase and subsequently the NS3 serine protease. An additional HCV protein (F) of unknown function is proposed by a ribosomal frameshift in the sequence encoding the N-terminal region of the polyprotein (10).
Humoral antibody responses to core, NS3, NS4A/4b, and NS5a are typically detected during serological diagnosis (47). However, a key target for antibody-induced viral neutralization includes the envelope glycoproteins E1 and E2 (14, 22, 24, 61). Surrogate models using soluble E2 binding to cell surface molecules have identified potential cellular receptor candidates such as human TAPA-1 (CD81), scavenger receptor class B type 1, glycosaminoglycans, and, more recently, DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin [CD209]) as well as L-SIGN (DC-SIGNR; liver and lymph node specific [CD209L]) (3, 9, 16, 17, 43, 44, 59, 71). The low-density lipoprotein receptor has also been associated with HCV infection by endocytosis studies on a variety of cell lines (3). However, since HCV exhibits extensive genetic heterogeneity, especially within its glycoproteins, the presence of E1/E2 antibodies in sera of chronically infected patients and chimpanzees suggests that the responses are likely limited and isolate restricted. In fact, hypervariable region 1 at the N terminus of E2 is proposed as a target of E2 neutralization, but the virus may generate "immune-escape" hypervariable region 1 variants (46). Nevertheless, studies in chimpanzees and patients favor a strategy combining the induction of cellular immune response with the generation of antibody responses to E1/E2, although generating immune responses to other viral proteins may also be important (14, 15, 27, 31, 75). The role of CD4+ T-helper response is also of significance in HCV infection since the major histocompatibility complex (MHC) class II genotype (HLA DRB1*1101/DQ0301) has been associated with spontaneous clearance of infection (76). Furthermore, strong T-helper 1 (Th1) CD4+ responses appear to protect from chronic infection (69, 70). During chronic infection, however, patients appear to mount weak or strong CD4/CD8 responses that subsequently wane and the effector functions of which appear impaired (29, 75, 78). Hence the development of efficient technologies capable of inducing strong Th1 CD4+ and CD8+ T-cell responses is probably essential for optimal HCV prophylactic or therapeutic vaccine design. Characterization of immune responses in chimpanzees points to the requirement of vaccine candidates that can stimulate vigorous, sustained, and multispecific Th1 intrahepatic CD8+ and CD4+ T-cell responses that appear associated with resolution of acute infection (15, 75).
Recombinant E1/E2 immunization of chimpanzees, the only true nonhuman permissive infectious model, demonstrates that neutralizing antibodies can protect against low doses of homologous HCV challenge but not heterologous virus (14, 37). However, as confirmed in more recent studies, the duration of viremia was significantly shortened following reinfection, and E2 subunit vaccines could reduce liver disease (61). Furthermore, in intravenous drug users, previously infected individuals appear less likely to develop new viremia, suggesting a positive role for immunity against HCV pathology (50). HCV vaccines may therefore reduce the common problem of HCV chronicity and morbidity or mortality associated with HCV-related liver disease. In this endeavor, many approaches to more recent HCV vaccine strategies have attempted to improve immunogenicity to antigens and protective efficacy of subsequent immune responses using chimpanzees and smaller animal models. Examples of such strategies include incorporation of HCV proteins into recombinant viruses, naked DNA vaccines with or without boosting with recombinant proteins to improve cytotoxic T-lymphocyte (CTL) responses, virosome or liposome carriers for immunogenic peptides to enhance their delivery to cells, and use of HCV-like particles to mimic virions (11, 33, 51, 55, 62). Use of the more conserved HCV core proteins among HCV genotypes as an adjuvant for envelope glycoprotein vaccine has similarly proven useful in rhesus macaque studies (60).
Recently, we have described the usefulness of vesicular stomatitis virus (VSV) as a tool for delivering HCV immunity in mice (23). VSV vectors have been shown to exhibit significant promise in vaccine studies against respiratory syncytial virus, human papillomavirus, and AIDS-related diseases (40, 64, 65, 68). More recently, further strategies have been developed to generate safer nonpropagating viruses that lack the VSV glycoprotein (G) that is essential for infectivity (66). Recombinant VSV (rVSV) lacking the G gene (G) can be generated by supplying the G protein in trans. Resultant viruses are therefore capable of highly tropic infectivity for one cycle, but progeny viruses are incapable of infecting other cells since they lack the G gene in their genome. Here we report that despite the inability of progeny viruses (G) to undergo recurrent cycles of infection, they induce robust immune responses in vaccine studies compared to their replication-competent counterparts.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Replication-defective VSVG was generated by inserting a linker encoding the restriction enzyme sites MluI, BsiWi, PmeI, and XhoI into pVSV-XN2 using the MluI and XhoI restriction sites. The MluI restriction site in pVSV-XN2 is located after the transcriptional start signal of the G gene but before the ATG, and the XhoI site is located 3' of the G gene (42). This caused the entire G gene to be replaced with the multiple cloning site linker, creating the pVSVG vector. To construct VSVG-C/E1/E2, the core, E1, and E2 region of HCV (as described above) was amplified by PCR with the BsiWi and XhoI restriction sites at the 5' and 3' ends, respectively, and inserted into the pVSVG plasmid.
The VSVG and VSVG-C/E1/E2 viruses were made as described above with the following alterations. To construct the virus, a plasmid expressing the VSV G protein (pVSVG) was transfected 24 h before the vTF7-3 infection and again with the pVSVG, pBl-N, -P, and -L transfection that follows the vaccinia virus infection. This provides G protein, which is no longer encoded in the parent plasmid, so that infectious virus can be assembled. To grow the virus, BHK cells were transfected with pVSVG 24 h before infection so the resulting virions would have the G protein on the surface and be capable of infection. Any cell that is subsequently infected without previously expressing the G protein will not yield infectious virus.
Growth curves and viral titration.
VSV infections were performed (also see below) by infecting cells at the indicated MOI in serum-free medium for 1 h. The inoculum was aspirated and replaced with complete medium containing 10% FBS. Viral titers were determined by conducting 10-fold serial dilutions of each sample and then infecting confluent BHK cells as described above. The cells were then overlaid with 1% low-melting-temperature agarose in complete DMEM. Plaques were then counted 24 h postinfection after being stained with crystal violet; the titers are presented as PFU/ml. Titrations of replication defective VSV were conducted on BHK cells previously transfected with pVSVG (helper cells). One-step growth curve analysis of VSVG or VSVG-C/E1/E2 was performed in BHK helper cells at an MOI of 10. Culture medium (100 µl) was removed at each indicated time point, and the titer was determined on BHK helper cells as described above.
Recombinant vaccinia virus. vvHCV.s (51) and vv-WR (Western Reserve) (39) were gifts kindly provided by Jake Liang from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). VV stocks were propagated in HeLa cells, and standard vaccinia virus plaque assay was performed in the BSC-1 cell line.
Antibodies. Conformation-dependent mouse anti-E2 monoclonal antibody (MAb) H33 was kindly provided by Jean Dubuisson (Unité Hépatite C, CNRS-UPR2511, Institute de Biologie de Lille Cedex, France), as was the conformation-independent anti-E2 MAb, H52. Conformation-independent MAb to E1, A4, was also provided by J. Dubuisson as a gift from Harry Greenberg (Stanford University). Human anti-E2 MAbs CBH-7 and CBH-8C were obtained from Steven Foung (Stanford University). Polyclonal rabbit anti-E2 was provided by Charles Rice (Rockefeller University). Mouse core MAb was purchased from ViroStat, Portland, Maine.
Cell culture. Huh-7, BHK, HeLa, and BSC-1 cells were propagated in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 100 IU/ml (each) penicillin and streptomycin (DMEM-C). Mouse splenocytes were incubated in RPMI supplemented as described above and also with 0.2 ng/ml mouse interleukin-2 (IL-2) (Endogen, Rockford, IL) and corresponding peptide. Mouse tumor cell line CT26 was maintained as described above, and CT26-hghE2t, which expresses a genotype 1b HCV E2 (aa 384 to 719) protein was grown in DMEM-C and 350 µg/ml G418 selection medium (73).
Radiolabeling HCV glycoproteins. Huh-7 or BHK cells grown in 150-mM dishes were infected with VSV-C/E1/E2 or control VSV-GFP at an MOI of 10 in serum-free DMEM. After 1 h, the cells were washed in PBS and complete growth medium was added, as described previously. Following a 5-h infection period, cells were depleted of methionine and cysteine for 30 min and 600 µCi [35S]Met/Cyst was added. The infection was allowed to proceed for a maximum of 12 h. Cells were then scraped and lysed (0.5% NP40 in 10 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 20 mM iodoacetamide, and 1 µg of proteinase inhibitors). The lysate was clarified by centrifugation in an Eppendorf centrifuge for 10 min and then precleared with 2.5 µl of normal mouse immunoglobulin G (IgG; Santa Cruz Biotechnology, Santa Cruz, CA) complexed to protein G (Invitrogen, Carlsbad, CA) in immunoprecipitation (IP) buffer (0.2% NP-40 in 10 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA). HCV E1 and E2 were coimmunoprecipitated with 10 µl (approximately 10 µg) of H33 MAb-protein G with continual shaking at 4°C. After a minimum of 4 h, the immunocomplexes were centrifuged and the pellets were washed and centrifuged several times in IP buffer. For the final wash, the complexes were equally divided into two tubes and the pellet was resuspended in either reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (200 mM Tris, pH 6.8, 0.5% SDS, 10% glycerol, 0.01 g bromophenol blue, 2% ß-mercaptoethanol) or nonreducing buffer (SDS sample buffer without ß-mercaptoethanol). After boiling in the respective SDS-PAGE sample buffer for 5 min, the complexes were separated on 10% polyacrylamide gel. The gel was treated with NAMP100 Amplify (Amersham Biosciences, Piscataway, NJ) and then dried prior to exposure to autoradiograph film.
Immunoblotting.
Infection procedures and immunoprecipitation prior to Western analysis were carried out as described for radiolabeling experiments. Briefly, Huh-7 or BHK cells were infected at an MOI of 10 for 1 h with live VSV-C/E1/E2, VSV-GFP, or nonpropagating VSVG-C/E1/E2 or VSVG. The inoculum was removed, the cells were washed, and growth medium was added to allow the infection to proceed for 12 h. Cells were then harvested as before, and immunoprecipitations were performed using the panel of MAbs. Immunocomplexes were resuspended in SDS-PAGE sample buffer (with or without ß-mercaptoethanol) and then run on 10% polyacyrlamide gel. Proteins were then transferred to nitrocellulose membranes (Hybond-ECL; Amersham) using a vertical slab gel unit apparatus (model SE400; Hoefer Scientific Instruments, San Francisco, CA) and revealed using anti-E2 rabbit polyclonal antibody (diluted 1/1,000) followed by a mouse anti-rabbit immunoglobulin conjugated to horseradish peroxidase (HRP; diluted 1/5,000; Santa Cruz Biotechnology, CA). Immunoblots were then analyzed by enhanced chemiluminescence detection (ECL; Amersham, Piscataway, NJ) as recommended by the manufacturer. To detect E1, the mouse anti-E1 A4 antibody (diluted 1/1,000) was used followed by goat anti-mouse antibody (1/5,000) conjugated to HRP. Core protein was detected using a mouse anti-core antibody (Virostat, Portland, Maine) diluted 1/500 followed by the HRP-conjugated goat anti-mouse antibody. For detection of E1 and E2 heterodimers, the membranes were incubated in prewarmed stripping buffer (50 mM Tris, 2% SDS, 0.7% ß-mercaptoethanol) for 30 min and then extensively washed in PBS-0.05% Tween followed by reincubation with antibody and chemiluminescent detection.
Immunization of mice.
Female BALB/c mice (aged 6 to 8 weeks; Jackson Laboratories, Bar Harbor, Maine) were immunized intravenously (i.v.) or by intraperitoneal (i.p.) injection with VSVC/E1/E2, VSVG-C/E1/E2, or control virus (VSV-GFP or VSVG) at 6 x 106 PFU in PBS or with PBS alone. A 100-µl total volume of PBS-virus or PBS alone was administered twice during a 3-week interval (day 1 and day 14). Bleeding of mice before or after treatment was undertaken using the microhematocrit tube method. The splenocyte isolation procedure is described below. Mice to be challenged with CT26-hghE2t cancer cells (or control CT26) were given a second boost on day 28 and 2 weeks later received cancer cells (as described below). In viral challenge experiments, 1 x 107 recombinant vaccinia virus PFU (vvHCV.S or vv-WR) were administered intraperitoneally 2 weeks following the second boost.
HCV core and E2 ELISA. HCV core (genotype 1a) protein expressed in Saccharomyces cerevisiae (ViroStat, Portland, Maine) or full-length E2 protein (genotype 1a) purified following baculovirus expression (ImmunoDiagnostics, Inc., Woburn, MA) was diluted (0.5 µg) in coating buffer (50 mM Na2HPO4 [pH 9.6]) and bound overnight at 4°C to Nunc-Immuno MaxiSorp surface plates. Wells were washed several times in PBS-0.05% Tween 20 and blocked with PBS-10% heat-inactivated FBS (HI-FBS) at room temperature (RT). Subsequently, the plates were washed again prior to addition of mouse serum (1:100) or control antibody (anti-core; 1:500; and anti-E2, 1:1,000) in PBS-10% HI-FBS for 2 h at RT. Following the wash steps, goat anti-mouse HRP-conjugated antibody (1:4,000 in PBS-10% HI-FBS) was added to test sera and control wells for 1 h at RT. The wells were then washed, and substrate was added for 30 min in the dark, after which the reaction was stopped using 2 N H2SO4. The plates were read using an enzyme-linked immunosorbent assay (ELISA) plate reader at 450 nm (subtracted from 570 nm).
Peptides. The core (aa 133 to 142), E1 (aa 315 to 322), and E2 (aa 570 to 584) peptides have been described previously (23). HCV (genotype 1b) CTL peptides (Genemed Synthesis, Inc., South San Francisco, CA) (core, LPRRGPRLGVRATR [aa 37 to 50] and RRGPRLGVRATRKT [aa 37 to 52]; E2, SGPSQKIQLV [aa 405 to 414] and PPQANWFGCTWMNSTGFTKT [aa 544 to 563]) and a control human immunodeficiency virus peptide (RIQRGPGRAFVTIGK [aa 308 to 322]) were also selected for additional enzyme-linked immunospot (ELISPOT) analysis (12, 56).
ELISPOT assay.
Nunc Maxisorp plates (Nunc) were coated by overnight incubation at room temperature with a 5-µg/ml solution of anti-mouse IFN- MAb (BD Biosciences, PharMingen, San Diego, CA). The plates were washed (PBS-0.1% Tween), and wells were blocked with RPMI-5% FCS for 1 h at room temperature. Splenectomies were performed on mice sacrificed 21 days postimmunization, and the spleens were removed to a petri dish containing cold sterile PBS. After homogenization, splenocytes were transferred to 50-ml conical tubes and centrifuged at 1,800 rpm for 5 min. The pellets were resuspended in PBS followed by cold sterile water, the tubes were inverted for several seconds, 10x PBS was added, and the suspensions were vortexed prior to centrifugation. Pellets were resuspended in 30 ml of RPMI 1640 (Invitrogen, Carlsbad, CA), 1 mM sodium pyruvate, 2 mM L-glutamine (Invitrogen, Carlsbad, CA), and 10% heat-inactivated FCS (HyClone, Logan, UT), and the cell suspension was poured through a sterile cell container with a 40-µm pore diameter (Falcon, BD-Biosciences, Bedford, MA). Harvested cells (from 1 x 106 to 2.5 x 105) from each mouse set were seeded in triplicate into 96-well plates precoated with IFN- MAb. Cells were stimulated with 0.2 ng/µl of mouse IL-2 (Endogen, Rockford, IL) and pulsed with 10 µg/ml of core, E1, or E2 peptide (described above) for at least 36 h at 37°C. In addition, cells were plated as mock (nonpulsed) controls for each mouse group. For E2-specific CTL responses in CT26-hghE2t tumor-challenged mice, splenocytes were restimulated with mitomycin C-treated (25 µg/ml) cancer cells for 1 week at a ratio of 10:1 splenocytes to CT26-hghE2t cancer cells. The cell medium was removed, and plates were washed extensively with PBS and PBS-0.1% Tween followed by addition of 0.5 µg/ml of anti-IFN- MAb (BD Biosciences, PharMingen, San Diego, CA) to each well. Following a 90-min incubation at room temperature and additional washes in PBS-0.1% Tween and PBS alone, 100 µl of 0.2 µg/ml streptavidin-alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA) was added to each well. The wells were incubated for 1 h at room temperature and washed, and 3% type 1 low-electroendosmosis agarose (Sigma, St. Louis, MO) and 2.3 mM 5-bromo-4-chloro-3-indolyl phosphate in AMP buffer (75 mg MgCl2 hexahydrate, 50 µl Triton X-405, 500 mg NaN3, 47.9 ml 2-amino-2-methyl-1-propanol [Sigma, St. Louis, MO], pH 10.25) were added to wells. The developed spots were then counted with the aid of an inverted microscope the next day. Any background ELISPOTS from mock wells were subtracted from the experimental groups; the results are presented as mean ELISPOTS per million cells ± standard deviation for each group.
Lymphoproliferative assay. Splenectomies were performed as described above for ELISPOT analysis. Splenocytes were plated in triplicate at dilutions of 1 x 106 to 2.5 x 105 cells in RPMI-10% FBS and 100 IU/ml (each) penicillin and streptomycin in a 96-well format. The cells were stimulated with HCV (genotype 1a) core protein (Virostat, Portland, Maine) at 0.1- and 0.01-µg/ml concentrations, and control cells were stimulated with medium alone for 5 days. The cells were pulsed with 1 µCi of [3H]thymidine per well for at-least 18 h prior to the 5-day incubation. The cells were harvested (Filter maid harvester; Perkin-Elmer, Fremont, CA), and radiation counts were subsequently analyzed.
Tumor challenge experiments.
For the CT26-hghE2t and CT26 cancer cell challenge (73), mice were prevaccinated with replication-defective VSVG-C/E1/E2 or VSV-G (eight mice per group). A third PBS group (eight mice) was also included. Two weeks after the second boost, 2 x 106 CT26-hghE2t or CT26 cells were injected subcutaneously into each mouse group. The cancer growth was measured every 3 days, and mice were sacrificed when cancers reached approximately 20 mm in diameter or if cancers became highly necrotized.
Viral challenge of mice.
Female BALB/c mice (six per group) immunized (days 1, 14, and 28) with replication-defective VSVG-C/E1/E2 or VSV-G were challenged with 1 x 107 PFU of vvHCV.s (51) or control wild-type vv-WR (39) by intraperitoneal injection 2 weeks after the last immunization. In addition, no immunized mice were infected with vvHCV.S or vv-WR as controls. Mice (six per group) immunized with replication-competent VSV-C/E1/E2 or VSV-GFP were also challenged as described above. All mice were sacrificed 5 days after challenge, and their ovaries were harvested. Following freeze-thaw and homogenization, vaccinia virus titers were determined in BSC-1 cells by plaque assay.
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RESULTS |
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G-C/E1/E2 and expression of HCV core E1/E2.
We have previously demonstrated that replication-competent VSV can be genetically engineered to express HCV structural antigens and that this strategy can be used to generate immune responses in BALB/c mice (23). To evaluate the use of a nonpropagating form of rVSV vector in HCV vaccine strategies, the entire G gene from the parent pVSV-XN2 encoding recombinant VSV was deleted (as described in Materials and Methods) to create the pVSVG vector. The entire HCV structural region containing core, E1, and E2 of HCV genotype 1b (aa 1 to 746) was subsequently cloned into pVSVG (Fig. 1A). Nonpropagating VSVG-C/E1/E2 or control VSVG virus was recovered by expression of VSV N, P, and L genes in BHK cells harboring expression of VSV G. Infectious VSVG-C/E1/E2 was plaque purified, and its growth properties were compared to those of VSVG (Fig. 1B). Virus titers peaked exponentially between 4 and 12 h of infection and then reached a plateau 24 h postinfection at 5 x 106 and 1 x 108 PFU/ml for VSVG-C/E1/E2 and VSVG, respectively. VSVG titers were 1.5 logs higher than those of VSVG-C/E1/E2, possibly due to slight attenuation in the latter virus due to the addition of longer heterologous gene products (6). However, initial VSVG-C/E1/E2 titers were generally comparable to those previously reported for the replication-competent VSV-C/E1/E2, VSV-GFP, and parental VSV-XN2 (23). To confirm HCV gene expression, BHK cells were infected with VSVG-C/E1/E2 or VSVG at an MOI of 10 for approximately 24 h, at which point a cytopathic effect was clearly visible (data not shown). The cells were harvested and lysed as described previously, and SDS-PAGE analysis followed by immunodetection of target proteins was performed (Fig. 1C). This study confirmed that both VSVG and VSVG-C/E1/E2 lacked the VSV G protein. A trace amount of residual G protein was detected, however, plausibly from the original infection of cells. Subsequently, significant HCV C/E1/E2 expression was confirmed in VSVG-C/E1/E2-infected BHK cells following immunoblot analysis. Analysis of equal amounts of total cell lysates demonstrated a similar level of correctly sized proteolytically processed HCV C, E1 (35 kDa), and E2 (62 to 68 kDa) protein expression (Fig. 1C). VSVG-C/E1/E2 expression followed by Western blotting confirmed detection of the immature (22 kDa) and mature (19 kDa) HCV core products that have been described in other studies (38, 80). These data would suggest that nonpropagating VSVG-C/E1/E2 expresses heterologous HCV proteins to levels similar to those of its replication-competent counterpart.
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68-kDa product under reducing conditions (Fig. 2A). The specificity of VSV-expressed E2 binding to the anti-E2 H33 or anti-E2 H52 was emphasized by the fact that these antibodies did not react to proteins from VSV-GFP-infected cells following similar treatment. Furthermore, mouse IgG did not precipitate E2 from VSV-C/E1/E2-infected lysates, as expected (Fig. 2A). Very small amounts of more highly reactive bands indicative of uncleaved E1/E2 and C/E1/E2 were also evident but were not observed in the control lanes. Previous studies have shown that following HCV glycoprotein expression, disulfide-linked E1/E2 products can exist as unproductive, misfolded protein products that appear as large-molecular-weight aggregates under nonreducing SDS-PAGE (18, 20, 21, 57). However, functional noncovalently linked E1/E2 oligomers appear at their respective sizes of approximately 35 kDa and 68 kDa when examined under similar conditions (18, 53, 58). VSV-expressed HCV glycoproteins were therefore immunoprecipitated with anti-E2 H33 and anti-E2 H52 and examined under nonreducing conditions. Our data revealed that anti-E2 H33 bound strongly to E2 of the expected size (68 kDa), indicating that a significant amount of VSV-expressed E2 product was indeed conformationally authentic (Fig. 2B). In contrast, a weaker signal was seen using the nonconformationally sensitive anti-E2 H52. Since for each IP, equal amounts of lysate were used and equivalent volumes of eluted product were analyzed by SDS-PAGE, the difference in signal was unlikely due to variation in preparation or loading. Plausibly, the anti-E2 H52 antibody may exhibit some capacity to bind both conformationally authentic E2, as evidenced by the presence of some 68-kDa E2 protein. Misfolded E2 aggregates, in contrast, would appear as higher-molecular-weight products when examined under nonreducing SDS-PAGE conditions or become lodged in loading wells (Fig. 2B). Collectively, our data indicate that correctly sized, conformationally authentic VSV-expressed E2 products can be observed following nonreducing SDS-PAGE analysis of anti-E2 H33 immunoprecipitates.
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To further confirm the authenticity of our E1/E2 products, we next utilized conformation-dependent human anti-E2 CBH-7 and CBH-8 MAbs (hMAbs) (32). Huh-7 cells were thus infected with VSV-C/E1/E2 (or VSV-GFP), and lysates were subjected to coimmunoprecipitation analysis as described above. CBH-7- or CBH-8-precipitated E2 glycoproteins were confirmed by immunoblotting using rabbit anti-E2 polyclonal antibody under reducing or nonreducing SDS-PAGE conditions (Fig. 2D and E, upper panels). To detect E1, immunoblots were reprobed with anti-E1 A4. Our findings illustrate that VSV-expressed E1 could indeed be coimmunoprecipitated using anti-E2 CBH-7 or CBH-8 under reducing or nonreducing conditions, presumably since it is appropriately bound to E2 (Fig. 2D and E, lower panels).
To examine the authenticity of VSVG-C/E1/E2-expressed E1/E2 products, Huh-7 cells were infected with VSVG-C/E1/E2 or VSVG at an MOI of 10 for 12 h and the lysates were subjected to coimmunoprecipitation analysis as described above. CBH-7- or CBH-8-precipitated E2 glycoproteins from VSVG-C/E1/E2-infected lysates were confirmed by immunoblotting using rabbit anti-E2 polyclonal antibody under nonreducing SDS-PAGE conditions (Fig. 2F, upper panels). The blots were then reprobed with anti-E1 A4 to demonstrate the presence of noncovalently bound E1 (Fig. 2F, lower panel). Collectively, our data illustrate that VSV-expressed HCV E1 and E2 can for the majority form noncovalently linked heterodimers as determined by antibodies generated to native HCV envelope glycoproteins (32). Thus, the VSV-C/E1/E2 and VSVG-C/E1/E2 system appears capable of efficiently expressing HCV structural proteins, including E1/E2, a proportional amount of which form correctly processed heterodimers.
Generation of humoral immune responses by VSVG-C/E1/E2.
Following generation of nonpropagating VSVG-C/E1E2 and evaluation of VSV-expressed E1/E2 products with conformationally sensitive MAbs, we next evaluated the potential of VSVG-C/E1/E2 as a vector to generate anti-HCV immune responses and compared the responses to those of the replication-competent (VSV-C/E1/E2) virus counterpart. First, immunoblot analysis confirmed that the replication-defective VSVG-C/E1E2 expressed HCV core, E1, and E2 at similar levels in BHK cells (MOI of 10; Fig. 3A to C). Subsequently, BALB/c mice were injected twice (day 1 and day 14) with 6 x 106 PFU of replication-competent VSV-C/E1/E2, VSV-GFP, or nonpropagating VSVG-C/E1/E2 or VSVG by either i.p. or i.v. administration. Control mice were injected with an equal volume (0.1 ml) of PBS. The serum of immunized mice was analyzed for antibody to HCV core or E2 by ELISA at day 0 and day 21 postinoculation. These data revealed that only mice injected with VSV-C/E1/E2 or VSVG-C/E1/E2 exhibited potent and highly specific anti-HCV core antibodies following i.p. or i.v. immunization (Fig. 3D, E, F, and G). No significant cross-reacting antibodies to HCV core protein were detected in mouse serum isolated from control or VSVG-immunized mice. However, i.p. routes of administration appeared to result in stronger antibody responses to the HCV core. Our data confirm that nonpropagating VSVG-C/E1/E2 can be used to express similarly high levels of HCV antigens compared to its replication counterpart, VSV-C/E1/E2, and to elicit robust anti-HCV core antibodies in mice models.
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G-C/E1/E2 (i.p. or i.v.) also mediated clear anti-HCV E2 responses following two immunizations as described above (Fig. 4). Although the E2 glycoprotein is highly variable between subtypes and genotypes (46), our data demonstrate that VSV-induced HCV E2 (genotype 1b) antibodies can react with a genotype 1a antigen. The level of anti-E2 response was similar using either VSV-C/E1/E2 or VSVG-C/E1/E2 via the i.v. route (Fig. 4C and D), but VSVG-C/E1/E2 generated marginally more anti-E2 antibody than VSV-C/E1/E2 via the i.p. route of administration, for reasons that remain unclear (Fig. 4A and B). We may have detected even stronger anti-HCV E2 responses in VSV-C/E1/E2 or VSVG-C/E1/E2 mouse sera in the presence of an HCV 1b E2 antigen in our ELISA. Nevertheless, the data presented clearly illustrate the usefulness of nonpropagating VSV expressing HCV structural antigens in generating anti-HCV core and E2 antibody responses in mice. In addition, intraperitoneal administration of recombinant VSV appears to deliver marginally better humoral immunity than intravenous injection.
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, whereas IgG2b switching requires transforming growth factor ß (72). Anti-HCV core-positive sera from three mice were therefore tested in an ELISA format, and isotype-specific secondary antibodies were used to detect immunoglobulin (IgG) subtypes. Previous studies of patients have shown that high levels of IgG2a are typical of Th1 responses and correlate with HCV clearance (77). Our findings in BALB/c animals illustrated high levels of both IgG1 and IgG2a anti-HCV core responses following i.p. immunization with VSVG-C/E1/E2 (Fig. 5). Although potent IgG1 responses are also shown and are typically indicative of Th2-type responses, the high levels of IgG2a indicate that VSVG-C/E1/E2 immunization of mice can stimulate antibody subtypes indicative of Th1-type responses. Collectively, our data would indicate that VSVG-C/E1/E2 is able to elicit robust antibody responses to all of the structural proteins of HCV.
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G-C/E1/E2.
Next we analyzed cellular immune responses associated with our immunization model. Specifically, we were interested in evaluating whether our vaccine regimens could generate Th1-type CTL responses, as demonstrated by IFN- release by CD8+ T cells. These T-cell subsets have been shown to play an important role in HCV infection (30). Accordingly, splenectomies were performed on day 21 in mice that had been immunized with either VSV-C/E1/E2 or VSVG-C/E1/E2 (i.p. or i.v.). Splenocytes from mice were pulsed with 10 µg/ml of genotype 1b core, E1, or E2 peptide with IL-2 and analyzed for IFN- production by ELISPOT assay (Fig. 6). Our results indicate that mice immunized with either VSV-C/E1/E2 or VSVG-C/E1/E2 generated statistically significant (P = <0.05) CTL activity against a panel of HCV structural peptides as compared with control viruses. We found that, in general, higher CTL responses to HCV C, E1, and E2 were observed in VSVG-C/E1/E2-immunized mice than in VSVC/E1/E2-immunized mice (the exception being E1 following i.v. injection of VSV-C/E1/E2). Interestingly, the removal of the VSV G protein appeared to modulate a more responsive CTL response to HCV antigens and also a general increase in nonspecific CTL immunity following i.p. injection, for reasons that are yet unclear (Fig. 6B and E). These results indicate that the VSVG-C/E1/E2 vaccine strategy can stimulate CTL responses to HCV C, E1, and E2. Furthermore, enhanced CTL responses are achieved with nonpropagating VSVG-C/E1/E2 compared to replication-competent VSV-C/E1/E2.
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G-C/E1/E2 intraperitoneally immunized animals following stimulation of splenocytes with the HCV core protein (Fig. 6F). Although we did not directly study CD4+ responses in this study, the splenocyte proliferation observed will represent both CD8 and CD4 cells following MHC I and MHC II class presentation. However, since CD8 cells are dependent upon the proliferation of CD4 cells as they require an exogenous source of IL-2 to initiate proliferation (28), the total lymphoproliferative responses observed should include HCV core-specific CD4+ T-cell subsets induced by VSVG-C/E/1/E2. In addition, our notion that CD4 T cells are induced in our system is corroborated by the induction of strong IgG antibody responses (Fig. 3, 4, and 5). The characterization of these total lymphoproliferative responses is warranted in further studies. However, here we show that lymphoproliferative responses were only observed in VSVG-C/E1/E2-immunized mice and not using control viruses. Overall, our data suggest that VSVG-C/E1/E2 can induce IFN- CTL activity to multiple CD8+ epitopes in HCV structural antigens and total lymphoproliferative responses to HCV core antigen. Our approach may therefore offer a potentially useful and safer approach to delivering HCV immunity using VSV.
Immunization with VSVG-C/E1/E2 can inhibit the growth of tumors expressing HCV E2.
To evaluate whether the immune responses generated from our VSVG-C/E1/E2 could be protective against tumor cells expressing HCV E2 protein, we utilized a mouse colon cancer model expressing HCV-E2 (CT26-hghE2t) and looked for protection against malignant disease in immunized BALB/c mice (73). Eight mice per group were intravenously injected with 6 x 106 PFU of VSVG-C/E1/E2, VSVG, or PBS alone (three inoculations, days 1, 14, and 28). Two weeks following the second boost, 2 x 106 control CT26 or CT26-hghE2t cells in log phase were subcutaneously injected into immunized mice or a control nonimmunized group. The number of mice that presented with palpatable tumors was examined every third day, and measurement of average tumor diameter as well as calculation of tumor volume (according to the formula 0.5 x length x width2 (52) began on the 8th day postinsertion of tumor cells (Fig. 7). All control (PBS only) mice grew CT26 and CT26hghE2t tumors that remained present throughout the experiment (data not shown). In mice that were vaccinated with VSVG or VSVG-C/E1/E2, all animals (8/8 per group) that received control CT26 challenge developed tumors by day 8 (Fig. 7A and B). Tumor measurements demonstrated that wild-type CT26 cancer grew exponentially from day 8 to day 20 in all groups without any significant difference between VSVG-C/E1/E2- and VSVG-immunized mice (Fig. 7A and B). Median CT26 tumor diameters in VSVG- and VSVG-C/E1/E2-vaccinated mice were similar on day 8 (5.18 versus 5.4 mm, respectively) and increased rapidly over the course of the experiment (13.7 mm for VSVG and 13.2 mm for VSVG-C/E1/E2 by day 20) (Fig. 7E). These tumors remained present throughout the experiment, and together with the PBS-treated mice, these mice were eventually sacrificed due to tumor size. All VSVG-immunized mice also presented CT26-hghE2t tumors between days 8 and 14 postchallenge. However, two animals in this group were found to clear the tumor (Fig. 7C). Significantly, only 3/8 animals inoculated with VSVG-C/E1/E2 mice exhibited any form of tumor by day 26 (Fig. 7D). Furthermore, the median CT26-hghE2t tumor diameter of VSVG-immunized mice was much greater (12 mm) than that in VSVG-C/E1/E2-vaccinated mice (2.31 mm) (Fig. 7F). In addition, mice that had been immunized with VSVG and VSVG-C/E1/E2 showed a significant difference (P = 0.00039) in the tumor growth kinetics over time between these two groups following CT26hghE2t challenge (as determined by statistical analysis using Student's t test). Our data indicate a significant correlation for protection against CT26-hghE2t challenge in mice immunized with VSVG-C/E1/E2.
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G-C/E1/E2-generated CTL activity to E2, which could eliminate CT26-hghE2t growth, IFN- ELISPOT analysis was carried out in vaccinated mice. For the CT26-hghE2t analysis, two surviving mice from the VSVG-C/E1/E2 or VSVG group that had been immunized as described above and challenged with CT26-hghE2t tumor cells were sacrificed on day 30. Splenocytes from mice were stimulated with mitomycin C-treated CT26-hghE2t cells (or the irrelevant HeLa cell control) at a ratio of 10:1 splenocytes to cancer cells for 1 week. For E2-specific CTL analysis, splenocytes were stimulated with E2 peptide as previously described (Fig. 7G). This analysis showed that while VSVG-C/E1/E2 mice had slightly elevated levels of CT26-hghE2t IFN--producing splenocytes, larger tumor-bearing VSVG-immunized mice also exhibited IFN- reactivity to CT26-hghE2t cancer for reasons that remain unclear. However, it is possible that VSVG exerts oncolytic activity to lyse tumor cells and generate some CTL activity to E2 (4, 5, 25). Interestingly, there was a clear difference in the level of CTL activity to E2 peptide, which may have accounted for enhanced protection in VSVG-C/E1/E2 mice compared with the VSVG group (Fig. 7G). Immunoblot analysis of CT26-hghE2t cells was performed and confirmed expression of HCV E2 glycoprotein prior to insertion of the cancer cells into mice (Fig. 7H). These results indicate that CTL-mediated IFN- activity to CT26-hghE2t cells can occur in mice challenged with this tumor and, more specifically, that E2 CTL activity generated from VSVG-C/E1/E2-immunized mice may facilitate protection against HCV-associated tumor formation.
VSVG-C/E1/E2 immunization of BALB/c mice protects against vvHCV.S challenge.
The above data indicated that VSVG-C/E1/E2 immunization could protect against a tumor model expressing HCV E2. Subsequently we expanded our studies to examine the potential use of our vaccine strategy to neutralize a surrogate viral HCV challenge model. Mice immunized as described above for the tumor experiments were challenged with 1 x 107 PFU of recombinant vaccinia virus-expressing HCV-C/E1/E2 (vvHCV.S) or with a control vaccinia virus-wild-type Western Reserve strain (vv-WR) (39, 51). Intraperitoneal injection of vvHCV.S or vv-WR was undertaken 2 weeks following the last boost with VSVG-C/E1/E2 or VSVG. Control PBS-injected mice were also administered the same dose of challenge virus. Five days later, ovaries were harvested from mice and freeze-thawed several times subsequent to plaque titer analysis (Fig. 8A). The mean (five mice per group) vvHCV.S titer observed in control PBS mice was 2.28 x 109 PFU/ml, and that for VSVG-immunized mice was 2.98 x 109 PFU/ml. However, in mice immunized with VSVG-C/E1/E2, the mean vvHCV.S titer was 1.18 x 107 PFU/ml and was 2 logs lower than that in VSVG-immunized mice (P = 0.0039) and PBS-treated mice (P = 0.010). There were no differences in viral titers in any of the groups who were challenged with a nonspecific vv-WR (Fig. 8B). These findings indicate that the VSV-expressed HCV structural antigens can inhibit the replication of vvHCV.S in VSVG-C/E1/E2-immunized mice. The specificity of this protection is illustrated by the lack of neutralization against vv-WR challenge in VSVG-C/E1/E2-immunized mice (Fig. 8B). We also observed vvHCV.S virus titer reduction in animals immunized with VSV-C/E1/E2 (Fig. 8C). The experiments were performed as described above for VSVG-C/E1/E2, except that we also evaluated the potential effect of ovary size against final vvHCV.S titers. We found that in VSV-C/E1/E2-immunized animals, there was over a 2-log reduction (P = 0.022) in the ratio of residual vvHCV.S titers per milligram of ovaries as compared to animals immunized with VSV-GFP. The results are indicative of the protective immune responses induced by VSV-C/E1/E2 or VSVG-C/E1/E2 and are not due to variations in weights of ovaries sampled. Collectively, our data indicate that VSVG-C/E1/E2 could present a promising and safer alternative strategy for immunotherapy against HCV.
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G-C/E1/E2) was generated and evaluated as a safer alternative vaccine strategy in murine models. We report that VSVG-C/E1/E2 expressed high levels of the HCV core, E1, and E2 structural proteins, a significant quantity of which were authentically processed as noncovalently linked HCV E1/E2 oligomers, as demonstrated by binding to conformationally sensitive mouse or human MAbs. In this report, we utilized several previously described mouse or human conformation-dependent antibodies to examine the authenticity of VSV-expressed HCV glycoproteins (18, 26, 32, 57). These antibodies included human mAbs CBH-7 and CBH8-C, which block binding of E2 to the cellular receptor CD81 (32). CBH-7 and CBH8-C were isolated from an HCV 1b-infected individual and have been reported as being a positive indicator for the correct processing of VSV-expressed HCV E1/E2 noncovalent heterodimers. Such data would indicate that VSV-expressed HCV glycoproteins may generate humoral responses that could be effective at preventing bona fide HCV infection, at least against similar genotypes. Furthermore, our findings that VSV-induced anti-HCV E2 antibodies were cross-reactive between genotypes 1a and 1b is encouraging for vaccine purposes, since these genotypes are the most prevalent worldwide (45). Interestingly, nonpropagating VSVG-C/E1/E2 was found to stimulate humoral and cellular immune responses in mice that were comparable to those of live VSV-C/E1/E2 without the prerequisite for adjuvant enhancement. This strategy has several features that are beneficial over replication-competent approaches and seem to generate robust immune responses. First, the removal of the VSV-G gene from the genome results in the reduction of the highly antigenic VSV glycoprotein, allowing immune responses to respond more efficiently against HCV structural antigens. Second, this technology has the benefit of being a safer viral approach that still can initiate both humoral and cellular immunity. Although nonpropagating, it is likely that this viral system may still facilitate innate intracellular responses that control subsequent adaptive immunity through release of cytokines and "cross-priming." Current, E1/E2 subunit vaccines appear to generate humoral responses but lack the capability of inducing CTL responses (14, 37, 60). However, core protein-based nonclassical immunostimulating complex approaches have been shown to generate long-lived CD4+ and CD8+ Th0-type responses in rhesus macaques (60). Although currently our studies in BALB/c mice are in preliminary stages, the humoral and cellular immune responses against HCV structural proteins using a nonpropagating VSV approach provide an excellent platform for further studies to enhance and review the immune response further in both transgenic human HLA-A 2.1 (AAD) mice and nonhuman primate models.
To date, vaccine approaches to HCV have failed to produce sterilizing immunity, and with this factor in mind, here we utilized two surrogate challenge models to examine the protective nature of the nonpropagating VSVG-C/E1/E2 strategy using mouse models. In the first challenge model, we utilized a CT26-hghE2t mouse colon cancer model expressing HCV-E2. VSVG-C/E1/E2-immunized mice appeared protected from CT26-hghE2t tumors since the growth of the cancer was restricted compared to that in control groups and CT26 tumors. This effect was clearly due to E2 immunization since control groups were not protected against CT26-hghE2t tumors and there was no protection against wild-type CT26 challenge. Furthermore, ELISPOT analysis illustrated that CD8+ CTL activity specific to E2 may in part be responsible for this protection. However, we cannot rule out antibody-dependent cell-mediated cytotoxicity, a process frequently mediated by IgG2a binding to FcR on macrophages and natural killer cells (1, 41, 73). In our study, we also observed IgG2a responses to HCV core and anticipate that similar responses to HCV E2 may be present to facilitate antibody-dependent cell-mediated cellular cytotoxicity to CT26-hghE2t tumor. Intriguingly as VSV exhibits oncolytic activity itself (reviewed in reference 7), it may be interesting to see if replication-competent VSV-C/E1/E2 or even VSVG-C/E1/E2 can be used therapeutically to treat animals bearing CT26-hghE2t tumors. Whether through immune enhancement or potentially by direct oncolytic activity, these studies may provide a basis for feasibility studies into HCV cancer vaccines against potentially HCV-induced B-cell non-Hodgkin's lymphomas or hepatocellular carcinoma (19, 54, 67).
The only true infectious in vivo model organism with which to examine HCV vaccines before trials in humans is the chimpanzee. However, the ability to titrate recombinant vvHCV.S or vv-WR in murine ovaries offers another useful measure of protective immunity by potential prophylactic HCV vaccines. In this study, we noticed a significant reduction in vvHCV.S plaque titers of mice that had been immunized with VSVG-C/E1/E2 compared to VSVG- or PBS-immunized control groups. The lack of complete neutralization of vvHCV.S may be due to the heterologous nature of the recombinant viral challenge. However, others have demonstrated the usefulness of recombinant vaccinia virus challenge models in improving vaccine approaches in mice and nonhuman primates against HCV and human papillomavirus (48, 51). The goals for vaccine approaches are to potentially achieve optimal humoral and cellular (strong CD4+ and CD8+) responses to engage HCV infection before infection can outpace the immune system, as seems to occur during persistence (79). Additional hope comes from the fact that HCV immunotherapy may prevent chronic infection and also liver disease that appears to be associated with potentially compromised T-cell responses and their effector functions (63). Using our experimental models, we describe positive humoral and cellular (total lymphocyte proliferation and CD8) immune responses that include markers associated with Th1-type (IgG2a and IFN-) immunity. Since these responses play an important role in resolving HCV infection (15, 30, 75), our findings here and subsequent observations from two different challenge systems provide the basis for further study of VSVG-C/E1/E2-based vaccination and immunotherapy to combat HCV infection.
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ACKNOWLEDGMENTS |
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This work was supported by NIH funding (NIH R01A1053670).
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FOOTNOTES |
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REFERENCES |
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