Virology 334 (2005) 255 – 263 www.elsevier.com/locate/yviro
Endoplasmic reticulum targeting sequence enhances HBV-specific cytotoxic T lymphocytes induced by a CTL epitope-based DNA vaccine Wei Xu, Yiwei Chu, Ruihua Zhang, Huanbin Xu, Ying Wang, Sidong XiongT Department of Immunology, Shanghai Medical College of Fudan University; E-Institutes of Shanghai Universities, Immunology Division, Shanghai, PR China Key Laboratory of Molecular Medicine of Ministry of Education, Shanghai Medical College of Fudan University; E-Institutes of Shanghai Universities, Immunology Division, Shanghai, PR China Received 23 November 2004; returned to author for revision 20 December 2004; accepted 28 January 2005
Abstract CD8+ T cells play a critical role in protective immunity against Hepatitis B Virus (HBV). Epitope-based DNA vaccines expressing HBVdominant CTL epitopes can be used as candidate vaccines capable of inducing cytotoxic T Lymphocytes (CTL) responses. A plasmid DNA encoding a CTL epitope of HBV core antigen, HBc18–27, was constructed. Intramuscular immunization of C57BL/6 mice with this DNA vaccine resulted in successful induction of HBV-specific CTL responses. In order to promote transportation of the peptide into endoplasmic reticulum (ER) to bind to MHC class I molecules for optimal class I antigen presentation, an ER targeting sequence (ERTS) was fused with the C18–27 encoding gene. ERTS fusion significantly enhanced specific CD8+ T cell responses in terms of CTL cytolysis as well as IFN-g secretion. This enhancement was correlated with promoted epitope presentation on target cell surface. We report here an enhanced immunogenicity of an epitope-based DNA vaccine using an ER targeting signal sequence, which has significant implications for future design of therapeutic HBV vaccine. D 2005 Elsevier Inc. All rights reserved. Keywords: Epitope; DNA vaccine; Hepatitis B virus; CTL; Endoplasmic reticulum targeting sequence
Introduction Delivery of plasmid DNA into living animals leads to expression of the protein of interest in vivo and effective induction of both humoral and cellular immunity to the expressed protein. Especially the ability of DNA vaccine to induce specific CTL responses makes it a promising candidate to confer protection against viral infections and tumor challenges (Nagata et al., 2004). Most currently available vaccines including DNA vaccines are based on dnaturalT forms of various pathogens or selected antigenic components of the pathogen. Although * Corresponding author. Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, 200032, PR China. Fax: +86 21 54237749. E-mail address:
[email protected] (S. Xiong). 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.01.040
effective against some pathogens, they have thus far been unable to deal with most types of tumors and chronic viralinfectious diseases, such as caused by HIV and HBV. One of the possible reasons may lie in that whole protein antigen is composed of various epitopes, and that dominant epitopes usually determine the immunogenicity of the whole protein. Therefore, antigenic variation of a specific epitope can result in immune escape (Chen and Oon, 1999; Klenerman and Zinkernagel, 1998; Rimmelzwaan et al., 2004). While irrelevant or suppressive epitopes within the same protein could interfere with the function of dominant epitopes, as a result making whole protein vaccine generate unwanted responses such as antibody-mediated enhancement of viral diffusion (Takada and Kawaoka, 2003). Meanwhile, CTL responses to a dominant epitope can recognize potential epitope variants which are absolutely requested for vaccines design against highly-mutating viruses (Charini et al.,
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2001). Therefore, identification of dominant CTL epitopes within a protein and development of CTL epitope-based vaccines could be promising to counteract chronic viral diseases (Goulder et al., 2000; Zhong et al., 2003). Single or multi-epitopes-based DNA vaccines have been successfully developed since 1996 resulting in efficient induction of epitope-specific cytotoxic T lymphocytes (CTLs) against various pathogens (Ciernik et al., 1996; Lindinger et al., 2003; Nakamura et al., 2003; Subbramanian et al., 2003). However, their capabilities to elicit potent immune responses need to be improved, since the immunogenicity of an epitope is usually poor. Chronic viral hepatitis caused by Hepatitis B virus (HBV) infection remains a significant world health problem. HBV-specific CTLs play a crucial role in the control of HBV infection, through direct lysis of virus-infected cells and release of cytokines at the site of infection (Bertoletti et al., 1997; Guidotti et al., 1999). Most importantly, effective HBV-specific CD8+ T cell responses proved to inhibit virus replication independent of liver damage (Maini et al., 2000a, 2000b) while inadequate CTL responses and nonantigenspecific T cells infiltration might contribute to liver pathology (Bertoletti and Maini, 2000). Therefore, it is vital to stimulate or boost HBV-specific CTLs for design of prophylactic or therapeutic HBV vaccines. And the relative contribution of virus-specific CTLs to viral control may be significantly determined by their epitope specificity (Webster and Bertoletti, 2001). When strong HBV core antigen (HBc)-specific CTLs could be always detected lysing infected hepatocytes during the recovery period of acute HBV infection, their reduction or even absence in chronic infection indicates an important correlation of protection with HBc antigen. Therefore, HBc protein-based DNA vaccines have been largely evaluated its ability to induce specific CTL responses (Bo¨cher et al., 2001; Kwissa et al., 2000; Thermet et al., 2004). Moreover, mutant viruses may evade immune system due to the loss of a CTL epitope within the HBc gene indicating a dominant CTL epitope could determine the immunogenicity of the HBc antigen (Lee et al., 2001). C18–27(FLPSDFFPSV), an HBcderived CTL epitope, represents so far the most dominant CTL epitope within the HBV genome (Maini et al., 2000a, 2000b). ~N90% of HLA-A2-positive patients with acute HBV infection produce a CTL response against HBc18–27, which is not detectable in HLA-A2-positive patients with chronic hepatitis, suggesting that a vigorous CTL response to this epitope may contribute to viral clearance during HBV infection (Missale et al., 1993). HBc18–27 epitope can bind to and prime different HLA-A2 subtypes (Maini et al., 2000a, 2000b; Missale et al., 1993) and it could activate HLA class II restricted HBc-specific T-cell response which plays a central role in anti-HBV immunity (Bertoletti et al., 1997). Based on these premises, C18–27 was selected as the target gene of our epitope-based DNA vaccine; we reasoned that focusing CTL responses to this dominant epitope would be very helpful for HBV clearance.
Since CTL induction requires endogenous peptides loaded onto major histocompatibility complex (MHC) class I molecules on surfaces of antigen-presenting cells (APCs) and target cells, peptide transport into the endoplasmic reticulum (ER) is therefore very critical where it is complexed with newly synthesized MHC class I molecules (Moron et al., 2004). To target CTL epitopes into the ER, the C18–27 coding gene was fused with an ER targeting signal sequence (ERTS) derived from the Adenovirus E3 leader sequence which has been demonstrated to facilitate epitope transport into ER and therefore enhance specific immunity induction (Anderson et al., 1991; Ciernik et al., 1996; Restifo et al., 1995). Here, we report an epitope-based DNA vaccine targeted against the HBc18–27 peptide and demonstrate its efficacy in inducing specific CTL responses against HBV in an H-2b mouse model. Furthermore, an ER targeting signal sequence (ERTS) was inserted upstream the C18–27 sequence in order to promote CTL induction by enhancing intracellular antigen presentation efficiency.
Results Construction of pECK-C18 and pECK-E3-C18 A modified plasmid expression vector containing hCMV IE1 promoter, BGH polyA signal sequence was constructed as pECK. Oligonucleotides coding for a CTL epitope, HBc18–27, were synthesized and cloned into pECK, yielding plasmid pECK-C18 (Fig. 1A). Fusion of ER targeting sequence derived from adenovirus E3 leader sequence in frame with C18–27 gene generated plasmid pEC K -E3-C18 (Fig. 1B). Epitope expression in vitro In vitro transfection efficiency of pECK-C18 was evaluated in C2C12 skeletal muscle cells. FuGENETM 6–DNA complex was used to transfect C2C12 cells. Peptide expression in the culture supernatant was analyzed by Dot Immunoblot Assay. C18–27 peptide expression reached a maximum around 24 h after transfection, then decreased slightly, still had 50% of the maximum expression at 72 h (Fig 2). In vitro expression of pECK-C18 showed that transfectants secreted transgenic peptides at 24, 48 and 72 h posttransfection, implying that C18–27 epitope cloned in pECK was correctly transcripted, translated, and secreted. Fusion of ER targeting sequence with C18–27 encoding gene hardly changed the expression kinetics of C18–27 epitope. Induction of C18–27-specific CTL responses by C18–27-based DNA vaccine To demonstrate whether the C18–27 CTL epitope cloned in pECK vector was immunogenic, 6- to 8-week-old
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Fig. 1. Schematic presentation of pECK-C18 and pECK-ERTS-C18 plasmid.
C57BL/6 female mice were inoculated intramuscularly with two doses of 100 Ag pECK-C18 DNA. Mice inoculated with empty pECK DNA were used as control. 10 days after the last immunization, splenocytes were isolated and expanded as CTLs by in vitro stimulation with C18–27 peptides-pulsed syngeneic feeder cells for 6 days. They were then tested for cytolytic activity against 51Cr labeled EL4 cells prepulsed with or without C18–27 peptide. Under these test conditions, only peptide-pulsed EL4 cells were lysed; whereas no or low lysis was detectable using EL4 or HBs208–215 control peptide-pulsed EL4 as target cells indicating CTLs were successfully generated and were C18–27 specific. The cytolysis of non-restimulated splenocytes was very low, only 2.83% and 0.4% for pECK-C18 and pECK-immunized mice at E/T ratio 10/1 (figure not shown). Cytotoxic assay showed that intramuscular delivery of pECK-C18 into C57BL/6 mice resulted in efficient induction of CTLs specific against HBV core antigen. The specific cytolysis amounted to 16.21% at E/T ratio 10:1 compared to 2.48% for control mice (Fig. 3).
Fig. 2. In vitro C18–27 peptide expression after DNA transfection. 2 Ag DNA complexed with FuGENE6 were added to C2C12 cells, and synthesized C18–27 peptide was used as positive control. Expressed peptides in the supernatant were detected by DIA 24, 48, and 72 h posttransfection. (A) pECK-E3-C18 transfected C2C12 cell supernatant; (B) pECK-C18 transfected C2C12 cell supernatant; (C) pECK transfected C2C12 cell supernatant; (D) C18–27 peptide.
C18–27-specific CTL responses are enhanced by upstream fusion of ERTS with C18–27 To further investigate whether ER targeting signal sequence (ERTS) fusion could effectively enhance the immunogenicity of C18–27 encoding gene, two doses of 100 Ag pECK-E3-C18 DNA were intramuscularly administrated to C57BL/6 mice. C18–27-specific CTL responses in mice immunized with DNA construct harboring ERTS-C18–27 fusion gene were significantly higher than that in mice treated with DNA containing C18–27 gene only ( P b 0.05). Specific cytolysis of CTLs derived from pECK-E3-C18-, pECK-C18-, and pECK-immunized mice were 24.06%, 17.20%, and 0.048%, respectively at E/T ratio 10:1 (Fig. 4). Enhancement of CTL epitope immunogenicity is correlated with increased amounts of presented peptides If the variation of the immunogenicity of C18–27 epitope associated with or without ERTS was the result of different efficiencies of the CTL epitope binding to MHC I molecule
Fig. 3. Immunogenicity of C18–27 epitope-based DNA vaccine in C57BL/6 mice. C57BL/6 mice were intramuscularly immunized with two doses of pECK (circles) or pECK-C18 (squares) DNA with 3 weeks intervals. 10 days after the last immunization, splenocytes were isolated and stimulated with C18–27-pulsed syngeneic feeder cells in vitro for 6 days and used as effector cells. Cytotoxicity was assayed in a 51Cr release assay against EL4 target cells in the presence (filled symbols) or absence (open symbols) of C18–27 peptide. *P b 0.05.
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Fig. 4. HBc-specific CTLs induced by pEC -E3-C18 DNA vaccine. Splenocytes from immunized mice were stimulated with C18–27-pulsed syngeneic feeder cells for 6 days and tested for cytotoxic activity in a 4-h 51 Cr release assay against allogeneic EL4 targets prepulsed with (filled symbols) the same peptide or without (open symbols). The E/T ratio used was 80:1 to 10:1. Representative CTL responses induced by pECK (triangle), pECK-C18 (circles) and pECK-E3-C18 (squares) DNA immunization were indicated. *P b 0.05.
in ER, then differences in levels of epitope presentation should be detectable in cells transfected with different DNA constructs. To test this idea, EL4 cells were transfected with empty vectors, DNA vectors expressing either C18–27 or ERTS-C18–27 fusion protein for 24 h and served as stimulators. Splenocytes from pECK-E3-C18-, pECK-C18-, or pECK-immunized mice were restimulated in vitro with C18–27 peptides and used as responders. Specific proliferation assays were conducted showing that SI of splenocytes stimulated with pECK-E3-C18-pretransfected EL4 was significantly greater than that of splenocytes stimulated with pECK-C18-pretransfected EL4 ( P = 0.0024 and P = 0.0222 for pECK-C18 and pECK-E3-C18 immunization group, respectively), indicating that fusion of ERTS increased antigen presentation levels effectively (Fig. 5). This result was paralleled with the elevated CTL responses induced by pECK-E3-C18 vaccine indicating that an ER targeting sequence at the amino-terminus of a CTL epitope could enhance the immunogenicity of the epitope through elevating epitope presentation efficiency. It is unlikely that this difference in C18–27 presentation is attributed to differences in gene expression among stimulator cells, since C18–27 peptide expression in EL4 cells 24 h after DNA transfection showed no significant difference whether fused or not with ERTS (Fig. 2).
Fig. 5. T cell proliferative responses to C18–27 peptide presented on surfaces of target cells. Splenocytes stimulated with C18–27-pulsed syngeneic feeder cells in vitro for 6 days were incubated with mitomycin-treated EL4 cells (5), mitomycin-treated EL4 cells transfected the day before with pECKC18 (q) or pECK-E3-C18 (n) DNA. ConA stimulation ( ) was used as a positive control. Each bar represents the average values of 3 mice, measured in duplicate. * a: P = 0.0024 compared to pECK-C18 transfected EL4 stimulation of pECK-C18 immunized mice; * b: P = 0.0222 compared to pECK-C18 transfected EL4 stimulation of pECK-E3-C18-immunized mice.
DNA constructs were tested for their ability to secrete IFN-g into the culture supernatant. It was found that restimulated T cells derived from pECK-E3-C18-immunized mice produced at least 2 times higher levels of IFN-g than those derived from pECK-C18-treated mice (Fig. 6), confirming that ERTS fusion at the amino terminus of the CTL epitope could largely increase epitope-specific CD8+ T responses induced by our CTL epitope-based DNA vaccine.
Discussion By virtue of the sustained in vivo antigen synthesis and the comprised stimulatory CpG motifs, DNA vaccine is known for induction of a full range of immune responses, including neutralizing antibodies and CTLs, resulting in anti-pathogen protection. Substitution of whole protein encoding gene with specific epitope encoding sequence is an alternative way to improve immunological focusing and
ERTS contributed to increased IFN-c production by restimulated T cells Since HBc18–27-based DNA vaccine was demonstrated effective to induce strong C18–27-specific CTL responses, specific Th1 responses generated by this DNA vaccine were also evaluated in an IFN-g secretion assay. In vitro restimulated splenocytes from mice immunized with various
Fig. 6. IFN-g production of in vitro restimulated splenocytes. Splenocytes derived from pECK- (5), pECK-C18 (q) or pECK-E3-C18 (n) immunized mice were restimulated in vitro with C18–27-pulsed syngeneic feeder cells for 96 h before the culture supernatants were collected and tested for IFN-g levels by ELISA Assay. *P b 0.05.
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reduce unwanted responses. Epitope-based approach brings vaccines with increased safety, the opportunity to rationally engineer epitopes for increased potency and breadth, and the ability to focus immune responses on conserved epitopes (Sette and Fikes, 2003). The ability of CD8+ T cells to directly recognize virusinfected cells and activate anti-viral pathways in the infected hepatocytes through secretion of cytokines makes these cells the obvious candidate for expansion through a vaccine therapy for HBV infection (Webster and Bertoletti, 2001). CTL responses also play an important role in limiting virus replication without causing inflammatory disease (Bertoletti et al., 2003). Therefore, induction of CTL responses specific against HBV represents a promising strategy to protect against HBV infection. However, current widely used HBV vaccines for humans, whole-killed, subunit vaccines, or recombinant protein, are processed solely as exogenous antigens, and often result in poor induction of cell-mediated immunity (Davis and McCluskie, 1999), therefore limiting their use in therapeutic applications. Synthetic polypeptide vaccine owns poor immunogenicity and high cost of manufacturing. Moreover, adjuvant as Alum (currently licensed for human use) for protein-based vaccine has a strong Th2 bias that dramatically abrogates CTL responses (Petrovsky and Aguilar, 2004). Therefore, a CTL epitopebased DNA vaccine seems to have many advantages over traditional protein-based vaccines with regard to CTLs induction. It has become a promising approach for prophylaxis and therapy of hepatitis B (Thermet et al., 2003). Here, we reported the intramuscular immunization of C57BL/6 mice with a CTL epitope-based DNA vaccine carrying a minigene coding for a single CTL epitope, C18–27, derived from HBV core protein and demonstrated its efficacy to induce HBV-specific CTLs. We reasoned that this highly conserved CTL epitope, rather than the whole protein, is more rational for CTL induction. However, the specific cytolysis of CTLs induced with DNA construct with C18–27 gene only is not very high in our study. One reason is that the immunogenicity of an epitope lacking necessary flanking residues is usually poor, even in the form of DNA vaccine. The other reason might be that C18–27 was discovered as an HLA-A2-restricted peptide, not an H-2b-restricted peptide. Since peptide binding with an appropriate MHC molecule could influence the immunodominance of the peptide (Takada and Kawaoka, 2003), the species-specificity of the peptide binding to MHC molecules has become a challenge in the development of epitope-based vaccines (Moron et al., 2004). So we compared the amino acid sequence of C18–27 with that of H-2Db/H-2Kb binding CTL epitopes reported to date, natural, or predicted (Engelhard, 1994; Kuhober et al., 1996; Schirmbeck et al., 1998; Zhong et al., 2003). The carboxyl-terminal peptide residue is an important determinant of peptide-MHC I molecule binding. Most H-2Db or H-2Kb molecules favor aliphatic residues like I, L, sometimes V (Zhong et al., 2003) as the carboxyl-terminal peptide residue (P8/9/10). P2
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residues are also important for the binding; G, L and S are most often seen at this position. Furthermore, amino acid at position 5/6 (P5/6) is highly conserved and acts as a strong determinant of peptide-MHC I binding. H-2Kb molecule prefers CTL epitopes with F or Y residue at P5. Therefore, C18–27 epitope (FLPSDFFPSV) possesses L at P2, F at P6 and V at P10 which makes it a potential H-2b-binding epitope. HBc92–100 peptide (NMGLKFRQL) and HBs208–215 peptide (ILSPFLPL) were reported as both immunodominant H-2Kb-restricted epitopes (Kuhober et al., 1996; Schirmbeck et al., 1998). The former possesses F and L at P6 and P9, the latter has L at P2, F at P5, and L at P8. Both of them hold similar amino acid compositions as HBc18–27 does. Finally, it was experimentally demonstrated by us that it bound to murine (H-2b) MHC class I molecules to a sufficient level to induce HBc-specific T cell responses in C57BL/6 mice (H-2b). Therefore, poor immunogenicity of the HBc18–17 epitope is the reason for low CTLs induction by our C18–27 epitope-based DNA vaccine. In order to increase the potency of single epitope-based vaccine, a variety of methods have been described effective to enhance anti-virus or anti-tumor immunity, including the selection of the most immunodominant epitope (Melief et al., 2002), the use of long peptides (Zwaveling et al., 2002), the combination of CTL epitope with an Th epitope (Baird et al., 2004) or with co-stimulatory molecules (Shankar et al., 2001) and use of various adjuvants (Andersen et al., 2000). A complementary approach entails the direct delivery of vaccine into intracellular compartments associated with MHC class I or class II molecules, as a means to increase epitope uptake as well as epitope presentation in APCs or the target cells (Takada and Kawaoka, 2003). Special targeting genes or leader signal sequences derived from viral genomes have been largely employed to this aim by mimicking the highly efficient infection process of viruses. HIV tat antigen helped delivery of antigen into lysosome (Chikh et al., 2001), and Influenza virus HAderived amphipathic polypeptides have been used to direct cytosolic localization of the antigen which is necessary for CTL induction (Plank et al., 1994). Targeting antigens into the MHC class I-restricted presentation pathway is a prerequisite for the induction of CTL responses, there are two distinct routes by which a CTL peptide gains access to MHC class I molecules (Gromme and Neefjes, 2002). The first involves proteasome degradation and transportation via TAP into ER; and the second invokes a proteasome- and TAP-independent ERtargeting pathway (Leifert et al., 2004). Therefore, targeting peptides into ER might be a promising means to increase CTLs induction by the epitope-based DNA vaccine. Several studies have shown that specific signal sequences at the N-terminus of a protein could facilitate its entry into ER in a TAP-independent manner and its eventual presentation on the cell surface, where it can be recognized by CD8+ T cells (Anderson et al., 1991; Bacik et al., 1994; Rice et al., 1999). In particular, an
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ER-targeting sequence derived from the adenovirus E3 leader sequence was demonstrated to enhance CTL responses induced by a DNA vaccine harboring mutant p53 or HIV gp120 encoding gene (Ciernik et al., 1996). In our study, a similar ER-targeting signal sequence was fused with the HBc18–27 epitope encoding gene, and an enhancement of CTL induction due to promotion of epitope presentation was again validated. Besides, a Cterminal KDEL sequence derived from some toxins, also called ER retrieval signal, could efficiently target exogenous peptides into ER lumen therefore enhance T cell immune responses (Lee et al., 1998). Other strategies proposed to enhance delivery of CTL epitopes into the MHC I antigen presentation way include orienting protein delivery towards the proteasome (Rodriguez et al., 1998), promoting translational ubiquitination of the antigen (Wu and Kipps, 1997), and the use of other functional protein domains capable to enhance the class I epitope presentation. A GMI-binding B subunit of Escherichia coli heat-liable toxin (EtxB) was reported to improve the class I epitope presentation when conjugated to either of the two immunodominant MHC I epitopes (De Haan et al., 2002). All these strategies to direct CTL epitopes into class I antigen processing and presentation way resulted in successful enhancement of CTLs induction or Th1 immune responses as has also been demonstrated by our study. In our study, the enhanced induction of CTLs could be probably ascribed to the promotion of epitope presentation on surface of target cells or APC by the ERTS which was fused to the CTL peptide, since pECK-E3-C18 transfected EL4 cells stimulated more expanded T cells to proliferate than pECK-C18 transfected EL4 cells did (Fig. 5). These data also support the belief that the immunogenicity of a CTL epitope in DNA vaccine can be optimized by improving the level of epitope presentation (Livingston et al., 2001). Other possible strategies to increase epitope presentation include viral vector usage and modulation of flanking residue of specific CTL epitopes (Moron et al., 2004). Interferons are the most effective available drugs for treatment of chronic HBV infection (Lee et al., 2001). It has been demonstrated that successful CTL responses use a noncytopathic mechanism of viral clearance to completely abolish HBV replication mainly by secreting cytokines IFN-g and TNF-a (Bertoletti and Maini, 2000). These two cytokines are able to selectively degrade replicating genomes of HBV. To evaluate the potential of CTLs to produce IFN-g, in vitro expanded CTLs after 96 h of specific stimulation were tested for their ability to secrete IFN-g in the culture supernatant. As expected, IFN-g secretion was largely increased in mice immunized with DNA harboring C18–27 gene compared with the vector control and reached the highest level in mice treated with ERTS-C18–27 fusion gene vaccine. These results indicate that the CTL epitope-based DNA vaccine we constructed not only induces successful HBc-specific
CD8+ T cell responses, but also has a therapeutic potential. In this study, we have devised a genetic epitope vaccine vector that allows the efficient expression of the HBcderived C18–27 CTL epitope, resulting in the successful induction of HBV-specific cellular immunity, which was further improved by an N-terminal fused ER-targeting sequence. Our CTL epitope-based DNA vaccine may thus have a great potential as a future prophylactic or even therapeutic HBV vaccine.
Materials and methods Animals Female C57BL/6 mice, 6–8 weeks old, were obtained from Laboratory Animal Centers (Fudan University, Shanghai). The mice were housed under specific pathogen-free conditions. Treatment of the animals conformed to the ARVO Statement on the Use of Animals in vaccine research. Plasmids and primers pECK plasmid DNA vector was derived from pUC19 plasmid (Invitrogen) and modified by adding HCMV IE1 promoter/enhancer complex and BGH polyA sequence. Oligonucleotide GATCT GCCAC CATGT TTTTG CCTTC TGACT TCTTT CCTTC TGTTT AGTAA coding for the aa 18–27 of HBc antigen, with additional KOZAK, ATG and double stop codons, and its antisense analogue were synthesized, annealed and inserted into the BglII site of pECK vector thus generating pECK-C18 plasmid. To construct the inserts for pECK-E3-C18, the following oligonucleotides were annealed, extended with Klenow and dNTPs, and amplified by PCR: 5V-GAAGA TCTGC CACCA TGAGG TACAT GATTT TAGGC TTGCT CGCCC TTGCG GCAGT CTGCA GCGCT GCCTT TTTGC CTTCT GACTT C-3V and 3V-CGGAA GACTG AAG AA AGGAA GACAAA TCATT CTAGA AG-5V. The resulting oligonucleotide duplexes encoding for C18–27 peptide preceded by ER targeting sequence/ERTS (adenovirus E3 leader sequence) were digested with BglII and cloned into the unique site BglII of pECK, resulting in pECK-E3-C18 plasmid. All inserts were sequenced after construction. Endotoxin-free plasmids were obtained using Endofree plasmid mega kit (Qiagen, Germany). Cells, peptides and antibodies C3H C2C12 skeletal muscle cell is a gift from Prof. Maurizio Zanetti (UCSD, USA). C57BL/6 EL4 (H-2b) thymic lymphoma cells were obtained from ATCC. HBc18~27 peptide with amino acid sequence FLPSDEFPSV was synthesized (Sbsbio Co Ltd., P.R. China).
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Epitope expression in vitro 5 105 C2C12 cells were transfected with 2 Ag of plasmid DNA in 6-well plate at 37 8C and 5% CO2 in RPMI 1640 containing non-serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin for 48 h, followed by changing the medium to fresh complete medium (1640 containing 10% FBS). Cells were cultured for 3 days before assay. Transfection of pECK, pECK-C18 and pECK-E3-C18 using FuGENE 6 (Boehringer Mannheim) was carried out to the manufacturer’s protocol. C18–27 synthesized peptide was used as a positive control. Peptide detection by dot immunoblot assay Peptide detection was carried out using a dot immunoblot assay according to standard protocols (Loi et al., 1997). 20 Al of cell culture supernatants were spotted onto a 0.22-Am nitrocellulose membrane (Amersham). The membrane was air dried at room temperature and fixed at 37 8C for 60 min. After washing, it was incubated for 60 min in a blocking solution containing 0.3% bovine serum albumin (BSA) in 0.1 M Tris buffer (pH 7.4). Then, it was incubated overnight with rabbit-anti-HBc polyclonal antibody (Dako, USA), followed by a biotinylated secondary antibody (VECTASTAIN ABC Reagents, USA) for 30 min. The membrane was then transferred to a solution of streptavidin peroxidase for 30 min and revealed by DAB substrate. The spot was taken as photographs, scanned into computer, and adjusted for equal brightness and contrast using PhotoShop. DNA intramuscular immunization For DNA immunization, groups of 6–8-week-old C57BL/6 (n=6) mice were pretreated by injecting 100 Al of 0.25% bupavokein (Sigma, St. Louis, MO) into the tibialis anterior muscle. After 24 h, the same muscle was injected with 100 Ag plasmid DNA diluted in phosphate buffer saline (PBS) and boosted with 100 Ag DNA 3 weeks later. For CTL assays, mice were sacrificed 10 days after the second immunization.
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final concentration of 20 U/ml was changed at 2- to 3-day intervals. For in vitro restimulation, splenocytes from C57BL/6 mice stimulated with an irrelevant Kb-restricted control peptide derived from HBsAg208–215 (ILSPFLPL) did not generate cytolytic responses above background level. CTL assay A standard chromium release assay was used to monitor CTL activity. Briefly, 5 106 EL4 (H-2b) cells were labeled with 7.4 106 Bq 51Cr (Amersham, USA) for 1.75 h then pulsed with 20 Ag/ml C18–27 peptide for 2 h at 37 8C and used as target cells. They were mixed at appropriate ratios with restimulated effectors in duplicate and incubated at 37 8C for 4 h. Supernatants were harvested and radioactivity was counted in a gamma counter (LKB, Gaithersburg, MD). Each animal was analyzed independently. Percent-specific lysis was calculated using the equation: 100 [(experimental release spontaneous release) / (maximum release spontaneous release)]. Results are presented as peptide-specific lysis, calculated by subtracting the percent specific lysis of control EL4 target cells from the percent specific lysis of the peptide-pulsed EL4 target cells at the same E/T ratio. Spontaneous release was determined by incubating labeled target cells with RPMI 1640 and ranged between 10 and 15% in various experiments. Maximum release was determined by lysing the target cells with 5% SDS. Lymphocyte proliferation In vitro stimulated CTLs (6 days after C18–27-pulsed syngeneic feeder cells stimulation) were titrated into 96well round-bottomed plates (Costar) at a concentration of 5 106/ml as responder T cells, 1.5 106/ml pECK-E3C18-, pECK-C18- or non-transfected (24 h) EL4 cells were inactivated and used as stimulators. The cultures were incubated for 72 h at 37 8C in 5% CO2, pulsed with 3.7 104 Bq [3H]-thymidine (Amersham, USA) per well during the last 18 h of incubation, and then were harvested. [3H]-thymidine uptake was determined by standard liquid scintillation.
CTLs expansion in vitro IFN-c measurements CTLs were prepared from spleens of DNA immunized C57BL/6 and used either directly or after 6 days restimulation in vitro in CTL assay. Splenocytes were resuspended in RPMI 1640 medium containing 10% FBS, antibiotics and l-glutamine. Feeder cells were syngeneic spleen cells depleted of erythrocytes, subjected to 20 Gy of irradiation, resuspended in RPMI 1640 containing 20 Ag/ml C18–27 peptide, incubated for 2 h at 37 8C, washed, and resuspended in RPMI 1640 at a concentration of 1 106/ml. CTLs were obtained by culturing 5 106 immune splenocytes with 1 106 feeder cells in flat-bottom 24-well tissue culture plates for 6 days, where the medium supplied with IL-2 to a
IFN-g was determined by enzyme linked immunosorbent assay (ELISA) according to the manufacturerTs directions (Pharmingen). Briefly, ELISA plates (Costar) were coated with R4-6A2, a rat monoclonal antibody specific for murine IFN-g (Pharmingen) overnight at 4 8C, and then blocked for 2 h at room temperature with 10% fetal bovine serum (FBS) in PBS. Culture supernatants of splenic cells which had been restimulated with C18–27 peptide-pulsed feeder cells for 96 h (mentioned above) were added to the plate. After washing, the second biotinylated monoclonal antibody specific for murine IFN-g (Pharmingen) was added. HRP-
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coupled strepavidin and TMB were used to detect biotinylated antibody. The absorbance is read at 450 nm using ELX800 ELISA plate reader (Biotek, USA). Statistical analysis Data are shown as means and standard deviations; differences were analyzed by Student’s t test and a probability of less than 0.05 was taken as significant.
Acknowledgments This work was funded in part by National Natural Science foundation (30400396), National High Technology Research and Development Program of China (2004AA215242), Major State Basic Research Development Program of China (2001CB510005) and China outstanding youth funds (39925031) also E-Institutes of Shanghai Universities, Immunology Division funds.
References Anderson, K., Cresswell, P., Gammon, M., Hermes, J., Williamson, A., Zweerink, H., 1991. Endogenously synthesized peptide with an endoplasmic reticulum signal sequence sensitizes antigen processing mutant cells to class I-restricted cell-mediated lysis. J. Exp. Med. 174, 489 – 492. Andersen, M.H., Pedersen, L., Becker, J.C., Straten, P.T., 2000. Identification of a cytotoxic T lymphocyte response to the apoptosis inhibitor protein surviving in cancer patients. Cancer Res. 61, 869 – 872. Bacik, I., Cox, J.H., Anderson, R., Yewdell, J.W., Bennink, J.R., 1994. TAP (transporter associated with antigen processing)-independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxylterminus of the peptide. J. Immunol. 152, 381 – 387. Baird, M., Wilson, R., Young, L., Williman, J., Young, S., Wilson, M., Slobbe, L., Lockhart, E., Buchan, G., 2004. Bystander help within a polyepitope DNA vaccine improves immune responses to influenza antigens. Scand. J. Immunol. 60, 363 – 371. Bertoletti, A., Maini, M.K., 2000. Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection? Curr. Opin. Immunol. 12, 403 – 408. Bertoletti, A., Southwood, S., Chesnut, R., Sette, A., Falco, M., Ferrara, G.B., Penna, A., Boni, C., Fiaccadori, F., Ferrari, C., 1997. Molecular features of the hepatitis B virus nucleocapsid T-cell epitope 18–27: interaction with HLA and T-cell receptor. Hepatology 26, 1027 – 1034. Bertoletti, A., Maini, M., Williams, R., 2003. Role of hepatitis B virus specific cytotoxic T cells in liver damage and viral control. Antiviral Res. 60, 61 – 66. Bfcher, W.O., Dekel, B., Schwerin, W., Geissler, M., Hoffmann, S., Rohwer, A., Arditti, F., Cooper, A., Bernhard, H., Berrebi, A., RoseJohn, S., Shaul, Y., Galle, P.R., Lohr, H.F., Reisner, Y., 2001. Induction of strong hepatitis B virus (HBV) specific T helper cell and cytotoxic T lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic HBV infection. Eur. J. Immunol. 31, 2071 – 2079. Charini, W.A., Kuroda, M.J., Schmitz, J.E., Beaudry, K.R., Lin, W., Lifton, M.A., Krivulka, G.R., Necker, A., Letvin, N.L., 2001. Clonally diverse CTL response to a dominant viral epitope recognizes potential epitope variants. J. Immunol. 167, 4996 – 5003.
Chen, W.N., Oon, C.J., 1999. Mutation bhot spotQ in HLA class I-restricted T cell epitope on hepatitis B surface antigen in chronic carriers and hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 262, 757 – 761. Chikh, G.G., Kong, S., Bally, M.B., Meunier, J.C., Schutze-Redelmeier, M.P., 2001. Efficient delivery of antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8R T cells. J. Immunol. 167, 6462 – 6470. Ciernik, F., Berzofsky, J.A., Carbone, D.P., 1996. Induction of cytotoxic T lymphocytes and antitumor immunity with DNA vaccines expressing single T cell epitopes. J. Immunol. 156, 2369 – 2374. Davis, H.L., McCluskie, M.J., 1999. DNA vaccines for viral diseases. Microbes Infect. 1, 7 – 21. De Haan, L., Hearn, A.R., Rivett, A.J., Hirst, T.R., 2002. Enhanced delivery of exogenous peptides into the class I antigen processing and presentation pathway. Infect. Immun. 70, 3249 – 3258. Engelhard, V.H., 1994. Structure of peptides associated with MHC class I molecules. Curr. Opin. Immunol. 6, 13 – 23. Goulder, P.J., Brander, C., Annamalai, K., Mngqundaniso, N., Govender, U., Tang, Y., He, S., Hartman, K.E., O’Callaghan, C.A., Ogg, G.S., Altfeld, M.A., Rosenberg, E.S., Cao, H., Kalams, S.A., Hammond, M., Bunce, M., Pelton, S.I., Burchett, S.A., McIntosh, K., Coovadia, H.M., Walker, B.D., 2000. Differential narrow focusing of immunodominant human immunodeficiency virus gag-specific cytotoxic T-lymphocyte responses in infected African and caucasoid adults and children. J. Virol. 74, 5679 – 5690. Gromme, M., Neefjes, J., 2002. Antigen degradation or presentation by MHC class I molecules via classical and non-classical pathways. Mol. Immunol. 39, 181 – 202. Guidotti, L.G., Rochford, R., Chung, J., Shapiro, M., Purcell, R., Chisari, F.V., 1999. Viral clearance without destruction of infected cells during acute HBV infection. Science 284, 825 – 829. Klenerman, P., Zinkernagel, R.M., 1998. Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature 394, 482 – 485. Kuhober, A., Pudollek, H.P., Reifenberg, K., Chisari, F.V., Schlicht, H.J., Reimann, J., Schirmbeck, R., 1996. DNA immunization induces antibody and cytotoxic T cell responses to Hepatitis B core antigen in H-2b mice. J. Immunol. 156, 3687 – 3695. Kwissa, M., Unsinger, J., Schirmbeck, R., Hauser, H., Reimann, J., 2000. Polyvalent DNA vaccines with bidirectional promoters. J. Mol. Med. 78, 495 – 506. Lee, R.S., Tartour, E., van der Bruggen, P., Vantomme, V., Joyeux, I., Goud, B., Fridman, W.H., Johannes, L., 1998. Major histocompatibility complex class I presentation of exogenous soluble tumor antigen fused to the B-fragment of Shiga toxin. Eur. J. Immunol. 28, 2726 – 2737. Lee, Y.S., Yoon, S.J., Kwon, T.K., Kim, Y.H., Woo, J.H., Suh, M.H., Suh, S.I., Baek, W.K., Kim, H.J., Ahn, S.Y., Choe, B.K., Park, J.W., 2001. Immune response induced by immunization with Hepatitis B virus core DNA isolated from chronic active hepatitis patients. Immunol. Lett. 78, 13 – 20. Leifert, J.A., Rodriguez-Carreno, M.P., Rodriguez, F., Lindsay Whitton, J., 2004. Targeting plasmid-encoded proteins to the antigen presentation pathways. Immunol. Rev. 199, 40 – 53. Lindinger, P., Mostbock, S., Hammerl, P., Hartl, A., Thalhamer, J., Abrams, S.I., 2003. Induction of murine ras oncogene peptide-specific T cell responses by immunization with plasmid DNA-based minigene vectors. Vaccine 21, 4285 – 4296. Livingston, B.D., Newman, M., Crimi, C., McKinney, D., Chesnut, R., Sette, A., 2001. Optimization of epitope processing enhances immunogenicity of multiepitope DNA vaccines. Vaccine 19, 4652 – 4660. Loi, P.K., Mcgraw, H.F., Tublitz, N.J., 1997. Peptide detection in single cells using a dot immunoblot assay. Peptides 18, 749 – 753. Maini, M.K., Boni, C., Lee, C.K., Larrubia, J.R., Reignat, S., Ogg, G.S., King, A.S., Herberg, J., Gilson, R., Alisa, A., Williams, R., Vergani, D., Naoumov, N.V., Ferrari, C., Bertoletti, A., 2000a. The role of virus-
W. Xu et al. / Virology 334 (2005) 255–263 specific CD8+ cells in liver damage and viral control during persistent Hepatitis B virus infection. J. Exp. Med. 191, 1269 – 1280. Maini, M.K., Reignat, S., Boni, C., Ogg, G.S., King, A.S., Malacarne, F., Webster, G.J., Bertoletti, A., 2000b. T cell receptor usage of virusspecific CD8 cells and recognition of viral mutations during acute and persistent hepatitis B virus infection. Eur. J. Immunol. 30, 3067 – 3078. Melief, C.J., Van Der Burg, S.H., Toes, R.E., Ossendorp, F., Offringa, R., 2002. Effective therapeutic anticancer vaccines based on precision guiding of cytolytic T lymphocytes. Immunol. Rev. 188, 177 – 182. Missale, G., Redeker, A., Person, J., Fowler, P., Guilhot, S., Schlicht, H.J., Ferrari, C., Chisari, F.V., 1993. HLA-A31- and HLA-Aw68-restricted Cytotoxic T cell responses to a single Hepatitis B virus nucleocapsid epitope during acute viral hepatitis. J. Exp. Med. 177, 751 – 756. Moron, G., Dadaglio, G., Leclerc, C., 2004. New tools for antigen delivery to the MHC class I pathway. Trends Immunol. 25, 92 – 97. Nagata, T., Aoshi, T., Uchijima, M., Suzuki, M., Koide, Y., 2004. Cytotoxic T-lymphocyte-, and helper T-lymphocyte-oriented DNA vaccination. DNA Cell Biol. 23, 93 – 106. Nakamura, Y., Suda, T., Nagata, T., Aoshi, T., Uchijima, M., Yoshida, A., Chida, K., Koide, Y., Nakamura, H., 2003. Induction of protective immunity to Listeria monocytogenes with dendritic cells retrovirally transduced with a cytotoxic T lymphocyte epitope minigene. Infect. Immun. 71, 1748 – 1754. Petrovsky, N., Aguilar, J.C., 2004. Vaccine adjuvants: current state and future trends. Immunol. Cell. Biol. 82, 488 – 496. Plank, C., Oberhauser, B., Mechtler, K., Koch, C., Wagner, E., 1994. The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. J. Biol. Chem. 269, 12918 – 12924. Restifo, N.P., Bacik, I., Irvine, K.R., Yewdell, J.W., McCabe, B.J., Anderson, R.W., Eisenlohr, L.C., Rosenberg, S.A., Bennink, J.R., 1995. Antigen processing in vivo and the elicitation of primary CTL responses. J. Immunol. 154, 4414 – 4422. Rice, J., King, C.A., Spellerberg, M.B., Fairweather, N., Stevenson, F.K., 1999. Manipulation of pathogen derived genes to influence antigen presentation via DNA vaccines. Vaccine 17, 3030 – 3038. Rimmelzwaan, G.F., Boon, A.C., Voeten, J.T., Berkhoff, E.G., Fouchier, R.A., Osterhaus, A.D., 2004. Sequence variation in the influenza A virus nucleoprotein associated with escape from cytotoxic T lymphocytes. Virus Res. 103, 97 – 100. Rodriguez, F., An, L.L., Harkins, S., Zhang, J., Yokoyama, M., Widera, G., Fuller, J.T., Kincaid, C., Campbell, I.L., Whitton, J.L., 1998. DNA
263
immunization with minigenes: low frequency of memory CTL and inefficient antiviral protection are rectified by ubiquitination. J. Virol. 72, 5174 – 5181. Schirmbeck, R., Wild, J., Reimann, J., 1998. Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen. Eur. J. Immunol. 28, 4149 – 4161. Sette, A., Fikes, J., 2003. Epitope-based vaccines: an update on epitope identification, vaccine design and delivery. Curr. Opin. Immunol. 15, 461 – 470. Shankar, P., Schlom, J., Hodge, J.W., 2001. Enhanced activation of rhesus T cells by vectors encoding a triad of costimulatory molecules (B7-1 ICAM-1, LFA-3). Vaccine 20, 744 – 755. Subbramanian, R.A., Kuroda, M.J., Charini, W.A., Barouch, D.H., Costantino, C., Santra, S., Schmitz, J.E., Martin, K.L., Lifton, M.A., Gorgone, D.A., Shiver, J.W., Letvin, N.L., 2003. Magnitude and diversity of cytotoxic-T-lymphocyte responses elicited by multiepitope DNA vaccination in rhesus monkeys. J. Virol. 77, 10113 – 10118. Takada, A., Kawaoka, Y., 2003. Antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications. Rev. Med. Virol. 13, 387 – 398. Thermet, A., Rollier, C., Zoulim, F., Trepo, C., Cova, L., 2003. Progress in DNA vaccine for prophylaxis and therapy of hepatitis B. Vaccine 21, 659 – 662. Thermet, A., Robaczewska, M., Rollier, C., Hantz, O., Trepo, C., Deleage, G., Cova, L., 2004. Identification of antigenic regions of duck hepatitis B virus core protein with antibodies elicited by DNA immunization and chronic infection. J. Virol. 78, 1945 – 1953. Webster, G., Bertoletti, A., 2001. Quantity and quality of virus-specific CD8 cell response: relevance to the design of a therapeutic vaccine for chronic HBV infection. Mol. Immunol. 38, 467 – 473. Wu, Y., Kipps, T.J., 1997. Deoxyribonucleic acid vaccines encoding antigens with rapid proteasome-dependent degradation are highly efficient inducers of cytolytic T lymphocytes. J. Immunol. 159, 6037 – 6043. Zhong, W.M., Reche, P.A., Lai, C.C., Reinhold, B., Reinherz, E.L., 2003. Genome-wide characterization of a viral cytotoxic T lymphocyte epitope repertoire. J. Biol. Chem. 278, 45135 – 45144. Zwaveling, S., Ferreira Mota, S.C., Nouta, J., Johnson, M., Lipford, G.B., Offringa, R., van der Burg, S.H., Melief, C.J., 2002. Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides. J. Immunol. 169, 350 – 358.
