Molecular characterization of woodchuck interleukin 15 (wIL-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (WHV)

www.elsevier.com/locate/issn/10434666 Cytokine 32 (2005) 296e303

Molecular characterization of woodchuck interleukin 15 (wIL-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (WHV) Baoju Wang a,b, Beate Lohrengel a, Yinping Lu a, Zhongji Meng a,b, Yang Xu a,c, Dongliang Yang b, Micheal Roggendorf a, Mengji Lu a,c,* a

Institut fu¨r Virologie, Universita¨tsklinikum Essen, Hufeland Str. 55, 45122 Essen, Germany Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Jiefang Avenue 1095, 430030 Wuhan, China c Department of Microbiology, Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13, 430030 Wuhan, China b

Received 16 June 2005; received in revised form 12 September 2005; accepted 3 November 2005

Abstract Interleukin 15 (IL-15) is a member of the four-helix bundle cytokine family and has T cell growth factor activity. IL-15 plays a unique role in both innate and adaptive immune cell homeostasis, particularly for the development of NK cells and CD8 þ memory cells. It may be useful for stimulation of specific immune responses in chronic viral infection such as hepatitis B virus infection. The woodchuck model is an informative animal model for studies on hepadnavirus infection and therapeutic interventions. Here, the complete coding sequence of woodchuck IL-15 (wIL-15) was cloned and sequenced. wIL-15 shows a high homology (>70%) to its counterparts of other mammalian species. His-tagged recombinant wIL-15 protein was expressed and purified and showed the ability to promote the proliferation of activated mouse splenocytes and woodchuck peripheral blood lymphocytes. Further, examination of mRNA amounts in liver samples of woodchucks by semi-quantitative RTPCR showed a slightly increased expression of wIL-15 in woodchuck livers during chronic woodchuck hepatitis virus infection. This available information will provide a basis for further studies on the function of IL-15 in the context of acute and chronic hepadnavirus infection and its potential therapeutic use for chronic hepatitis B virus infection in the woodchuck model. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Interleukin-15; Woodchuck; Intrahepatic expression

1. Introduction Interleukin 15 (IL-15) was originally discovered due to its ability to promote T cell proliferation [1e3]. It has been shown that IL-15 shares biological activities with IL-2 and is a member of four-helix bundle cytokine family like IL-2. Unlike IL-2, IL-15 is produced by macrophages and non-lymphoid (stromal and epithelial) cells [4e6]. The IL-15 expression is tightly * Corresponding author. Institut fu¨r Virologie, Universita¨tsklinikum Essen, Hufeland Str. 55, 45122 Essen, Germany. Tel.: þ49 201 723 3530; fax: þ49 201 723 5929. E-mail address: [email protected] (M. Lu). 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2005.11.007

controlled at the multi-levels of transcription, translation, and intracellular trafficking [7e15]. IL-15 plays unique roles in both innate and adaptive immune cell homeostasis and particularly for maintenance of NK cells and CD8 memory cells [16e23]. Due to its biological activities, IL-15 represents one of potential therapeutics for cancers [24e28] and chronic infections with human immunodeficiency virus [29e37]. Hepatitis B virus (HBV) causes acute and chronic hepatitis and leads to the development of cirrhosis and hepatocellular carcinoma (HCC) [38]. According to the estimation of World Health Organization, about 350 million people worldwide are chronically infected with HBV. Currently, the interferon therapy is the only available treatment leading to virus elimination,

B. Wang et al. / Cytokine 32 (2005) 296e303

however, it is only successful for a minority of patients and associated with severe side effects [39]. Nucleoside analogues like lamivudine suppress HBV polymerase activity and therefore block the HBV replication cycle. However, treatment with nucleoside analogues do not clear HBV from infected hepatocytes [40]. Thus, there is a great need for new therapeutic approaches to treat chronic HBV infection. Yet, IL-15 has not been experimentally tested for its potential use against HBV infection. Woodchuck hepatitis virus (WHV) is a member of the family Hepadnaviridae and has strong similarities with HBV in morphology, genomic structure, replication, and the natural history of infection [41,42]. Its natural host, North-American eastern woodchuck (Marmota Monax) represents a useful animal model to investigate immune response, pathogenesis, and antiviral therapy of HBV infection [43]. Therefore, it provides the opportunity to study IL-15 in the context of hepadnavirus infection. In the present work we cloned and sequenced the complete coding sequence of woodchuck IL-15 (wIL-15). Recombinant wIL-15 protein was expressed in E. coli and purified. The biological activity of recombinant wIL-15 was demonstrated by its ability to stimulate the proliferation of mouse splenocytes and woodchuck peripheral blood mononuclear cells (PBMCs). A slightly increased expression of wIL-15 in liver samples from chronically WHV-infected woodchucks was found by semi-quantitative RT-PCR. These data provide a basis for further evaluation of the value of IL-15 for treatment of chronic HBV infection in the woodchuck model. 2. Materials and methods 2.1. Purification of RNAs from woodchuck PBMCs and liver tissues Woodchucks were purchased from North Eastern Wildlife (Ithaca, NY, USA). Blood was drawn from anesthetized woodchucks via the vena saphena and collected in EDTA monovetts (Sarstedt, Numbrecht, Germany). PBMCs were separated by Ficoll-Paque density gradient centrifugation (Pharmacia, Freiburg, Germany) and suspended in 0.9% NaCl, aliquot of 4
106 cells were stored at 80  C before RNA extraction. Autopsy liver tissues of woodchucks were snap frozen in liquid nitrogen and stored at 80  C until RNA extraction. Naive woodchucks were negative for WHsAg, anti-WHV core antigen (anti-WHc), and anti-WHs antibodies. Chronically WHVinfected woodchucks were positive for WHsAg, anti-WHc, but negative for anti-WHs. Three woodchucks were experimentally infected with 106 WHV genome equivalents and sacrificed to take liver samples. Total RNAs from woodchuck PBMCs and liver tissues were extracted using the RNeasy Mini Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, cell pellets (4
106 cells) or liver tissue powder (30 mg) was lysed in 0.6 ml of lysis buffer of the RNA extraction kit. Lysates were applied onto the RNeasy spin column for centrifugation. RNAs bound to the spin column were washed twice with supplied wash buffer and eluted with 40 ml of sterile

297

RNase-free water. Purified RNAs were quantified by optical density (OD260) and stored in small aliquots at 80  C. 2.2. RT-PCR amplification of wIL-15 cDNA from woodchuck PBMC mRNA, cloning and sequencing of wIL-15 cDNAs Primers used to generate the complete coding sequence of wIL-15 from woodchuck PBMCs mRNA are listed in Table 1. The primers IL-15-3 and IL-15-4 were designed according to the conserved sequence among mammalian IL-15 cDNAs and are located in the 5#- and 3#-untranslated regions of IL-15, respectively. wIL-15 cDNAs were synthesized by incubating 1 mg RNA from woodchuck PBMCs with moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaitherburg, MD, USA) and primer (IL-15-4) at 42  C for 60 min and used for PCR with primers IL15-3 and IL15-4. PCR was performed over 30 cycles of 94  C 1 min, 50  C 1 min and 72  C 2 min, followed by an extension at 72  C for 6 min. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. PCR products were cloned into pCR2.1 vector according to the manufacturer’s instruction (Invitrogen, San Diego, CA, USA). Cloned cDNAs were sequenced by a commercial service (MWG, Munich, Germany) using an automated DNA sequencer (Applied Biosystems) with Sanger’s fluorescent dideoxy chain termination methods. 2.3. Construction of expression plasmids pQE30-wIL15 and pQE60-wIL15 Woodchuck IL-15 specific primers (IL15-s, as, 2s, and 2as) were designed according to the obtained sequences of wIL-15 cDNA, covered the predicted coding sequence of the putative mature protein (nt 145e486). Restriction sites for BglII, NcoI, EcoRI, and HindIII endonucleases were introduced into the primers (Table 1) for construction of the expression plasmids pQE30-wIL15 and pQE60-wIL15. The strategy of construction of the expression plasmids was shown in Fig. 1. PCR was performed under the conditions mentioned above by using Table 1 Primers used for RT-PCR and cloning of woodchuck IL-15 fragments Name

Polarity

Nucleotide sequence

nt position

IL15-3 IL15-4 IL15-s IL15-as IL15-2s IL15-2as IL15D1 IL15D2

sense antisense sense antisense sense antisense sense antisense

5#-gcc ata gcc agc tcw tct tca a-3# 5#-gta cct taa taa cag aaa ca-3# 5#-aac cat gga ctg gga aga tgt aag aaa g-3# 5#-aaa gat cta gga ggg ttg atg aac att tg-3# 5#-cgg gat cca act ggg aag atg taa gaa ag-3# 5#-gcg aag ctt agg agg gtt gat gaa cat ttg-3# 5#-ctt caa tcc agt gct atg tg-3# 5#-cct cta tgt ctc cat ctc tg-3#

266e287a 847e828a 146e165b 486e466b 145e165b 486e466b 32e51b 325e306b

a The nt positions for IL15-3 and 4 are referred to IL-15 sequence of human (HSU14407). b The nt positions for IL15-s, as, 2s, 2as, D1, and D2 are referred to wIL-15 sequence (AY426605). The underlined parts of primers IL15-s, as, 2s, and 2as indicate the specific cleavage sites of endonucleases BglII, NcoI, EcoRI, and HindIII, respectively.

298

B. Wang et al. / Cytokine 32 (2005) 296e303

Fig. 1. The alignment of the deduced aa sequence of wIL-15 precursor with its mammalian counterparts. The putative signal peptide is present in italic letters. It has a hydrophobic area from the aa position 10 to 42 (shaded sequences). The matured wIL-15 has four potential helical regions at aa 49e75, aa 88e101, aa 106e116, and aa 135e155 (underlined). Four cysteine sites (shaded positions) are conserved in mammalian IL-15 sequences.

a cDNA clone (K34) as the template. The plasmids pQE30wIL15 and pQE60-wIL15 were constructed by ligation of the corresponding PCR products and vectors digested with EcoRI and HindIII or with BglII and NcoI, respectively. 2.4. Expression and purification of recombinant woodchuck IL-15 The E. coli strain JM109 was transformed with the expression plasmids pQE30-wIL15 and pQE60-wIL15, respectively. E. coli was cultured to a density of A600z0.6. IPTG was added to a final concentration of 2 mM. Cells were sedimented at 4 h after induction. The protein was purified with HisTrap Kit (Amersham, Braunschweig, Germany) under denature conditions according to manufacturer’s instructions. The pooled fractions containing wIL-15 were desalted and refolded by using centrifugation filter device according to manufacturer (Amicon). E. coli lysates and purified recombinant proteins were separated by 15% SDS-PAGE and stained with Commassie blue. Western blot analysis was carried out with horseradishperoxidase-conjugated Ni-NTA according to the manufacturer (Qiagen) to detect recombinant IL-15. Protein concentrations were determined by the Bradford microassay procedure (BioRad). 2.5. Stimulation of proliferation of woodchuck PBMCs by recombinant wIL-15 To assess biological activity of wIL-15, mouse splenocytes or PBMCs from naı¨ve woodchuck were harvested according to routine procedures. Cell counting was performed with a Thoma hemocytometer. Five
104 of mouse splenocytes or woodchuck PBMCs were cultured in flat-bottom 96-well microtiter plates at 37  C in a humidified atmosphere containing 5%

CO2. Two hundred ml of culture medium supplemented with 10% fetal calf serum (Gibco BRL) was added to each well. RMPI-1640 complete medium or AIM V medium (Gibco BRL) were used for mouse splenocytes and woodchuck PBMCs, respectively. Recombinant wIL-15 was added in concentrations from 0.064 to 2 mg/ml under the stimulation of different concentrations of ConA (1 mg/ml, 0.5 mg/ml). Proliferation of mouse splenocytes and woodchuck PBMCs to different concentration of wIL-15 without ConA activation served as negative control. Spontaneous proliferation in complete medium served as a background control. Lymphoproliferation to wIL-15 was assessed after stimulation for 5 days including at 24 h pulse labeling with 1 mCi of [2-3H] adenine (Amersham). Results of triplicate cultures are presented as the mean stimulation index (the total counts per minute (cpm) for wIL-15 stimulated splenocytes divided by the total cpm of background controls).

2.6. Real-time RT-PCR analysis of wIL-15 mRNA expression in liver tissue The wIL-15 mRNA expression levels in liver tissues were quantitatively analyzed using a LightCycler RNA Amplification Kit SYBR Green I (Roche Diagnostics GmbH) according to the manufacturer instructions. Forward and reverse primers (IL15D1 and IL15D2) were shown in Table 1. One microgram of total RNA from woodchuck liver tissues was used for Real-time RT-PCR analysis. Briefly, RT was performed for 30 min at 55  C, followed by PCR (40 cycles of 5 s at 94  C, 10 s at 55  C, and 13 s at 72  C). Melting curve analysis was performed to ensure the specificity of the PCR product. PCR products were also run on agarose gels to correlate the expected product length. A standard curve was constructed using

B. Wang et al. / Cytokine 32 (2005) 296e303

299

Fig. 2. Cloning and expression of wIL-15. A. PCR cloning of wIL-15 cDNA. Primers IL15-3 and IL15-4 were designed according to the conserved sequence among IL-15 cDNAs of human, bovine, rabbit, monkey, and murine, located in 5#-UTR and 3#-UTR, respectively. B. Construction of the expression plasmids pQE30-wIL15 and pQE60-wIL15 for wIL-15. The predicted coding sequences (nt 145e486) of the mature protein were amplified and cloned into the expression vectors. The expression of the recombinant wIL-15 was under the control of tac promoter. A His-tag was introduced at the C- or N-terminal of the recombinant wIL-15.

dilutions with defined copy numbers of a plasmid containing the wIL-15 cDNA fragment. 3. Results 3.1. Cloning and sequence analysis of wIL-15 cDNA Total RNAs from woodchuck PBMC were purified and subjected to RT-PCR using primers IL15-3 and IL15-4 (Fig. 2, Table 1). A specific PCR product of 562 bp was generated and cloned into pCR2.1. Four clones were selected for sequencing. An open reading frame of 489 bp was identified and represents the complete coding sequence of wIL-15. The sequence information was submitted to Genbank (accession number AY426605). The deduced amino acid (aa) sequence of wIL-15 has a length of 162 aa residues. The complete coding sequence of wIL-15 shows high homologies with the IL-15 sequences of other species, ranging from 77% to 87% on the Table 2 Comparison of wIL-15 sequence with the respective sequences of other species on the nucleotide and amino acid levels Species

Rat Mouse Cow Cat Pig Monkey Human

Homology (%) nt

aa

77.91 78.12 84.25 84.46 84.46 86.09 86.91

69.75 70.37 73.46 74.69 75.93 79.63 79.01

Accession number of sequences used for alignment: Rat, U69272; Mouse, U14332; Cow, U42433; Cat, AF108148; Pig, U58142; Monkey, U03099; Human, U14407.

nucleotide (nt) level and from 69% to 80% on the aa level (Table 2). The alignment with known mammalian IL-15 sequences revealed that the wIL-15 precursor has a putative signal peptide of a length of 48 aa and the mature protein of 114 aa. Four cystein residues Cys83, Cys90, Cys133, and Cys136 are present, indicating that two intramolecular disulfide bridges are conserved. In addition, the mature wIL-15 may have a four-helix bundle-like secondary structure as GarnierOsguthorpe-Robson prediction suggests the presence of four helixes at the regions aa 49e75, aa 88e101, aa 106e116, and aa 135e155 (Fig. 2) [44]. 3.2. Expression of recombinant wIL-15 and analysis of its biological activity To construct the prokaryotic expression vectors of wIL-15, the sequence encoding the mature part of wIL-15 (nt 145e 486) was amplified by using primers IL15-s, IL15-as, IL152s, and IL15-2as (Table 1). The amplified PCR products were digested with respective restriction enzymes and cloned into the expression vectors pQE30 and pQE60, resulting into the expression vectors pQE30-wIL15 and pQE60-wIL15 (Fig. 2B). pQE30-wIL15 encodes a recombinant wIL-15 with a N-terminal hexahistidine tag, while the product from pQE60-wIL15 contains a C-terminal hexahistidine tag (Fig. 2B). The incubation of E. coli harboring pQE30-wIL15 with IPTG led to the production of a protein at a molecular weight of about 12.5 kDa, corresponding to the recombinant wIL-15 protein (Fig. 3A). The recombinant wIL-15 was reactive with horseradish-peroxidase-conjugated Ni-NTA in Western blotting, consistent with the fact that it contains an N-terminal hexahistidine tag. Additionally, since this protein was detectable in Western blot with Ni-NTA conjugates, it appeared to be expressed at a low level in E. coli without

300

B. Wang et al. / Cytokine 32 (2005) 296e303

incubation with IPTG (Fig. 3A). However, E. coli harboring pQE60-wIL15 expressed wIL-15 protein at a low level without regards to the presence of IPTG, that was only detectable by Western blot with horseradish-peroxidase-conjugated NiNTA. The recombinant wIL-15 protein expressed in E. coli with pQE-wIL15 was purified through a Ni-column to a high purity and confirmed with NTA-Ni complex in Westernblot (Fig. 3B). The purified wIL-15 protein was then desalted and refolded by Amicon centrifugation filter device to regain its biological activity. The biological activity of recombinant wIL-15 was determined by testing its ability to promote proliferation of activated mouse splenocytes and woodchuck PBMCs. Mouse splenocytes were incubated with increasing concentrations of recombinant wIL-15 ranging from 64 pg/ml to 1 mg/ml. The proliferative activity of mouse splenocytes was assessed at day 5 post pulse labeling cells with 1 mCi/ml of [methyl-3H] thymidine. At the concentration of 1 mg/ml of ConA, mouse splenocytes responded to recombinant wIL-15 in a dose-dependent manner (Fig. 4). SI values for mouse splenocytes proliferation reached >10 at 40 ng/ml of recombinant wIL-15. On the contrary, recombinant wIL-15 was not able to stimulate the proliferation of mouse splenocytes (SI<1) either without or in presence of 0.5 mg/ml of ConA. Similarly, woodchuck PBMCs were stimulated with increasing amounts of wIL-15 (Fig. 4B), in the presence of 0.5 or 1 mg/ml of ConA. Again, the activity of wIL-15 depends on the presence of ConA. This result is consistent with the fact that IL-15 can only support activated lymphocytes. 3.3. Qualitative and semi-quantitative analysis of wIL15 expression in liver tissues of naı¨ve, acute- and chronic-infected woodchucks IL-15 mRNA expression level in woodchuck liver was qualitatively analyzed by RT-PCR, and then was semi-quantitatively

analyzed by Real-time PCR. Primers IL15 D1/D2 were designed based on wIL-15 sequence to amplify a fragment of 293 bp (Table 1). Eight liver samples from 3 naı¨ve, 3 acutely WHV-infected, and 2 chronically WHV-infected woodchucks were tested. The qualitative RT-PCR with all 8 samples led to positive results, indicating that wIL-15 mRNA was expressed in liver tissues regardless of the status of WHV infection. Consistently, real-time RT-PCR generated comparable results. wIL15 mRNA was clearly expressed in all 8 liver tissues with >1000 copies per mg of total RNA. The wIL-15 mRNA was detected in 1050 to 3580 copies/mg of total RNA in liver tissues of naı¨ve and acutely WHV-infected animals. There was a slight increase of wIL-15 mRNA to 4300 and 5440 copies/mg of total RNA in samples from chronic carriers (Table 3).

4. Discussion In this paper, we characterized the cDNA of wIL-15 by cloning and expression. The nucleotide sequence of the wIL15 cDNA showed high similarities to the sequences of IL-15 of other mammalian species. The critical structure features of IL-15 protein are well-conserved in the wIL-15. A hydrophobic segment from aa 10 to aa 42 corresponds to the signal peptide. The putative mature wIL-15 has four regions corresponding to the helices at the positions aa 49e75, aa 88e 101, aa 106e116, and aa 135e155. Four cysteine residues at aa positions 83, 90, 133, and 136 are conserved among 8 mammalian IL-15 sequences. The conservation of structural features reflects the conservation of functions like support of T-cell proliferation. In concordance with the high homology among the mammalian IL-15, we found that human IL-15 supported woodchuck PBMC proliferation and IFN-g production of woodchuck PBMCs [45]. As demonstrated in the present work, wIL-15

Fig. 3. Expression and purification of recombinant wIL-15. A. SDS-PAGE analysis of lysates of E. coli expressing recombinant wIL-15 protein. 1 and 2: E. coli was transformed with pQE30-wIL15; 3 and 4: E. coli was transformed with pQE60-wIL15. Lane 1 and 3 show the samples of E. coli incubated with 2 mM of IPTG for 4 h. In lane 2 and 4 lysates of E. coli grown without IPTG were used as control. In the lower part, Western blot analysis of recombinant wIL-15 proteins using HRP conjugated Ni-NTA is shown. B. Purification of recombinant wIl-15 by HisTrap Kit. E. coli lysates containing recombinant wIL-15 proteins were loaded on the column. After wash, recombinant wIL-15 proteins bound to the column were eluted and desalted. The samples were analyzed by SDS-PAGE and Western blot by using horseradish-peroxidase-conjugated Ni-NTA. Lane 1 to 3: fractions eluted from the Ni column. Lane 4: recombinant wIL-15 after desalting.

B. Wang et al. / Cytokine 32 (2005) 296e303

301

Fig. 4. Stimulation of ConA-activated mouse splenocytes and woodchuck PBMCs by recombinant wIL-15. Mouse splenocytes (A) and woodchuck PBMCs (B) were cultured in the presence of different concentrations of wIL-15 (0.064 to 2000 ng/ml). ConA was added to the cultures: 1 mg/ml, diamond; 0.5 mg/ml, square; negative control, Triangle. Cells were cultured for 5 days including 24 h pulse with 1 mCi of [methyl-3H] thymidine or [2-3H] adenine, respectively and then harvested. The proliferation of mouse splenocytes or woodchuck PBMCs to the stimulation of wIL-15 was presented as stimulation index.

was able to stimulate mouse splenocytes, indicating the conserved function of IL-15. Due to its important regulatory functions, the expression of wIL-15 itself is tightly regulated at multiple levels including the translational and posttranslational steps. This fact may explain our failed attempt to express wIL-15 by transfection of mammalian cells. We cloned wIL-15 into an expression vector based on pcDNA3. After the transfection of various cell lines and at different time points, no significant wIL-15 activity in culture supernatants could be detected by testing the stimulation of woodchuck PBMCs and mouse splenocytes (Lu et al., unpublished results). In contrast, other woodchuck cytokines like interferon-a, -g, and TNF-a could be detected by the same approach. Presumably, the expression of wIL-15 in mammalian cells is kept down by the control mechanisms. The level of wIL-15 expression in liver samples from woodchuck with different infection status was assessed with RT-PCR. In chronic carrier woodchucks, wIL-15 transcription seems to be slightly enhanced. The enhanced expression of wIL-15 may be due to the continuous presence of inflammatory

stimuli. However, the wIL-15 expression was not elevated at the level of transcription during the acute phase of WHV infection. It should be monitored for the course of WHV infection to learn more about the possible involvement of wIL-15 in regulation of specific immune responses. The specific T-cell response to HBV is supposed to be critical for the outcome of HBV infection. Particularly, HBVspecific CD8 þ T-cells play a decisive role for the clearance of HBV during primary infection [46]. Both HBV-specific CTL and Th responses in chronically HBV-infected patients are low or not detectable [47]. The recent concept to treat chronic HBV infection by immunomodulation led to different experimental approaches [48e50]. However, sustained HBVspecific immune responses are difficult to induce in chronically infected patients. Due to the ability of IL-15 to support T-cells and NK cells, it could be a component to improve specific cellular immune responses by immunomodulation. Our work provides the basis for further experiments to test immunotherapeutic approaches based on IL-15 in the woodchuck model.

Table 3 wIL-15 mRNA expression in woodchuck liver tissues Group

Naı¨ve woodchuck

Animal

N1

N2

N3

A1

Acute-infected woodchuck A2

A3

WHV carrier C1

C2

Qualitative RT-PCR Real-time RT-PCR (copies/mg RNA)

þ 2440

þ 1580

þ 1130

þ 1050

þ 1500

þ 3580

þ 4300

þ 5440

302

B. Wang et al. / Cytokine 32 (2005) 296e303

Additionally, IL-15 appeared to be a suitable cytokine adjuvant for vaccinations [51e53]. Particularly, IL-15 has been shown to enhance the DNA primed immune responses to HBV large surface antigen in mice [53]. In previous work, we were able to demonstrate the usefulness of DNA vaccination in the woodchuck model [43]. The ability of IL-15 to enhance DNA vaccination against hepadnavirus infection could be tested in this model.

[15]

[16]

[17]

Acknowledgments We are thankful for technical assistance of Thekla Kemper and Barbara Bleekmann. We are grateful to Delia Cosgrove for proof reading of manuscript. References [1] Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, et al. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science 1994;264:965e8. [2] Bamford RN, Grant AJ, Burton JD, Peters C, Kurys G, Goldman CK, et al. The interleukin (IL) 2 receptor beta chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Proc Natl Acad Sci U S A 1994;91:4940e4. [3] Burton JD, Bamford RN, Peters C, Grant AJ, Kurys G, Goldman CK, et al. A lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Proc Natl Acad Sci U S A 1994;91:4935e9. [4] Waldmann TA, Tagaya Y. The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens. Annu Rev Immunol 1999;17: 19e49. [5] Reinecker HC, MacDermott RP, Mirau S, Dignass A, Podolsky DK. Intestinal epithelial cells both express and respond to interleukin 15. Gastroenterology 1996;111:1706e13. [6] Quinn LS, Haugk KL, Grabstein KH. Interleukin-15: a novel anabolic cytokine for skeletal muscle. Endocrinology 1995;136:3669e72. [7] Onu A, Pohl T, Krause H, Bulfone-Paus S. Regulation of IL-15 secretion via the leader peptide of two IL-15 isoforms. J Immunol 1997;158:255e62. [8] Tagaya Y, Kurys G, Thies TA, Losi JM, Azimi N, Hanover JA, et al. Generation of secretable and non-secretable interleukin 15 isoforms through alternate usage of signal peptides. Proc Natl Acad Sci U S A 1997;94:14444e9. [9] Nishimura H, Washizu J, Nakamura N, Enomoto A, Yoshikai Y. Translational efficiency is upregulated by alternative exon in murine IL-15 mRNA. J Immunol 1998;160:936e42. [10] Meazza R, Gaggero A, Neglia F, Basso S, Sforzini S, Pereno R, et al. Expression of two interleukin-15 mRNA isoforms in human tumors does not correlate with secretion: role of different signal peptides. Eur J Immunol 1997;27:1049e54. [11] Gaggero A, Azzarone B, Andrei C, Mishal Z, Meazza R, Zappia E, et al. Differential intracellular trafficking, secretion, and endosomal localization of two IL-15 isoforms. Eur J Immunol 1999;29:1265e74. [12] Kurys G, Tagaya Y, Bamford R, Hanover JA, Waldmann TA. The long signal peptide isoform and its alternative processing direct the intracellular trafficking of IL-15. J Biol Chem 2000;275:30653e9. [13] Nishimura H, Yajima T, Naiki Y, Tsunobuchi H, Umemura M, Itano K, et al. Differential roles of interleukin 15 mRNA isoforms generated by alternative splicing in immune responses in vivo. J Exp Med 2000;191:157e70. [14] Bamford RN, DeFilippis AP, Azimi N, Kurys G, Waldmann TA. The 5#-untranslated region, signal peptide, and the coding sequence of the

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25]

[26]

[27]

[28]

[29] [30]

[31]

[32]

[33]

carboxyl terminus of IL-15 participate in its multifaceted translational control. J Immunol 1998;160:4418e26. Musso T, Calosso L, Zucca M, Millesimo M, Ravarino D, Giovarelli M, et al. Human monocytes constitutively express membrane-bound, biologically active, and interferon-gamma-upregulated interleukin-15. Blood 1999;93:3531e9. Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieh M, Paxton R, et al. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 1994; 180:1395e403. Puzanov IJ, Bennett M, Kumar V. IL-15 can substitute for the marrow microenvironment in the differentiation of natural killer cells. J Immunol 1996;157:4282e5. Carson WE, Fehniger TA, Haldar S, Eckhert K, Lindemann MJ, Lai CF, et al. A potential role for interleukin-15 in the regulation of human natural killer cell survival. J Clin Invest 1997;99:937e43. Naora H, Gougeon ML. Enhanced survival and potent expansion of the natural killer cell population of HIV-infected individuals by exogenous interleukin-15. Immunol Lett 1999;68:359e67. Zhang X, Sun S, Hwang I, Tough DF, Sprent J. Potent and selective stimulation of memory-phenotype CD81 T cells in vivo by IL-15. Immunity 1998;8:591e9. Becker TC, Wherry EJ, Boone D, Murali-Krishna K, Antia R, Ma A, et al. Interleukin 15 is required for proliferative renewal of virus-specific memory CD8 T cells. J Exp Med 2002;195:1541e8. Goldrath AW, Sivakumar PV, Glaccum M, Kennedy MK, Bevan MJ, Benoist C, et al. Cytokine requirements for acute and Basal homeostatic proliferation of naive and memory CD8 þ T cells. J Exp Med 2002;195:1515e22. Oh S, Perera LP, Burke DS, Waldmann TA, Berzofsky JA. IL-15/IL15Ralpha-mediated avidity maturation of memory CD8 þ T cells. Proc Natl Acad Sci U S A 2004;101:15154e9. Waldmann TA. The IL-2/IL-15 receptor systems: targets for immunotherapy. J Clin Immunol 2002;22:51e6. Fehniger TA, Cooper MA, Caligiuri MA. Interleukin-2 and interleukin15: immunotherapy for cancer. Cytokine Growth Factor Rev 2002;13: 169e83. Kishida T, Asada H, Itokawa Y, Cui FD, Shin-Ya M, Gojo S, et al. Interleukin (IL)-21 and IL-15 genetic transfer synergistically augments therapeutic antitumor immunity and promotes regression of metastatic lymphoma. Mol Ther 2003;8:552e8. Klebanoff CA, Finkelstein SE, Surman DR, Lichtman MK, Gattinoni L, Theoret MR, et al. IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8 þ T cells. Proc Natl Acad Sci U S A 2004;101: 1969e74. Roychowdhury S, May Jr KF, Tzou KS, Lin T, Bhatt D, Freud AG, et al. Failed adoptive immunotherapy with tumor-specific T cells: reversal with low-dose interleukin 15 but not low-dose interleukin 2. Cancer Res 2004;64:8062e7. Kanai T, Thomas EK, Yasutomi Y, Letvin NL. IL-15 stimulates the expansion of AIDS virus-specific CTL. J Immunol 1996;157:3681e7. Umemura M, Nishimura H, Yajima T, Wajjwalk W, Matsuguchi T, Takahashi M, et al. Overexpression of interleukin-15 prevents the development of murine retrovirus-induced acquired immunodeficiency syndrome. FASEB J 2002;16:1755e63. d’Ettorre G, Forcina G, Lichtner M, Mengoni F, D’Agostino C, Massetti AP, et al. Interleukin-15 in HIV infection: immunological and virological interactions in antiretroviral-naive and -treated patients. AIDS 2002;16:181e8. Chitnis V, Pahwa R, Pahwa S. Determinants of HIV-specific CD8 T-cell responses in HIV-infected pediatric patients and enhancement of HIVgag-specific responses with exogenous IL-15. Clin Immunol 2003;107: 36e45. d’Ettorre G, Forcina G, Andreotti M, Sarmati L, Palmisano L, Andreoni M, et al. Interleukin-15 production by monocyte-derived dendritic cells and T cell proliferation in HIV-infected patients with discordant response to highly active antiretroviral therapy. Clin Exp Immunol 2004;135:280e5.

B. Wang et al. / Cytokine 32 (2005) 296e303 [34] Mastroianni CM, d’Ettorre G, Forcina G, Vullo V. Teaching tired T cells to fight HIV: time to test IL-15 for immunotherapy? Trends Immunol 2004;25:121e5. [35] Lum JJ, Schnepple DJ, Nie Z, Sanchez-Dardon J, Mbisa GL, Mihowich J, et al. Differential effects of interleukin-7 and interleukin-15 on NK cell anti-human immunodeficiency virus activity. J Virol Jun 2004;78:6033e42. [36] Mueller YM, Petrovas C, Bojczuk PM, Dimitriou ID, Beer B, Silvera P, et al. Interleukin-15 Increases Effector Memory CD8 þ T Cells and NK Cells in Simian Immunodeficiency Virus-Infected Macaques. J Virol 2005;79:4877e85. [37] Alpdogan O, van den Brink MR. IL-7 and IL-15: therapeutic cytokines for immunodeficiency. Trends Immunol 2005;26:56e64. [38] Hollinger FB, Liang TJ. Hepatitis B virus. In: Fields BN, Knipe DM, Howley PM, et al, editors. Fields Virology. 4th ed., vol. 2. Philadelphia: Lippincott Williams & Wilkins Publishers; 2001. p. 2971e3036. [39] Di Bisceglie AM, Rustgi VK, Kassianides C, Lisker-Melman M, Park Y, Waggoner JG, et al. Therapy of chronic hepatitis B with recombinant human alpha and gamma interferon. Hepatology 1990;11:266e70. [40] Dienstag JL, Perrillo RP, Schiff ER, Bartholomew M, Vicary C, Rubin M. A preliminary trial of lamivudine for chronic hepatitis B infection. N Engl J Med 1995;333:1657e61. [41] Summers J, Smolec JM, Snyder R. A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc Natl Acad Sci USA 1978;75:4533e7. [42] Roggendorf M, Lu M. Woodchuck hepatitis virus. In: Thomas TH, Zuckermann A, Lemon S, editors. Viral hepatitis. 3rd ed. Oxford: Blackwell Publishing Ltd; 2005. p. 210e24. [43] Roggendorf M, Lu M. The woodchuck: a model for studies on immunopathogenesis and therapy of hepadnaviral infection. In: von Weiza¨cker F, Roggendorf M, editors. Models for viral heptitis. Monogr virol. Basel: Karger (Review); 2005. p. 1e27.

303

[44] Pascarella S, Bossa F. A simple microcomputer program for predicting the secondary structure of proteins. Comput Methods Programs Biomed 1987;24:207e8. [45] Lu M, Lohrengel B, Hilken G, Kemper T, Roggendorf M. Woodchuck gamma interferon upregulates major histocompatibility complex class I transcription but is unable to inhibit the woodchuck hepatitis virus (WHV) replication in persistently WHV-infected woodchuck primary hepatocytes. J Virol 2002;76:58e67. [46] Thimme R, Wieland S, Steiger C, Ghrayeb J, Reimann KA, Purcell RH, et al. CD8(þ) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol 2003;77:68e76. [47] Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995;13:29e60. [48] Wen YM, Wu XH, Hu DC, Zhang QP, Guo SQ. Hepatitis B vaccine and anti-HBs complex as approach for vaccine therapy. Lancet 1995;345: 1575e6. [49] Couillin I, Pol S, Mancini M, Driss F, Brechot C, Tiollais P, et al. Specific vaccine therapy in chronic hepatitis B: induction of T cell proliferative responses specific for envelope antigens. J infect Dis 1999;180:15e26. [50] Pol S, Nalpas B, Driss F, Michel ML, Tiollais P, Denis J, et al. Efficacy and limitations of a specific immunotherapy in chronic hepatitis B. J Hepatol 2001;34:917e21. [51] Rubinstein MP, Kadima AN, Salem ML, Nguyen CL, Gillanders WE, Cole DJ. Systemic administration of IL-15 augments the antigen-specific primary CD8 þ T cell response following vaccination with peptidepulsed dendritic cells. J Immunol 2002;169:4928e35. [52] Xin KQ, Hamajima K, Sasaki S, Tsuji T, Watabe S, Okada E, et al. IL-15 expression plasmid enhances cell-mediated immunity induced by an HIV-1 DNA vaccine. Vaccine 1999;17:858e66. [53] Kwissa M, Kroger A, Hauser H, Reimann J, Schirmbeck R. Cytokinefacilitated priming of CD8 þ T cell responses by DNA vaccination. J Mol Med 2003;81:91e101.