Induction of virus-specific cytotoxic T lymphocytes by in vivo electric administration of peptides

Vaccine 19 (2001) 2190– 2196 www.elsevier.com/locate/vaccine

Induction of virus-specific cytotoxic T lymphocytes by in vivo electric administration of peptides Satori Uno-Furuta a,b, Shigenori Tamaki b, Yutaka Takebe c, Shiki Takamura a, Akira Kamei a,b, Gisen Kim a, Isao Kuromatsu a, Masahiko Kaito b, Yukihiko Adachi b, Yasuhiro Yasutomi a,* a

Department of Bioregulation, Mie Uni6ersity School of Medicine, 2 -174 Edobashi, Tsu Mie 514 -8507, Japan b Third Department of Internal Medicine, Mie Uni6ersity School of Medicine, Mie 514 -8507, Japan c Laboratory of Molecular Virology and Epidemiology, AIDS Research Center, National Institute of Infectious Disease, Tokyo 108 -8640, Japan Received 17 July 2000; received in revised form 23 August 2000; accepted 29 August 2000

Abstract Generally, major histocompatibility complex (MHC) class I presentation of peptide antigens only occur for proteins’ which are actively synthesized and processed intracellularly, so that immunization with a cytotoxic T lymphocyte (CTL) target peptide does not usually elicit effective CTL responses. In the present study, we explored the use of epitope peptides by in vivo electroporation to introduce directly into the cytoplasm for the vaccine elicitation of virus-specific CTLs in a mouse system. BALB/c mice were immunized with human immunodeficiency virus (HIV) env (P18, residues 311– 320) or hepatitis C virus (HCV) NS5 (P17, residues 2423–2434) with or without electric pulses. Effector cells against peptide-labeled target cells were elicited in mice immunized with peptides with electric administration but not without electric administration. Moreover, cytolytic activities of CTL against peptide-labeled target cells were enhanced by the addition of plasmid having the immunostimulatory sequence (ISS) or cDNA of the B7-1 molecule in electric administration of peptides. The results of the present study suggest that a peptide vaccine against a virus using electric administration is effective in eliciting virus specific CTLs. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: CTL; Peptide vaccine; Immunostimulatory sequence (ISS)

1. Introduction For the elicitation of virus-specific CTL, viral antigen (Ag) must be processed as peptide fragments to facilitate their association with MHC class I molecules for presentation to CD8+ T cells [1]. Since MHC class I processing of viral Ag occurs most efficiently in the course of intracellular synthesis of viral proteins, the approaches to virus-specific CTL induction that have been pursued most actively are those using live vectors [2]. However, recent studies have indicated that Ag can enter the MHC class I presentation pathway if introduced intracellularly directly into the cytoplasm, bypassing endosomal/lysosomal degradation [3,4]. * Corresponding author. Tel.: +81-59-2321111; fax: + 81-592315225. E-mail address: [email protected] (Y. Yasutomi).

Accordingly, various strategies using virus subunits formulated with various liposomes and proteoliposomes have been assessed for their ability to elicit virus-specific CTL [5–11]. CTLs are a critical component of protective immunity against viral infections. In a small number of carefully studied humans, a dramatic expansion of oligoclonal CD8+ peripheral blood lymphocytes populations during the primary phase of infection with HIV has been described [12]. Furthermore, clearance of the viremia associated with such a primary HIV infection has been shown to occur coincident with the emergence of HIV-specific CTL and prior to development of a detectable HIV neutralizing antibody response [13,14]. On the other hand, virus-specific CTL also plays a critical role in preventing the spread of HCV and clearing HCV during infection in infected individuals [15,16]. Moreover, overt neutralizing antibody response

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against HCV has not been detected in humans or chimpanzees infected with HCV [17,18]. From these observations, an effective virus vaccine should elicit virus-specific CTL responses in some cases. In the present study, we explored novel approaches of peptide vaccines without any kind of adjuvant to elicit virus-specific CTL by using strategies of in vivo electric administration of peptides. Moreover, enhancement of cytotoxic activities of virus-specific CTL by addition of B7-1 and immunostimulator sequence (ISS) DNA were also explored in this strategy of in vivo electric administration of peptides.

2. Materials and methods

2.1. Peptide synthesis The peptides used in this study included the HCV nonstructural protein 5 (HCV-NS5) CTL epitope (HCV-NS5, MSYSWTGALVTPCAAE; P17) [19] and HIV-env CTL epitope (HIV 308-322, RIQRGPGRAFVTIGK; P18) [20].

2.2. Plasmid Plasmid pCMV –LacZ, which is known to contain potent ISS has been described previously [21]. PCMV – LacZ contains the CMV IE1 promoter intron, the simian virus 40-intron, the E.coli LacZ cDNA, and the simian virus 40 polyadenylation site. This plasmid has various (ten) CpG-containing hexamers — six 5%GACGTC-3%, two 5%-AGCGCT-3% and two 5%AACGCT-3% hexamers. The plasmid DNA having murine B7-1 cDNA backbone is identical to pCMV – LacZ does not have 5%-AGCGCT-3% and 5%-AACGCT3% sequences [22]. Plasmid without cDNA of B7-1 was used as a control.

2.3. Immunization Six- to eight-week-old BALB/c mice were intramuscularly injected with the 50 mg of peptide with or without plasmid DNA twice or three times, and then the site of inoculation was immediately given an electric pulse by an Electric Square Porator (T820; BTX, San Diego, CA). Pulses were delivered to the muscle by using a pair of electrode needles. Eight electric pulses were administered at a rate of one pulse per s. Pulses were delivered to the gastrocnemius muscle, which had been injected with the peptide between the electrode needles separated by a distance of approximately 5 mm. The electric pulse was 99 ms in duration at a voltage of 50 V; the resistance was monitored with a graphic pulse analyzer (Optimizer 500, BTX, San Diego, CA) [23].

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2.4. Immunohistochemical analysis A biotin-conjugated peptide, P18 (obtained from Funakoshi Co. Ltd., Japan), mixed with plasmid DNA having murine B7-1 cDNA was injected and electroporated into the gastrocnemius muscle of mice, and the mice were euthanized on third days after inoculation. Cryostat sections (6-mm thick) of muscle were prepared, air-dried, and fixed in acetone for 5 min at 4°C. The sections were incubated with Block Ace (Dainippon Pharmaceutical, Japan) to prevent nonspecific reactions. Subsequently, they were incubated with a ratio of 1:250 diluted rat anti-mouse B7-1 mAb (Immunotech, France) for 30 min at 37°C. After washing, they were incubated with mixture of fluorescein-conjugated goat anti-rat IgG antibody (Caltag, CA, USA) and rhodamin-conjugated avidin (Boheringer Mannheim, Germany) for 30 min, and then washed, examined, and photographed under a fluorescence microscope.

2.5. Generation of CTL effector cells Effector cells were derived from spleen cells as precursor CTL. Aliquots of 5× 106 spleen cells were cocultured with 2.5×106 mitomycin C-treated autologous spleen cells labeled with peptide at 37°C in a CO2 incubator. The effector cells generated were harvested after 5 days in culture.

2.6. Cytotoxicity assay Target cells were MHC-matched (P815) tumor cells (2×106) incubated at 37°C in a 5% CO2 atmosphere with 10 mg/ml of the P17, P18 or control peptide (HCV E1 315 –322, [24]), or recombinant vaccinia virus (rVV) expressing HCV-NS or an irrelevant virus gene (2×107 PFU) for 16 h. Then target cells were washed and labeled with 51Cr. The 51Cr-labeled target cells were incubated for 5 h with effector cells. Spontaneous release varied from 5 to 10%. Percent lysis was calculated as [(experimental release−spontaneous release)/(100% release− spontaneous release)]× 100. All experiments were performed more than three times and each group consisted of five mice.

3. Results

3.1. Virus-specific effector cells are elicited in mice immunized with peptide by in 6i6o electric administration BALB/c mice were immunized twice with CTL epitope peptide, P17 or P18, with or without electric pulses delivered by an Electric Square Porator. Spleen cells obtained from these immunized mice were assessed for

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their ability to lyse P17 or P18 pulsed MHC-matched target cells. Effector cells against P17- or P18-labeled target cells were elicited in mice immunized with P17 or P18 with electric administration. These effector cells demonstrated low-level P17 or P18-specific lytic responses; however, no such effector cells were developed in mice immunized with the same peptides without in vivo electric administration or unimmunized control mice (Fig. 1).

3.2. Enhancement of cytolytic acti6ities against CTL epitope peptide-labeled target cells by addition of plasmid DNA containing ISS Previous studies have demonstrated that ISS has potent adjuvant activities in various DNA vaccine strategies [25]. We explored the enhancement of the effect of cytolytic activities against virus epitope peptide-labeled target cells by mixture with plasmid DNA having ISS, pCMV – LacZ, or control plasmid in the in vivo electric administration of the peptide. P17- or P18-specific lytic activity was assessed in spleen cells of these mice following in vitro P17 or P18 stimulation. Spleen cells obtained from mice immunized with a peptide mixture with pCMV – LacZ showed greater peptide-specific lytic activities than did spleen cells of the mice immunized with a peptide mixture with the control peptide (Fig. 2), and these effector cell responses were not detected without in vivo electroporation (data not shown). There was no difference in the peptide-specific lytic activities in the spleen cells of mice immunized with the control plasmid mixture and with the peptide alone (data not shown).

Fig. 1. Spleen cells from mice immunized with CTL epitopes by in vivo electric administration develop CTL epitope-specific lytic activity. Mice were administered with HCV-NS5 (P17) or HIV env (P18) CTL epitope peptides concomitantly with (circle) or without (triangle) in vivo electroporation. Unimmunized controls were square. Closed symbols indicate the lysis of target cells labeled with the CTL epitope peptide (P17 or P18), and open symbols indicate the lysis of control peptide-labeled target cells. The data represent the mean percentage of the specific lysis values obtained from five mice.

Fig. 2. Addition of plasmid DNA containing ISS enhance cytolytic activities against target cells labeled with CTL epitope peptides in electric administration of peptides. Closed symbols indicate the lysis of target cells labeled with the CTL epitope peptide (P17 or P18), and open symbols indicate the lysis of control peptide-labeled target cells. The data represent the mean percentage of the specific lysis values obtained from five mice.

3.3. Addition of plasmid ha6ing B7 -1 cDNA enhances peptide-specific cytotoxicity in in 6i6o electric administration of peptide B7-1 molecules can transduce costimulatory signals through interaction with CD28 [26 –28]. Several studies have demonstrated that enhanced effects of the activity of CTL are obtained by addition of B7-1 DNA in vaccine strategies against virus and tumor cells [29,30]. Based on these findings, we assessed the lytic activities of spleen cells against peptide-labeled target cells from mice administered peptides with plasmid DNA having B7-1 cDNA by in vivo electric stimulation. Cytolytic activities of spleen cells from mice immunized with peptides mixed with B7-1 cDNA by in vivo electric administration were much higher than those of spleen cells from mice immunized with the control plasmid by the same method (Fig. 3). These cytolytic activities were not elicited without in vivo electroporation in accordance with the results of the addition of ISS (data not shown).

3.4. Expression of the peptide and B7 -1 molecule by in 6i6o electroporation To confirm the expression of the peptide and B7-1 molecule by in vivo electroporation, tissues were examined by immunohistochemical analysis using double staining. Immunohistochemical examination of the gastrocnemius muscle of mice administered with biotin-

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conjugated P18 mixed with plasmid having cDNA of B7-1 by in vivo electroporation was performed 3 days after injection. The B7-1 molecule (Fig. 4a and c) and P18 peptide (Fig. 4b and d) were observed in the cells of fascia and connective tissue among muscle fascicles in the same area. These were only seen in the area between the electrode needles. Expression of the peptide and molecule were considered as the characteristics of in vivo electroporation, since an electric current generally flows into tissues with low electric resistance. These findings suggested that the peptide and plasmid might have been transferred into the same cells.

3.5. Peptide immunization by electric administration elicited effector cells that recognized processed 6iral core protein The capacity of in vivo electric administration of peptides to elicit effector cells that recognize virus-infected cells was assessed using target cells infected with rVV carrying the HCV – NS or control gene. As shown in Fig. 5, mice immunized with peptides mixed with plasmid by electric pulse demonstrated more lysis of vaccinia HCV –NS-infected target cells than did control vaccinia-infected target cells. Thus, peptides immunization of mice by using in vivo electric administration generated an effector T-cell response capable of recognizing endogenously processed viral protein. These effector cells were CD8+ and showed MHC class I restriction (data not shown). Fig. 4. Immunostaining of sections of muscle tissue from a mouse 3 days after electric administration of peptides mixed with plasmid having B7-1 cDNA. B7-1 (a, c) and P18 (b, d) were observed in the cells of fascia and connective tissue among muscle fascicles in the same area. Control sections immunized with empty plasmid (e) or peptide without electric stimulation (f) did not show any positive reactions.

4. Discussion

Fig. 3. Addition of plasmid having B7-1 cDNA enhances cytolytic activities against target cells labeled with CTL epitope peptides in electric administration of peptides. Closed symbols indicate the lysis of target cells labeled with the CTL epitope peptide (P17 or P18), and open symbols indicate the lysis of control peptide-labeled target cells. The data represent the mean percentage of the specific lysis values obtained from five mice.

Short peptides corresponding to known CTL determinants have been used to sensitize target cells for lysis by specific CTLs and to induce Ag-specific CTL responses in vivo. Administration of MHC class I-restricted epitope peptides from several viral pathogens formulated in CFA, IFA or Titer Max induced Ag-specific CD8+ CTL [31 –33]. The mechanisms of induction of CTLs by immunization with CTL epitope peptides formulated in these adjuvants are not clear. MHC class I Ag presentation generally occurs only for Ags, synthesized and processed within a cell. Delivery of exogenous Ags into the MHC class I pathway can be achieved by osmotic loading [3], covalent or noncovalent association lipid carriers [5,34], conjugation to latex beads [35], or by encapsulation of the antigen in liposomes [5–

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11,36]. Electroporation efficiently introduces foreign genes into living cells, but the use of this technique had been restricted to suspension of cultured cells only, since the electric pulses were administered in a cuvette-type electrode. Recently, however, various types of electrodes have been developed, and electroinjection has been applied to chemicals or foreign genes in vivo [37 – 40]. We previously demonstrated that electric administration was effective in eliciting immune responses in mice immunized with DNA [23]. In the present study, the elicitation of virus-specific CTLs by peptides administered by in vivo electric stimulation was considered by means of epitope peptides that were introduced directly into cells and processed MHC class I molecules. The present study showed the enhancement effect of induction of CTLs in peptide vaccine by this in vivo electric administration was easy to be given by the addition of plasmid DNA. Co-stimulatory molecules play a key role in the induction of T-cell immune responses [41]. The generation of the T-cell immune responses is a complex process that requires the engagement of T cells with professional APCs and the production of different cytokines which regulate the clonal expansion, and differentiation of antigen-reactive cells. Effective T-cell activation by APC requires two stimuli — the first signal originates from the binding of Ag-MHC and TCR molecules which confer specificity and the second signal is from costimulatory proteins on APCs. Amongst the most potent co-stimulatory proteins are B7 co-stimulatory molecule family [42,43]. Based on these findings, an attempt to enhance the effects of the vaccine by utilizing B7-1 cDNA has been reported [33,34,44]. These results indicated that tumor cells transfected with B71 cDNA for anti-tumor vaccine were effective both in vivo and in vitro in enhancing CTL activities against tumor cells [45,46]. Other systems using B7-1 for vaccines, however, were not perfect for enhancing the effects of the vaccine inducing CTL responses. DNA

Fig. 5. Spleen cells of P17 peptide-immunized mice lysed rVV-HCV NS-infected target cells. Spleen cells obtained from peptide-immunized mice were examined for lysis of target cells infected with rVV-HCV core or rVV-control. Effector: target ratio was 80:1.

vaccines via co-delivery of B7-1 molecule genes have not exhibited obvious enhanced effects of CTL responses in some cases [47,48]; however, greater Agspecific CTL responses through a mixture of recombinant vaccinia virus expressing a specific target Ag and recombinant vaccinia virus having the gene for B7-1 molecule immunization than through recombinant vaccinia virus expressing a specific target Ag alone have been reported [49,50]. Based on these findings, a combination of formulation of Ags and B7-1 molecules might be important for utilizing the addition of B7-1 molecules for enhancement of CTL responses in vaccine strategies. The present study showed that an enhanced effect of CTL activities could be obtained by the addition of B7-1 cDNA to electric administration of peptides. Recent studies have indicated that the mammalian immune system is stimulated by DNA containing the following CpG hexamers: 5%-GACGTC-3%, 5%AGCGCT-3% and 5%-AACGTT-3% [51]. These sequence motifs are rarely present in eukaryotic genomes but are common in prokaryotic genomes. CpG DNA and these oligonucleotides activate B cells, T cells, and macrophages to proliferate and to produce various types of Th1-associated cytokines, including IFNg,IL-12, IL-6, IL-18 and TNF-a [52 –55]. These data suggested that in the setting of genetic vaccination, ISS functions as an adjuvant and promotes a Th1 immune response to the antigen [56]. In the present study, we first reported enhancement of the effects of CTL activities by the addition of ISS-DNA in a peptide vaccine strategy. The pCMV –LacZ has various CpG-containing hexamers that include six 5%GACGTC-3%, two 5%-AGCGCT-3% and two 5%AACGCT-3% hexamers. However, the control plasmid does not have two 5%-AGCGCT-3% and two 5%AACGCT-3% hexamers. This result indicated the importance of these two sequences, 5%-AGCGCT-3% and 5%-AACGCT-3%, in accordance with the results of other DNA vaccine studies [51]. In our experiments, however, it was not clear which sequence or how many sequences are necessary to enhance the CTL activities in vivo. The use of peptide immunogens as vaccinations against HIV or HCV is attractive; however, traditional approaches in peptide vaccine have not elicited virus-specific CD8+ MHC class I-restricted CTL. The findings in this study provide a basis for the approach of a peptide vaccine capable of inducing antivirus CTL responses. Peptide immunization by in vivo electroporation caused no local or systemic damage and did not need any type of adjuvant to elicit virus-specific CTL. This novel approach to the administration of peptides may prove the usefulness to generate virus-specific CTLs.

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Acknowledgements We thank Kazunori Sato for his valuable advice and Kyoko Nohtomin for her technical assistance through this study. This work was supported by Grants-in-Aid 1998 and 1999 from the Mie Medical Research Foundation, the Ryoichi Naito Foundation for Medical Research, the Ministry of Health and Welfare of Japan, and the Ministry of Education, Science, Sports and Culture of Japan.

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