Original article CpG oligodeoxynucleotide inhibits HBV replication in a hydrodynamic injection murine model

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1 Antivirl Therpy 205; 20: (doi: 0.385/IMP2870) Originl rticle CpG oligodeoxynucleotide inhibits HBV repliction in hydrodynmic injection murine model Wei Hu, Hi Hung,2, Ting-Yu Zhng,2, Ying-Ying Mo, Xue-Jun Wng *, Sheng-Qi Wng,2 * Beijing Institute of Rdition Medicine, Beijing, Chin 2 Henn University of Trditionl Chinese Medicine, Zhengzhou, Chin *Corresponding uthor e-mils: xjwng@bmi.c.cn; sqwng@bmi.c.cn These uthors contributed eqully to this work Bckground: Chronic HBV infection is significnt public helth problem nd one mjor cuse of liver cirrhosis nd heptocellulr crcinom (HCC). HBV impirs the host immune system nd results in immunotolernce, which is mjor obstcle to HBV therpy. CpG oligodeoxynucleotide (ODN) is strong immunostimulnt which ctivtes the innte immune response rpidly nd hs been shown to be n efficient therpy gent in virl infection tretment. Here, we report the nti- HBV ctivity of CpG-826 in hydrodynmic injection murine model. Methods: CpG-826 ws dministrted intrperitonelly every other dy in HBV crrier mice estblished by til vein hydrodynmic injection of HBV plsmids. The serum concentrtions of HBV surfce ntigen (HBsAg), HBV e ntigen (HBeAg), HBV surfce ntibody (HBsAb), HBV core ntibody (HBcAb), interferon- (IFN-) nd interferon-g (IFN-g) were mesured by enzyme-linked immunosorbent ssy (ELISA). The ctivities of lnine minotrnsferse (ALT) were determined by ALT kit using Spectrmx Plus spectrophotometer. Heptic HBV DNA ws quntified by quntittive rel-time PCR. The expression of HBV core ntigen (HBcAg) in liver ws detected by immunohistochemistry. Drug toxicity of CpG-826 ws evluted by body weighting nd liver histopthology confirmtion. Results: CpG-826 dministrtion inhibited HBV repliction efficiently with significnt reduction of serum HBsAg nd HBeAg, heptic HBcAg nd HBV DNA levels. The serum levels of IFN-, IFN-g nd HBsAb incresed but the HBcAb level declined in the CpG-826 group compred to CpG-982 nd control groups. Results of ALT ctivity indicted no significnt difference mong CpG-826 group, CpG-982 nd control groups. Body weighting nd histopthology exmintion showed no obvious toxicity. Conclusions: Given the stimultion ctivity of host immune system, CpG ODN is promising strtegy for HBV therpy with more relevnt reserch needed. Introduction Heptitis B is mjor helth-cre problem cused by HBV with bout 350 million humn infections worldwide. HBV infection cn led to cute heptitis, chronic heptitis nd hs close reltionship with liver cirrhosis nd heptocellulr crcinom []. HBV infection nd the relted diseses hve serious effects on ptients helth nd there is still no specific drug for HBV clernce. Nowdys, the widely used gents for HBV infection include interferon (IFN) nd nucleoside/nucleotide nlogues (NA), but they hve disdvntges for serious side effects or virl relpse. Consequently, there is gret need for new nd efficient medicine development for HBV infection. Unmethylted CpG dinucleotides within prticulr sequence context (CpG motifs) re present in high frequency in bcteril DNA nd re responsible for the stimultion of the innte immune system through ctivtion of Toll-like receptor 9 (TLR9) [2 4]. Synthetic CpG oligodeoxynucleotides (CpG ODNs) contining these CpG motifs hve similr effects on TLR9 nd could initite the innte immune response with the secretion of series of proinflmmtory cytokines nd/or type I IFNs which ply importnt roles in innte nd dptive immune responses [5]. CpG ODNs hve been shown to be excellent ntivirl gents in vriety of studies recently for cytomeglovirus, influenz virus nd so on [6 9]. The inherent immune stimultion bilities of CpG ODNs mke it n excellent drug cndidte. Moreover, some improvements such s chemicl modifiction of nturl CpG ODN bckbones 205 Interntionl Medicl Press (print) (online) 289

2 W Hu et l. [0,] nd the ppliction of delivery system provide CpG ODNs with excellent properties for drug development [2,3]. CpG-826, s representtive of these CpG ODNs, ws shown to hve strong immunostimultory bilities in mice nd ws n effective vccine djuvnt ginst virl nd bcteril infections [4,5] nd cncer [6 8]. In this study, we evluted the nti-hbv effects of CpG-826 in n HBV hydrodynmic injection mouse model nd showed tht CpG-826 could inhibit HBV repliction significntly in vivo. In the future, it is necessry to select new types of CpG ODNs which cn be used s effective drugs or supplements for the existing nti- HBV drugs such s IFN nd NA drugs for humn beings. Methods Estblishment of n HBV crrier mice model mice (mle, 4 6 weeks old, 6 8 g, SLAC Lbortory Animl, Vitl River Lbortory Animl Technology Co. Ltd, Beijing, Chin) were chosen for the estblishment of n HBV crrier mouse model by the til vein hydrodynmic injection of 6.0 mg HBV expression vector (phbv.8) [9]. This vector contins.8 greter-thn-genome-length HBV replicon (genotype B) which cme from clinicl chroniclly HBV-infected ptient. phbv.8 ws dissolved in 0% mouse body weight of endotoxin-free phosphte-buffered sline () nd the injection procedure ws completed in 5 8 s [20,2]. Two dys lter, the mice were eqully divided into four groups with ten nimls in ech group nd the expression levels of heptitis B surfce ntigen (HBsAg) nd heptitis B e ntigen (HBeAg) were similr mong groups. In vivo tretment of CpG 826 Completely phosphorothiote-modified CpG-826 (5 -TCCATGACGTTCCTGACGTT-3 ) nd the control ODN CpG-982 (5 -TCCAGGACTTCTCTCAG- GTT-3 ) were synthesized by our own lb nd purified by HPLC. After grouping, the nimls were treted either with 0.2 ml buffer (negtive control) or with different doses of CpG-826 (0.3 mg/kg,.0 mg/kg, 3.0 mg/kg), respectively. The CpG-982 ws lso used s negtive control ODN in the other experiments to determine the specific ctivity of CpG The bove tretments were crried out vi the intrperitonel route every other dy, 2 in ll. Ech niml ws weighed fter the first tretment of CpG 826 nd then every other 3 or 4 dys until the end of the experiment. Serologicl nd biochemicl nlysis For detection of HBV ntigens, HBV ntibodies nd IFN-/IFN-g, the mouse blood smples were collected on different dys fter mking the mice model nd diluted properly with. The diluted blood ws centrifuged t 5,000 rpm for 5 min to get the serum smples. The levels of these chemicl prmeters were mesured by HBsAg/ HBeAg, heptitis B surfce ntibody (HBsAb)/ heptitis B core ntibody (HBcAb) quntittive ELISA kit (Tigsun Dignostics Co., Ltd, Beijing, Chin), nd IFN-/ IFN-g ELISA kit (Shnghi Westng Bio-tech Co., Ltd, Shnghi, Chin) ccording to the mnufcturer s protocols. The mesuring rnges of these kits re s following: HBsAg ( ng/ml), HBeAg ( ntionl clinicl unit [NCU]/ml), HBsAb (5 500 miu/ml), HBcAb (0. 2 NCU/ml), IFN- (5 2,000 pg/ml) nd IFN-g (2 400 pg/ml). For detection of lnine minotrnferse (ALT) ctivity, mouse vein serum smples were collected s described bove with no dilution. ALT ctivity ws mesured by ALT kit (BioSino Bio-technology nd Science Inc., Beijing, Chin) using Spectrmx Plus spectrophotometer (Moleculr Devices, Sunnyvle, CA, USA) ccording to the mnufcturer s instructions. The mesuring rnge of the kit is U/l. HBV DNA quntifiction The edge tissue of the liver (bout 0. g) ws removed fter the lst blood collection, lysed in ml (per 0. g) lysis solution (0.5% Nonidet P-40, 50 mm Tris-HCl, mm EDTA, ph 8.0) for 0 min t room temperture (RT) nd homogenized t 60 Hz for 90 s. The lyste ws centrifuged t,000 g for min to remove the nuclei. Cellulr debris in the superntnt ws clered by n dditionl centrifugtion t 4,000 g for 5 min. For elimintion of residul plsmid DNA nd unencpsidted HBV RNA in the superntnt, micrococcl nuclese (New Englnd Biolbs, Ipswich, MA, USA) digestion in 5 mm CCl 2 ws performed t 37 C overnight [22]. The digestion ws inctivted by EDTA (0 mm) nd HBV DNA in virl prticles ws extrcted by Column Virl DNAout kit (TIANDZ, Beijing, Chin) ccording to the mnufcturer s protocol. HBV DNA copies were quntified by commercil quntittive PCR kit (Puruikng, Shenzhen, Chin). Histomorphology of liver nd immunohistochemistry for HBcAg After blood collection finished, liver tissue ws removed, fixed in 4% neutrl buffered formlin, prffin embedded nd sectioned t 3 mm for hemtoxylin-eosin (HE) stining in the conventionl routine. The stined section ws exmined under microscopy in blinded mnner. To detect the repliction of HBV, the immunohistochemicl stining for HBcAg ws performed using rbbit nti-hbc ntibody (DAKO, Crpinteri, CA, USA) Interntionl Medicl Press

3 CpG ODN inhibits HBV repliction in murine model Sttisticl nlysis The dt re expressed s mens ± stndrd devition (sd) nd the sttisticl significnce ws determined by the Student s t-test or one-wy ANOVA. Difference ws considered sttisticlly significnt t P-vlue of <0.05. Results CpG-826 inhibited HBV repliction in dosedependent mnner in mice ELISA results of mouse til vein serum smples indicted tht CpG-826 could inhibit serum HBsAg nd HBeAg expression in dose-dependent mnner. Serum HBsAg nd HBeAg were bsiclly removed in CpG mg/kg-dose group 2 dys fter HBV modelling (Figure A nd B). The other two groups treted with CpG-826 lso showed significnt reduction of HBsAg nd HBeAg t the sme time. In nother experiment, there ws significnt reduction of HBsAg in ser of the CpG-826-treted group compred to the nd ODN control (CpG-982-treted) groups 4 dys fter HBV modelling (Additionl file ). The results showed specific inhibition of HBsAg expression by CpG-826. After the lst blood collection, liver tissue ws removed nd HBV DNA in heptocytes ws extrcted nd quntified by quntittive rel-time PCR. As shown in Figure C, HBV DNA repliction ws significntly reduced in heptocytes in three groups treted with CpG-826. HBV DNA nerly turned to be negtive in 3.0 mg/kg-dose group while more thn,000 reduction ws observed in the other two groups. In Additionl file, HBV DNA of vein ser ws lso specificlly inhibited by CpG-826 compred to the nd ODN control (CpG-982-treted) groups. The HBcAg levels in heptocytes were detected by immunohistochemicl stining when blood smpling ws complete. Both microscope observtion (Figure D) nd the following mesurement of percentges of HBcAgpositive liver cells (Figure E) indicted tht HBcAg expression in the liver tissues declined grdully s the Figure. CpG-826 intrperitonel tretment inhibited HBV repliction in dose-dependent mnner in mice A ng/ml, Copies/ml HBsAg Dy HBV DNA CpG-826 (3.0 mg/kg) CpG-826 (.0 mg/kg) CpG-826 (0.3 mg/kg) CpG-826 (0.3 mg/kg) CpG-826 (.0 mg/kg) CpG-826 (3.0 mg/kg) NCU/ml HBcAg Dy (HBV model) CpG-826 (0.3 mg/kg) CpG-826 (.0 mg/kg) CpG-826 (3.0 mg/kg) 0 0. HBeAg C D E 00 µm 00 µm 00 µm 00 µm 00 µm CpG-826 (0.3 mg/kg) CpG-826 (.0 mg/kg) CpG-826 (3.0 mg/kg) Percentge of liver cells HBcAg CpG-826 (0.3 mg/kg) CpG-826 (.0 mg/kg) CpG-826 (3.0 mg/kg) Different doses of CpG-826 were delivered every other dy nd mounted to 2 in ll. (A) Heptitis B surfce ntigen (HBsAg) nd (B) heptitis B e ntigen (HBeAg) expression levels in serum were mesured by ELISA. Ech smple ws diluted 20-fold. (C) At the end of the experiment, 6 dys fter the lst tretment of CpG 826, HBV DNA in heptocytes ws extrcted nd nlysed by quntittive rel-time PCR (qpcr). (D) Immunohistochemicl nlysis of heptitis B core ntigen (HBcAg) expression in liver t the end of the experiment. Originl mgnifiction: 320. (E) Mesurement of percentges of HBcAg-positive liver cells on immunohistochemicl sections. P<0.00. b P<0.0. NCU, ntionl clinicl unit;, phosphte-buffered sline. B b Antivirl Therpy

4 W Hu et l. dose of CpG-826 incresed until it ws lmost undetectble in the 3.0 mg/kg-dose group. Toxicity of CpG-826 tretment to mice Hispthologicl exmintion on HE-stined liver sections showed severe liver lesions, heptic lobule dmge, inflmmtory infiltrtes round vessels, disrrnged heptocytes with lymphocytic infiltrtion in control group. The hydrodynmic HBV mice hd milder symptoms s the dose of CpG-826 incresed nd there ws no pprent pthologicl chnge in 3.0 mg/kg-dose group compred to the mice (Figure 2). Mice were weighed from the first tretment of CpG-826 until the end of the experiment. Weight gin occurred in ll CpG-826-treted groups during the experiment. Although the 3.0 mg/kgdose nd.0 mg/kg-dose groups of mice weighed less thn the control group, there were no significnt differences between them (Additionl file 2). Mesurement of serum ALT ctivity lso showed no pprent difference mong CpG ODNs nd -treted groups in nother experiment (Additionl file 3). Influence of CpG-826 on levels of HBsAb/HBcAb nd IFN-/IFN-g CpG-826 could trigger prolifertion of B-cells nd is n effective vccine djuvnt, we found tht the time of occurrence of HBsAb ws dvnced to the fifth week in the CpG-826-treted group compred to the seventh week in nd CpG-982 control groups (Tble ). There were more HBsAb-positive mice with higher ntibody titres in the CpG-826 group in the seventh week when the experiment ws finished (Figure 3A). In ddition, the time of occurrence of HBcAb ws delyed in CpG-826 group (Tble 2) with much lower titres of HBcAb compred to the nd CpG-982 control groups (Figure 3B). Incresing IFN production is n importnt wy for CpG ODNs to induce innte immunity. Therefore, the levels of IFN- nd IFN-g in serum were mesured by ELISA fter the CpG ODNs dministrtion. As shown in Figure 3C nd 3D, secretions of IFN- nd IFN-g in the CpG-826 (3.0 mg/kg) group incresed significntly compred to the, nd CpG-982 control groups. The dt suggested tht tretment of CpG 826 fter HBV vector hydrodynmic injection could effectively enhnce the secretion of IFN- nd IFN-g. Discussion HBV develops suppression strtegies to impir the host immune system nd induces host immunotolernce [23 25]. For exmple, HBV suppresses the expressions Figure 2. Histopthologicl exmintion of liver structure (hemtoxylin-eosin stined) t the end of experiment 00 µm (HBV model) 00 µm CpG-826 (0.3 mg/kg) 00 µm 00 µm CpG-826 (.0 mg/kg) 00 µm CpG-826 (3.0 mg/kg) Originl mgnifiction, 200., phosphte-buffered sline. 292 AVT-4-OA-3233_Hu.indd Interntionl Medicl Press 8/05/205 09:6:5

5 CpG ODN inhibits HBV repliction in murine model Tble. The HBsAb in mice fter hydrodynmic injection of HBV plsmids nd tretment with ODNs Plsmids ODNs Dy 7 Dy 4 Dy 2 Dy 28 Dy 35 Dy 42 Dy 49 Untreted Untreted phbv phbv.8 CpG phbv.8 CpG mice were hydrodynmiclly injected with 6 mg of phbv.8 plsmids. The number (out of 6 nimls) for ech cohort showing nti-hbs-positivity is shown. HBsAb, heptitis B surfce ntibody; ODNs, oligodeoxynucleotides;, phosphte-buffered sline. Figure 3. Detection of HBsAb/HBcAb nd IFN-/IFN-g in serum A Anti-HBs Ab B Anti-HBc Ab, miu/ml 0 NCU/ml CpG-982 (3.0 mg/kg) CpG-826 (3.0 mg/kg) 0. CpG-982 (3.0 mg/kg) CpG-826 (3.0 mg/kg) C IFN-α D IFN-γ b pg/ml pg/ml CpG-982 (3.0 mg/kg) CpG-826 (3.0 mg/kg) 0 CpG-982 (3.0 mg/kg) CpG-826 (3.0 mg/kg) The levels of (A) heptitis B surfce ntibody (HBsAb) nd (B) heptitis B core ntibody (HBcAb) in serum were mesured t dy 49 by ELISA when the experiment ws finished. The levels of (C) interferon (IFN)- nd (D) IFN-g were mesured t dy 28 by ELISA fter phosphte-buffered sline () nd CpG ODN dministrtion ws finished. P<0.00. b P<0.0. NCU, ntionl clinicl unit. Antivirl Therpy

6 W Hu et l. Tble 2. The HBcAb in mice fter hydrodynmic injection of HBV plsmids nd tretment with ODNs Plsmids ODNs Dy 7 Dy 4 Dy 2 Dy 28 Dy 35 Dy 42 Dy 49 Untreted Untreted phbv phbv.8 CpG phbv.8 CpG mice were hydrodynmiclly injected with 6 mg of phbv.8 plsmids. The number (out of 6 nimls) for ech cohort showing nti-hbc positivity is shown. HBcAb, heptitis B core ntibody; ODNs, oligodeoxynucleotides;, phosphte-buffered sline. of IFN-/b, IFN-g nd inflmmtory cytokines of innte immune cells by disrupting pttern-recognition receptorsmedited ntivirl signlling pthwy [26,27]. Immune tolernce is thought to be key fctor for HBV persistent infection. Therefore, immunotolernce reversl, in ddition to virl repliction suppression nd host immunity enhncement, is n importnt trget for HBV therpy. Previous studies indicted tht CpG motifs cn stimulte the innte immune system directly by interction with TLR9 on humn B-cells nd dendritic cells, nd indirectly induce nd enhnce potent ntigen-specific Th-type immunity [2,3,5]. Becuse of their bility to potently intensify vertebrte immunity, synthetic CpG ODNs hve been studied for prevention nd tretment of different infections, nd the results hve shown tht CpG ODNs re sfe nd efficient immunostimultory gents for mny virl infections [6 9]. In previous nti-hcv infection reserch, the level of HCV RNA in ptients could be evidently reduced fter the tretment of CpG- 00 (ACTILON ) with no severe dverse rections except slight flu-like symptoms [28,29]. This indictes tht CpG ODN is n effective nti-heptitis virus gent. Some in vitro studies demonstrted tht the tretment of CpG ODN could stimulte the production of cytokines by peripherl blood mononucler cells nd inhibit HBV DNA repliction nd the secretion of HBsAg nd HBeAg in HepG cells [30], or could reduce the effective concentrtions of lmivudine for in vitro nti-hbv tretment when used with CpG ODNs together [3]. In the present study, we firstly estblished n HBV hydrodynmic injection mouse model for long-lsting HBV repliction. We showed tht there were very few HBsAb-positive mice seven weeks fter model construction (Tble ). This is probbly the reson why longterm HBV repliction could exist in this model (we hve detected HBV-positive expression for more thn one yer in other experiments; dt not shown). Previously, this model ws successfully used to evlute new short hirpin RNA scffold for HBV inhibition in our other report [2]. In this study, CpG-826 efficiently reduced HBV ntigen expression, inhibited HBV DNA repliction nd liver lesions fter dministrtion for 2 in this HBV model. Moreover, CpG-826 tretment hs no pprent toxicity for mice under the given doses. Although HBsAb occurrence time in the CpG- 826 group ws dvnced compred to the nd ODN control (CpG-982) groups, the occurrence time of HBcAb ws delyed nd there were still hlf of mice with no HBsAb detection in ll the -, CpG-982- nd CpG-826-treted groups when the experiment ws finished (Figure 3A): this seems prdoxicl but is very interesting. In the third week the HBsAg nd HBeAg levels reduced gretly nd there were no HBsAb nd HBcAb detected in the CpG- 826-treted group t tht time. This implies tht HBV possibly ws clered minly in n IFN-- nd IFN-g-dependent mnner (Figure 3C nd 3D) but not by the ntibody-dependent pthwy. A further experiment using nude mice my help to determine the exct mechnism. If CpG-826 could eliminte HBV by n ntibody-independent pthwy, it mens tht we could find the possible pthwys ctivted by CpG-826 to cler HBV covlently closed circulr (ccc)dna without cell injury (Additionl file 3), such s the A3A nd A3B molecules involved in HBV cccdna clernce reported recently [32]. However, more reserch is needed to determine the detiled mechnisms involved in this process. In subsequent study, we will use high-throughput sequencing techniques to further investigte the specific mechnism of CpG-826 for its HBV clernce ctivity in our HBV injection model. In conclusion, this is the first study to demonstrte tht tretment of CpG ODN lone could inhibit HBV repliction efficiently, specificlly nd sfely in the HBV hydrodynmic injection mouse model. The results re positive for the reserch nd development of new nd effective nti-hbv CpG ODN cndidtes in future for humn beings. Acknowledgements This work ws supported by the Chinese Science nd Technology Key Projects grnts 202ZX nd 202ZX , the Ntionl Nturl Science Foundtion of Chin nd the Ntionl High Technology Reserch nd Development Progrm of Chin 205AA (863 Progrm) Interntionl Medicl Press

7 CpG ODN inhibits HBV repliction in murine model Disclosure sttement All uthors declred no competing interests. Additionl files Additionl file : The specific inhibition of HBV repliction by CpG-826 cn be found t Addfile.pdf Additionl file 2: The body weight of mice during experiments cn be found t com/uplods/documents/3233_hu_addfile2.pdf Additionl file 3: The serum ALT levels of mice during experiments cn be found t com/uplods/documents/3233_hu_addfile3.pdf References. Dienstg JL. Heptitis B virus infection. N Engl J Med 2008; 359: Krieg AM, Yi AK, Mtson S, et l. CpG motifs in bcteril DNA trigger direct B-cell ctivtion. Nture 995; 374: Sto Y, Romn M, Tighe H, et l. Immunostimultory DNA sequences necessry for effective intrderml gene immuniztion. Science 996; 273: Klinmn DM, Yi AK, Beucge SL, Conover J, Krieg AM. CpG motifs present in bcteri DNA rpidly induce lymphocytes to secrete interleukin 6, interleukin 2, nd interferon gmm. Proc Ntl Acd Sci U S A 996; 93: Krieg AM. 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