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Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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15% in other regions of the world (2).
Approximately 60% of individuals exposed to HCV will develop chronic
infection and hepatitis; 20 to 40% will eventually progress to
cirrhosis and liver failure (3). More important, persistent HCV
infection is associated with a high risk of primary hepatocellular
carcinoma, particularly in the setting of hepatic fibrosis and
cirrhosis (4). Effective therapy of chronic HCV infection has been
limited, at best, and only IFN and ribavirin have been shown to exhibit
beneficial antiviral activity (5). Indeed, 
10 to 15% of individuals
treated with IFN alone will respond and eradicate HCV from the liver.
However, recent studies have revealed that individuals who recover from
acute HCV infection develop substantial CD4+ T cell
proliferative responses against the nonstructural proteins as compared
with those individuals who develop persistent HCV infection (6, 7).
This type of cellular immune response suggests that the nonstructural
proteins may be the more critical immunogens to eradicate persistent
viral infection from the host. In this context, direct injection of DNA
encoding for viral genes in combination with different facilitators
into the muscle or skin has been shown to induce broad-based humoral
and, more important, cell-mediated immune responses, and is especially
effective in generating protective cytotoxic T cell responses against a
variety of pathogens (8, 9, 10, 11). However, the generation of such
protective immune responses in humans remains to be established. In the present investigation, we evaluated in vitro and in vivo humoral and cellular immune responses generated by DNA-based immunization against the three different nonstructural proteins of HCV in a murine model. It was found that the cDNAs encoding for the NS3 serine protease and helicase and NS5 RNA-dependent RNA polymerase were particularly effective in generating high-level CD4+ and CD8+ activities against epitopes that reside on these nonstructural proteins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a source of viral genes, a plasmid designated pBRTM/HCV1-3011
covering the full-length open reading frame (ORF) of HCV was used to
clone into expression vectors (12). Constructs pAp031-NS3, pAp031-NS4,
and pAp031-NS5 were PCR-cloned after inserting engineered start and
stop codons as well as restriction enzyme sites using the following
primers: for NS3, 5'-GG TCT AGA TTG ATG GCG CCC ATC ACG GC-3'
(XbaI), 5' CAC ACG CGT TCA CGT GAC GAC CTC CAG GT 3'
(MluI); for NS4, 5'-G GTC TAG ATG AGC ACC TGG GTG CTC-3'
(XbaI) and 5'-CCA GGA TCC TCA GCA TGG AGT GGT ACA-3'
(BamHI); and for NS5, 5'-T CAG TCT AGA ATG TCC GGC TCC TGG
CTA AGG GA-3' (XbaI) and 5'-A GCT ACG CGT TCA CCG GTT GGG
GAG GAG GT-3' (MluI). After PCR amplification using a
high-fidelity PCR system (Boehringer Mannheim, Indianapolis, IN), the
cDNA fragments were inserted into the plasmid expression vector pAp031
containing a Rous sarcoma virus enhancer element and a CMV promoter
(Apollon, Malvern, PA). Constructs were transformed into DH5
cells, and plasmid DNA was subsequently purified by either 2x cesium
chloride centrifugation or with a Qiagen Giga kit using the Endofree
buffer system (Santa Clara, CA). Correct insertion of cDNAs coding for
of the nonstructural proteins was verified by sequencing analysis using
standard methods. To establish stable NS3-, NS4-, and NS5-expressing
cell lines as target cells for the CTL assays, the nonstructural
protein-encoding gene fragments were also cloned into the pcDNA3 and
pcDNA3.1/Zeo(-) expression vectors (Invitrogen, San Diego, CA)
with a neomycin selectable marker. An XbaI and
MluI fragment of NS3 and NS5 was subcloned into the
NheI/MluI site of Litmus-38 vector (New England
Biolabs, Beverly, MA), cut with EcoRI and SalI,
and ligated into the EcoRI/XhoI multiple cloning
site of pcDNA3 and pcDNA3.1/Zeo(-), respectively. An XbaI
and BamHI fragment containing NS4 was ligated into Litmus-29
(New England Biolabs), recut with KpnI and EcoRI,
and subsequently ligated into the pcDNA3 vector. Plasmids were
designated pcDNA3-NS3, pcDNA3-NS4, and pcDNA3.1/Zeo(-)-NS5.
In vitro expression
The HuH-7 human hepatoma cell line was transiently transfected with the various constructs by the calcium phosphate method to assess expression levels of HCV nonstructural proteins. In brief, cell lysates were prepared in modified RIPA buffer (0.15 M NaCl, 1% Nonidet P-40, 50 mM Tris, 0.5% deoxycholate, and 1% SDS) after metabolic labeling with [35S]methionine and cysteine for 4 h. Cell lysates were precleared with horse serum and subsequently bound to Sepharose A by preincubation overnight with polyclonal antisera WU 110 (NS3), WU 148/151 (NS4), and WU 115 (NS5) (12). After separating the proteins by SDS-PAGE, the gels were dried and exposed. NS5 protein expression was also determined by Western blot and immunofluorescence analysis using a murine mAb (Biogenesis, Sandown, NH). To generate stably transfected cell lines expressing NS3, NS4, and NS5, the syngenic BALB/c mouse myeloma derived cell line SP2/0 was transfected by electroporation with pcDNA3-NS3, pcDNA3-NS4, or pcDNA3.1/Zeo(-)-NS5. Cells growing in selection medium were cloned by limiting dilution (0.3 cell/well) and screened by the methods described above. However, attempts to clone stable NS4-expressing cell lines were unsuccessful.
Immunization protocol
Female BALB/c (H-2d) mice were maintained under standard pathogen-free conditions in the animal facility of the Massachusetts General Hospital. Mice were obtained from Charles River Laboratories (Wilmington, MA) and used at the age of 6 to 20 wk for the in vivo studies. A total of 100 µg of plasmid DNA in 100 µl of 0.9% NaCl was injected two and three times over five different sites into the quadriceps muscle of the mice. Booster injections were given into the opposite leg every 14 days. As a positive control for all immunologic experiments, 5 µg of recombinant NS3, NS4, and NS5 protein (Mikrogen, Munich, Germany) was injected i.p. in CFA at day 0 and boosted with the same amount of protein in 0.05% SDS after 4 and 8 wk. As negative controls for these experiments, empty plasmid vector and recombinant hepatitis B virus surface Ag (HBsAg) (Engerix, Smith Kline Beecham, Philadelphia, PA) were employed. All mice were sacrificed at 10 days after the last immunization.
Measurement of humoral immune responses
Levels of anti-NS3, NS4, and NS5 Abs were determined in the serum of each immunized animal by ELISA. In brief, microtiter plates (Microtest IIIM flexible assay plate, Falcon, Oxnard, CA) were coated with the above-described recombinant proteins overnight at 4°C (0.5 µg/well). After blocking with FBS for 2 h at 20°C, a 1/50 dilution of mouse serum was added to the plates and incubated at 20°C for an additional 1 h. After washing four times with PBS containing 0.05% Tween-20, a horseradish peroxidase-conjugated anti-mouse Ab (Amersham, Arlington Heights, IL) was applied at a 1/2000 dilution. Plates were washed following a 1-h incubation, and substrate was added for color development and read in an automatic reader.
Lymphoproliferation and cytokine release assays
Mice were anesthetized with isoflurane (Aerrane, Anaquest, NJ),
and spleen cells were harvested. E were removed by incubation in 0.83%
NH4Cl/0.17 M Tris (pH 7.4) for 5 min at 25°C. Spleen
cells were washed two times and cultured in triplicate using 96-well
round-bottom plates at 5 x 105 cells/well in 200 µl
DMEM (Cellgro Mediatech, Washington, DC) containing 10% FBS and 2-ME.
Cells were stimulated with recombinant nonstructural proteins NS3, NS4,
and NS5-4 at different concentrations (0, 0.01, 0.1, and 1 µg/ml). As
negative controls, effector cells were stimulated with recombinant HCV
core or HBsAg proteins at the same concentrations. After stimulation
for 3 days, [3H]thymidine was added (1 µCi/well). Cells
were incubated for an additional 18 h, and the
[3H]thymidine incorporation into DNA was measured after
harvesting. Incorporation of radioactivity was corrected for background
activity (
cpm). For determination of cytokine release, effector
cells were cultured as described above; IL-2, IL-4, and IFN-
levels
were measured in the culture supernatant by commercial kits according
to the manufacturer’s instructions (Endogen, Boston, MA).
CTL activity
Spleen cells derived from immunized mice were suspended in DMEM supplemented with 10% FCS and 2-ME (5 x 10-5M) and analyzed for cytotoxic activity following 5 days of in vitro stimulation. In vitro stimulation was performed in 25-ml flasks with a total volume of 12 ml. Murine rIL-2 was added once at a concentration of 5 U/ml, and responder cells (4 x 107) were cocultured with 2 x 106 irradiated (10,000 rad) syngenic SP2/0 cells stably expressing either the full-length NS3 or NS5 protein (SP2/NS3-3, SP2/NS5-21). After 5 days, cytotoxic effector lymphocyte populations were harvested and washed in serum-free medium; a 4-h 51Cr release assay was performed in 96-well round-bottom plates (total volume of 140 µl) using 51Cr-labeled SP2/NS3-3 or SP2/NS5-21. These cells (1 x 106) were incubated for 1 h with 100 µl of 51Cr (1 mC/ml) and subsequently washed three times in DMEM containing 10% FCS (4°C). Parental SP2/0 or SP2/19 cells expressing the HCV core protein were used as controls for Ag specificity of lysis and background activity. Assays for CTL activity were performed at lymphocyte E:T ratios of 100:1, 30:1, 10:1, and 3:1, respectively, using 1 x 104 51Cr-labeled target cells/well. T cell depletion experiments were conducted by incubating effector cells with either an anti-CD4+ or CD8+ mAb containing hybridoma supernatant (GK1.5 anti-CD4, rat or 3.155 anti-CD8, rat) for 30 min at 4°C; next, the cells were washed and then incubated at 37°C with complement (1/5 dilution of low-toxicity rabbit complement; Cedarlane Laboratories, Hornby, Canada) before performing the CTL assay described above.
Assessment of CTL activity in vivo
Mice were immunized i.m. three times with either Mock DNA or pApNS5 vector. Some animals were also immunized i.p with recombinant NS5 protein or a combination of both. Recombinant proteins (5 µg i.p.) were administered as a mixture of NS5-4 (aa 2622–2868) and NS5-12 (aa 2007–2268). At 1 wk after the last immunization with the various plasmid constructs or recombinant protein, 2 x 106 syngenic SP2/0-derived cells stably expressing NS5 were washed, resuspended in 200 µl PBS, and inoculated s.c. into the right flank. SP2/0 cells that stably expressed HCV core protein (SP2/19) were used as a control in selected animals. Tumor formation was assessed at 15 days postinoculation, and the number of animals with tumors and tumor weight was determined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HCV is a positive-strand RNA virus with a genome length of 9.5
kb. One large ORF encodes for a polyprotein precursor of 3000 aa
that is processed by a combination of host and viral proteases into
10 different structural and nonstructural proteins (12, 13, 14). We
cloned the genes encoding for the individual nonstructural proteins
with engineered start and stop codons into an expression plasmid driven
by a CMV promoter and a Rous sarcoma virus enhancer (pAp031). The
expression vector pcDNA3 containing a neomycin selection marker was
also used to generate stable SP2/0-derived cell lines (Fig. 1
a). The plasmid constructs
were sequenced across the cDNA inserts, and protein expression was
analyzed in vitro in HuH-7 cells after transient transfection and in
SP2/0 target cells after stable transfection, respectively. Signals
corresponding to proteins with molecular masses of 70 kDa for NS3,
30 kDa for NS4, and 125 kDa for NS5 were observed in cellular lysates
but not in supernatant from transfected cells (Fig. 1b).
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Specific Ab responses directed against all three nonstructural
proteins were found in all animals by ELISA following three
immunizations. No Ag-specific immune responses were detected in mice
immunized with mock DNA (Fig. 2a). As positive controls,
mice were vaccinated three times i.p. with recombinant NS3, NS4, and
NS5 proteins in combination with CFA; as expected, the mice
demonstrated a strong humoral immune response (data not shown).
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To investigate cell-mediated immune responses to the nonstructural
proteins, spleen cells were harvested and restimulated either with
recombinant Ag or with Ag expressed by stably transfected cell lines in
vitro. Substantial lymphocyte proliferation was induced by all
nonstructural proteins at different Ag concentrations as measured by
[3H]thymidine incorporation (Fig. 2
b).
Immunization with recombinant protein i.p. as a means of generating
maximum stimulation produced a 5- to 10-fold higher lymphocyte
proliferative rate for all three proteins (data not shown). The
cytokine profile determined after DNA-based immunization demonstrated a
classic Th1 response, with high levels of IFN- (Fig. 2c)
and IL-2 (Fig. 2d) secreted into the cell culture
supernatant. The cytokine release after incubation with recombinant NS3
could only be studied at a concentration of 0.1 µg/ml, since higher
concentrations of NS3 (1 µg/ml) were toxic to the cells. In contrast,
very little IL-4 production was observed after genetic immunization
with genes encoding for the HCV nonstructural proteins (Fig. 2e).
Because CTL responses are essential to eliminate virus from infected
cells, we studied the ability of splenocytes derived from immunized
mice to lyse syngenic SP2/0 murine myeloma target cells stably
expressing NS3 and NS5 proteins in a 51Cr release assay.
The NS3- and NS5-immunized mice exhibited a specific cytotoxic T cell
response after 5 days of in vitro stimulation, whereas low activity was
observed against SP2/0 or SP2/19 (stably expressing HCV core
protein) cells used as controls for target cell specificity (Fig. 3, a and b). To
demonstrate the phenotype of cells producing the specific lysis,
splenocytes were incubated with CD8+- or
CD4+-specific mAbs in the presence of complement. These
studies revealed that the cytotoxic activity was mediated by
CD8+ cells (Fig. 3c). We were unable to
establish SP2/0 cell lines stably expressing NS4 protein; therefore,
CTL activity was not measured against this HCV nonstructural protein.
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, a
and b). Indeed, 90 to 100% of mice immunized with mock DNA
or challenged with SP2/9 cells demonstrated tumor formation, confirming
the specificity of the CTL activity in this tumor model. It is
important to emphasize that immunization with recombinant NS5 protein
in CFA did not protect animals against tumor formation. To assess the
effect of a combination of DNA-based immunization and recombinant
protein vaccination, one group of animals was immunized with both.
There was partial but significant (p < 0.03)
protection against tumor formation, but combined immunization was not
as effective as immunization three times with a DNA construct encoding
for NS5 protein alone (Fig. 4a).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is not known whether the nonstructural proteins NS3, NS4, and NS5 are sufficiently immunogenic to generate broad-based and vigorous CTL responses in vivo. The genetic immunization approach was employed to test this hypothesis, since this technique has been shown previously to induce cellular immune responses of different levels against a variety of pathogens in animal model systems (9, 10, 11, 18, 19). The advantage of this method compared with immunizations with soluble recombinant proteins or peptides is its ability to induce a more Th1-like immune response with the production of inflammatory CD4+ T cell as well as cytotoxic T cell activity, presumably due to the intracellular processing of viral proteins into peptides and subsequent loading onto MHC class I molecules in transfected muscle cells as well as to yet to be defined interactions of the complex with APCs. In contrast, immunization with soluble protein primarily leads to a humoral immune response due to processing through the MHC class II pathway. Immunization with synthetic peptides has several disadvantages, since only a limited number of epitopes are available for stimulation of the host immune response. In contrast, all naturally occurring B and T cell epitopes encoded for each protein by the DNA construct of interest are presumably preserved for recognition by TCRs and consequently will generate very broad-based humoral and cellular immune responses (20).
During active viral replication, HCV has a very high mutation rate, and
several genotypes and subtypes have been described previously (13, 14).
In this regard, the Ags are processed intracellularly in infected
hepatocytes, and a large number of epitopes are presented to the immune
system. However, neutralizing Abs generated against the envelope region
of HCV have been found to be insufficient to provide protection and
tend to promote immunoselection of quasispecies (21). In this study, we
present evidence that DNA-based vaccination with plasmids encoding for
three different nonstructural proteins of HCV is capable of eliciting
Ag-specific immune responses in both effector pathways of the immune
system. It was noteworthy that all animals developed detectable Ab
responses after three immunizations. In this regard, these
nonstructural proteins are far better Ags to stimulate humoral immune
responses compared with previous studies by us using the HCV core
structural protein (22, 23). Similar to the findings of HCV core, the
humoral immune response to the NS3 protein was weak; therefore, it may
be necessary to activate APCs by the coadministration of
cytokine-expressing plasmids such as IL-2 and granulocyte macrophage
CSF to achieve optimal humoral and cellular immune responses, (23, 24).
Nevertheless, the generation of inflammatory CD4+ T cell
responses with a predominant Th1 phenotype was demonstrated for all
three plasmids encoding for NS3, NS4, and NS5. Most important, a
specific CD8+ CTL response was generated for NS3 and NS5
with lysis values that have been shown previously to induce protection
against a variety of pathogens in animal model systems (18, 19). It was
not possible to measure CTL responses to NS4, since we were unable to
establish stable NS4-expressing SP2/0 myeloma cell lines. However, CD4
T cell responses and IL-2 and IFN- release were in the range
observed for NS3 and NS5, and NS4 appears to be an attractive candidate
protein for this immunization approach as well. Since no small animal
model is currently available for HCV infection, we determined whether
the CTL responses generated by DNA-based immunization would protect
animals against tumor formation using syngenic SP2/0 tumor cells stably
transfected with a cDNA encoding for NS5 protein. Approximately 60% of
mice were protected against tumor formation, indicating the in vivo CTL
activity produced by this immunization approach. Moreover, tumor weight
in those animals that developed tumors was significantly reduced
compared with mice immunized with mock DNA or recombinant NS5 protein.
This study emphasizes the capability of assessing cellular immune
responses against HCV nonstructural proteins in an animal model as
measured by inhibition of tumor growth. It should now be possible to
determine the fine specificity of CTL epitopes with overlapping
peptides using these techniques.
In contrast to the data presented here, DNA immunization using a construct encoding for the HCV core structural protein produced less vigorous cellular and humoral immune responses (22, 23, 25). The envelope region has great sequence diversity among the various genotypes and may not be a good target region because of immunoselection of viral variants known to occur during natural viral infection (22, 25, 26). The NS3 gene encodes for a serine protease that cleaves the viral polyprotein precursor posttranslationally at several junctions and also serves as the viral helicase. The NS5 region encodes for the RNA-dependent RNA polymerase of the virus. Both genomic regions are believed to be highly important and critical for viral replication; therefore, these regions may serve as important molecular targets for antiviral approaches (27, 28, 29). Based on both previous clinical studies, which demonstrate the importance of the cellular immune response to the nonstructural proteins with respect to preventing persistent viral infection in humans (6, 7), and the experimental results presented here, which demonstrate that the nonstructural proteins are particularly potent candidates in generating cellular immune responses in mice, we are led to believe that DNA-based immunization with genes encoding for the HCV nonstructural proteins is an attractive approach for the construction of therapeutic and prophylactic vaccines against HCV. However, the clinical efficacy of DNA-based immunization in generating antiviral immune responses against HCV in humans remains to be established. Finally, it will be important in the future to determine whether different genotypes or subtypes of HCV may circumvent the immune responses induced by one genotype following DNA-based immunization.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jack. R. Wands, Molecular Hepatology Laboratory, MGH Cancer Center, Harvard Medical School, 149, 13th Street, Charlestown, MA 02129. E-mail address:
3 Abbreviations used in this paper: HCV, hepatitis C virus; HBsAg, hepatitis B virus surface Ag; ORF, open reading frame.
Received for publication May 1, 1998. Accepted for publication June 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-2b trials. Hepatology 26:83S.[Medline]
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