重组乙肝病毒核心基因DNA与表面抗原-抗体共免疫应答研究

目的:了解小鼠对乙肝病毒核心抗原(HBcAg)基因免疫及与乙肝表面抗原-抗体复合物(HBsAg-抗HBs)联合免疫的应答。方法:用表达乙肝病毒核心抗原N端144个氨基酸的重组质粒DNA(简称pHBc144)100μg及含1μgHBsAg的重组乙肝表面抗原-鼠抗体组建的复合物(简称sIC)免疫Balb/c小鼠。用ELISA法检测血清抗体IgG和IgG1、IgG2a亚类的效价。用半定量PCR法分别检测鼠脾细胞IFN-γ及IL-4的mRNA。结果:pHBc144及sIC联合免疫鼠的抗HBs IgG、IgG1、IgG2a效价均显著高于sIC组(P＜0．05)。同时小鼠还可产生抗HBc及由HBsAg、HBcAg特异诱生的IFN-γ及IL-4。但与sIC共免疫,小鼠对HBcAg诱生的IFN-γ mRNA有所降低。结论:可用HBcAg基因与sIC共免疫,以获得针对乙肝病毒2种结构蛋白的免疫应答。     

IL-2 preS DNA疫苗免疫正常小鼠和HBV转基因鼠的免疫应答研究

目的 探讨IL—2 preS DNA疫苗作为预防和治疗性疫苗的可行性及作用机理。方法 应用基因重组技术，构建人白细胞介素2(hIL-2)和前表面抗原(preS)的真核表达载体，将此重组载体用基因枪分别注射正常的BALB／c小鼠和HBV转基因小鼠，通过ELISA方法检测BALB／c小鼠和HBV转基因鼠的抗-preS2、HBsAg及抗-HBs抗体水平；荧光定量PCR方法检测HBV转基因鼠血清中HBV DNA拷贝数，并用免疫病理HE染色观察小鼠肝组织炎症活动度，同时检测肝功能指标。结果 ①基因枪注射真核表达质粒免疫正常小鼠后，100％小鼠能在第4、6周检测到抗preS1抗体，持续时间长达10周。②用IL-2 preS真核表达质粒基因枪肌肉注射方式优于正常肌肉注射和皮下注射的方式，且所需质粒量(10μg／只)仅为后者(100μg／只)的1／10。③在第4周高峰期检测IgG亚类，是诱导以TH1(IgG2a)细胞免疫为主的反应。④基因枪注射真核表达质粒(1μg／只)免疫转基因小鼠后，80％的小鼠产生了抗体，HBV DNA量下降，其中20％的小鼠HBsAg转阴。⑤HE肝组织染色显示：肝组织有明显的炎细胞浸润、肝细胞肿大、颗粒样变性、转氨酶升高。结论 IL-2 preS DNA疫苗能刺激小鼠机体产生体液和细胞免疫，可部分打破小鼠机体的免疫耐受，为新型乙肝疫苗和抗HBV持续性感染的特异性免疫治疗剂的设计和构建提供理论与实践依据。     

质粒DNA促进HBsAg-抗HBs复合型疫苗诱生的免疫应答的研究

目的　研究HBsAg 抗HBs 质粒DNA复合型疫苗的免疫原性及其诱生细胞免疫应答的类型。方法　分别以HBsAg、HBsAg 抗 HBs(IC)、pI/AmpHBs、IC pI/AmpHBs及IC pI/Amp免疫小鼠 ,检测抗 HBs的效价 ,分析抗 HBsIgG亚类 (ELISA) ;取免疫小鼠脾细胞 ,体外抗原刺激 ,用竞争性RT PCR方法检测IFN γ及IL 4mRNA转录水平。结果　IC pI/AmpHBs诱生的抗 HBs效价明显高于IC或pI/AmpHBs单独免疫组 ,其IgG2a/IgG1比值高于IC免疫组 ,而低于pI/AmpHBs免疫组。IC pI/AmpHBs免疫小鼠脾细胞在HBsAg刺激下 ,IFN γmRNA转录水平明显高于其他免疫组 ,其IFN γmRNA的T/C(目的片段Target/竞争片段Competitor)比值为IC免疫小鼠脾细胞的 10倍 ;IC pI/AmpHBs免疫小鼠脾细胞IL 4mRNA转录水平亦高于其他免疫组 ,其IL 4mRNA的T/C比值为IC免疫小鼠脾细胞的 2倍。结论　HBsAg 抗HBs 质粒DNA复合型疫苗在增强体液免疫应答的同时可诱导脾细胞IFN γ的表达水平增高。     

DNA疫苗诱导健康小鼠细胞免疫及HBV转基因小鼠抗-HBs产生

目的 :在HBVDNA疫苗成功地诱导健康小鼠体液免疫应答的基础上 ,探讨其作为抗 HBV治疗的可行性及作用机理。方法 :应用基因重组技术 ,构建编码HBV中蛋白 (preS2 +S)及人白细胞介素融合蛋白基因的真核表达质粒pS2 .S及pFP ,继经肌注免疫健康Balb C及HBV转基因 (Tg)小鼠并观察健康小鼠细胞免疫应答及HBVTg小鼠HBsAg的血清转换。结果 :1 体外HBsAg对DNA疫苗免疫后的T细胞的刺激呈浓度相关 ,HBsAg 30 μg ml时刺激pS2 .S免疫小鼠脾细胞增殖指数(5 6± 0 9)明显较pcDNA3 1组 (2 0± 0 5 )高。细胞培养上清液细胞因子水平检测结果 :免疫实验组IL 2及IFN γ的分泌水平明显较对照组高 ,而IL 4水平于各组影响不明显。 2 pS2 .S免疫小鼠局部引流淋巴结DCs诱导HBsAg致敏的T 细胞增殖指数(4 2 0 )较pcDNA3.1组 (2 5 5高 )。 3 高剂量的pS2 .S组与联合pFP组各有一只Tg小鼠分别于 2、4w开始发生HBsAg血清转换 ,且其抗体水平随时间而增长。结论 :结果表明HBVDNA疫苗能有效诱导HBsAg特异性的细胞免疫应答 ;HBV Tg小鼠初步实验结果为治疗型HBVDNA疫苗的深入研制提供了实验依据。     

抗乙肝药物ZY101对HBV转基因鼠体内抗病毒作用研究

目的:观察ZY101在HBV转基因BALB/c小鼠体内的抗HBV作用。方法:将30只雄性HBV转基因鼠随机分为阴性对照组(杜氏磷酸盐缓冲液,DPBS)、阳性对照组(每天一次灌胃给予拉米夫定150mg·kg-1)、ZY101低剂量组(1mg·kg-1)、ZY101中剂量组(3mg·kg-1)和ZY101高剂量组(10mg·kg-1),每组6只,阳性对照组每天一次灌胃给药,其他组单次尾静脉注射。药前、药后后1、2、3、4、5、6、7、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38 w采集血清,用ELISA试剂盒检测HBsAg、HBeAg的表达,用实时荧光定量PCR技术检测血清中HBV DNA的含量。药后38w处死所有动物,采集肝组织,检测肝组织中HBsAg和HBeAg表达及HBV DNA和RNA的含量。结果:拉米夫定每天灌胃给予HBV转基因小鼠150mg·kg-1能够明显降低血清HBV DNA水平(P0.05),但对HBsAg、HBeAg和HBV RNA无明显影响;ZY101分别单次尾静脉注射给予HBV转基因小鼠1、3、10mg·kg-1,能够降低血清及肝组织中HBsAg和HBeAg的水平(P0.05、P0.05、P0.01),且有剂量依赖性,但血清HBV DNA及肝组织HBV DNA、HBV RNA水平无显著影响。结论:在本实验条件下,拉米夫定每天灌胃给予HBV转基因小鼠150mg·kg-1能够降低血清HBV DNA水平;ZY101单次尾静脉注射给予HBV转基因小鼠1、3、10mg·kg-1,能够剂量依赖的降低HBsAg和HBeAg的表达水平。     

乙型肝炎疫苗联合B7-H1蛋白疫苗对HBV转基因小鼠抗HBsAg免疫应答的影响

目的:研究B7-H1蛋白疫苗对HBV转基因小鼠免疫应答的影响,探索治疗慢性乙型肝炎的新方法。方法:用不同剂量的乙型肝炎疫苗和B7-H1蛋白疫苗联合免疫HBV转基因小鼠,应用ELISA法检测转基因小鼠在不同时间点血清抗B7-H1抗体滴度,同时在免疫后第14周末处死小鼠取脾细胞,检测不同的免疫方法对小鼠脾细胞产生HBsAg特异性Th1类细胞因子(IFN-γ及IL-2)、对HBsAg特异性分泌IFN-γT细胞数量及对小鼠淋巴细胞增殖的影响。结果:成功完成小鼠的免疫计划,5周起血清中即能测到B7-H1抗体,同一时间点各组之间的抗体滴度值并无明显差异。加B7-H1蛋白免疫各组与相同剂量单用HBsAg蛋白免疫各组相比:IL-2均明显减低(P<0.05),分泌IFN-γT的T细胞数量下降,但脾淋巴细胞分泌IFN-γ的水平各组间无明显差异;MTT法测定的淋巴细胞增殖能力各组间也无明显变化。结论:B7-H1蛋白疫苗可诱导HBV转基因小鼠产生明显的抗B7-H1抗体应答,但不能增强抗HBsAg的免疫应答。较小剂量的HBsAg即可引起HBV转基因小鼠Th1类细胞因子(IFN-γ及IL-2)的分泌以及淋巴细胞增殖。     

表达HIV-1 gag-gp120嵌合基因的DNA疫苗在小鼠体内的免疫应答

目的 :检测表达HIV 1gag gp12 0嵌合基因的DNA疫苗在小鼠体内的免疫应答效果。方法 :将真核表达质粒pVAXGE肌肉注射BALB C小鼠 ,观察免疫小鼠脾T淋巴细胞亚群的数量、特异性CTL杀伤率及小鼠免疫后不同时间点血清中IgG抗体滴度。结果 :重组质粒pVAXGE免疫组小鼠脾淋巴细胞进行了增殖 ,脾特异性CTL杀伤率显著高于对照组 (P <0 0 1) ;小鼠免疫后于第 8周血清抗体达到最高。结论 :表达HIV 1gag gp12 0嵌合基因的DNA疫苗质粒可诱导BALB C小鼠发生免疫应答     

DNA免疫诱导小鼠产生抗人心肌肌钙蛋白T抗血清的研究

目的 通过基因免疫诱导BALB／c小鼠产生抗人心肌肌钙蛋白T(cTnT)的抗体应答，并比较不同免疫佐剂、预处理方法对免疫应答的影响。方法 构建cTnT的真核表达质粒pCI-cTnT，大量提取、PEG纯化质粒，通过肌肉注射免疫BALB／c小鼠并加强免疫，每隔2周取血，ELISA法分析抗cTnT的体液应答，免疫印迹检测抗血清的特异性。并比较布吡卡因预处理、CpG ODN核酸佐剂、弗氏不完全佐剂对抗体应答效果的影响。结果 接种pCI-cTnT质粒的小鼠血清中检测出抗cTnT特异性抗体，加强免疫使抗体滴度提高；使用CpGODN后免疫应答增强，弗氏不完全佐剂可明显增强免疫效果，而吡卡因预处理使应答水平下降；初次免疫后14周仍可测得较高的抗体应答。结论 采用含cTnT基因的质粒DNA免疫，成功地诱导小鼠产生抗cTnT的抗体；传统的弗氏不完全佐剂可作为核酸免疫的佐剂使用。     

同基因背景小鼠核蛋白诱导狼疮鼠模型

背景：自发性狼疮鼠模型不能对基因以外其他的致病因素进行研究。 目的：以同基因背景Balb/c小鼠核蛋白免疫小鼠后诱导狼疮鼠模型。 方法：选取4~6周SPF级Balb/c小鼠30只,等分为3组。V1组肌肉注射提取的同系Balb/c小鼠核蛋白,间隔3周免疫1次,共免疫4次;V2组注射等体积PBS;V3组为正常对照。检测小鼠末次免疫后3周的24 h尿蛋白、血清抗核抗体、抗双链DNA抗体、小鼠肾脏直接免疫荧光。 结果与结论：V1组小鼠24 h尿蛋白、血清抗双链DNA抗体、抗核抗体均明显高于V2组、V3组,且V1组小鼠肾小球有免疫球蛋白G免疫复合物沉积,可见肾小球轮廓,V2组、V3组未见肾小球轮廓,只见非特异性的微弱荧光。说明以同基因背景Balb/c小鼠核蛋白免疫Balb/c小鼠能够成功诱导狼疮鼠模型。     

IL-18和HIV-1 gag-gp120嵌合基因DNA疫苗联合免疫小鼠的免疫应答

目的 :探讨IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫小鼠的免疫应答。方法 :构建含IL 18的真核表达质粒pVAXIL18,将他与表达HIV 1gag gp12 0嵌合基因的核酸疫苗质粒pVAXGE共同肌注免疫BALB/c小鼠 ,检测免疫小鼠脾特异性CTL杀伤活性和血清抗体滴度。结果 :联合免疫组小鼠脾特异性CTL杀伤活性和血清抗体水平均显著高于单独免疫组 (P <0 .0 5 ) ,空白质粒对照组 (P <0 .0 1)和PBS对照组 (P <0 .0 1)。结论 :IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫可诱导小鼠产生特异性细胞和体液免疫 ,且IL 18发挥了免疫佐剂的作用。     

CpG ODN增强乙型肝炎表面抗原免疫小鼠的抗体产生

目的：探讨合成含CpG基序的寡核苷酸（CpG ODN)对重组乙型肝炎表面抗原（rHBsAg)及乙型肝炎疫苗增强小鼠特异性抗体产生的效应。方法：采用非纯系（Km)及纯系（Balb/c)小鼠作为免疫对象，经后腱胫骨前肌免疫2次，ELISA法检测血清乙型肝炎表面抗体（抗－HBs)效价。结果：加CpG ODN组，其抗-HBs效价均较单独注射rHBsAg和疫苗组明显增高，持续时间长，且纯系鼠的抗体效价明显高于非纯系鼠。结论：CpG ODN对小鼠抗－HBs产生具有增强作用，具与疫苗中的铝佐剂有协同效应。     

Mucosal Immunity Induced by Pneumococcal Glycoconjugate

Host defenses against Streptococcus pneumoniae involve opsonophagocytosis mediated by antibodies and complement. Because the pneumococcus is a respiratory pathogen, mucosal immunity may play an important role in the defense against infection. The mechanism for protection in mucosal immunity consists of induction of immunity by the activation of lymphocytes within the mucosal-associated lymphoid tissues, transport of antigen-specific B and T cells from inductive sites through bloodstream and distribute to distant mucosal effector sites. Secretory IgA is primarily involved in protection of mucosal surfaces. Mucosal immunization is an effective way of inducing immune responses at mucosal surfaces. Several mucosal vaccines are in various stages of development. A number of mucosal adjuvants have been proposed. CpG oligodeoxynucleotide (ODN) has been shown to be an effective mucosal adjuvant for various antigens. Mucosal immunity induced by intranasal immunization was studied with a pneumococcal glycoconjugate, using CpG ODN as adjuvant. Mice immunized with type 9V polysaccharide (PS) conjugated to inactivated pneumolysin (Ply) plus CpG produced high levels of 9V PS IgG and IgA antibodies compared to the group that received the conjugate alone. High levels of subclasses of IgG1, IgG2 and IgG3 antibodies were also observed in sera of mice immunized with 9V PS-Ply plus CpG. In addition, high IgG and IgA antibody responses were observed in sera of young mice immunized with 9V PS-Ply plus CpG or the conjugate plus non-CpG compared with the group received the conjugate alone. These results reveal that mucosal immunization with pneumococcal glycoconjugate using CpG as adjuvant can confer protective immunity against pneumococcal infection.     

Mucosal immunity induced by pneumococcal glycoconjugate

Host defenses against Streptococcus pneumoniae involve opsonophagocytosis mediated by antibodies and complement. Because the pneumococcus is a respiratory pathogen, mucosal immunity may play an important role in the defense against infection. The mechanism for protection in mucosal immunity consists of induction of immunity by the activation of lymphocytes within the mucosal-associated lymphoid tissues, transport of antigen-specific B and T cells from inductive sites through bloodstream and distribute to distant mucosal effector sites. Secretory IgA is primarily involved in protection of mucosal surfaces. Mucosal immunization is an effective way of inducing immune responses at mucosal surfaces. Several mucosal vaccines are in various stages of development. A number of mucosal adjuvants have been proposed. CpG oligodeoxynucleotide (ODN) has been shown to be an effective mucosal adjuvant for various antigens. Mucosal immunity induced by intranasal immunization was studied with a pneumococcal glycoconjugate, using CpG ODN as adjuvant. Mice immunized with type 9V polysaccharide (PS) conjugated to inactivated pneumolysin (Ply) plus CpG produced high levels of 9V PS IgG and IgA antibodies compared to the group that received the conjugate alone. High levels of subclasses of IgGI, IgG2 and IgG3 antibodies were also observed in sera of mice immunized with 9V PS-Ply plus CpG. In addition, high IgG and IgA antibody responses were observed in sera of young mice immunized with 9V PS-Ply plus CpG or the conjugate plus non-CpG compared with the group received the conjugate alone. These results reveal that mucosal immunization with pneumococcal glycoconjugate using CpG as adjuvant can confer protective immunity against pneumococcal infection.     

包裹天然骨架CpG ODN和HBsAg的非磷脂脂质体疫苗的免疫效果

非磷脂脂质体NovasomeR○(Np )是由Brij5 2、胆固醇和油酸组成 ,可作为同时传递佐剂和抗原的载体。我们将HBsAg与天然骨架CpGODN (phosphodiesterCpGODN ,pdCpGODN )包裹于Np后免疫BALB/c小鼠 ,检测其免疫效果。结果显示 ,包裹pdCpGODN和HBsAg的Np在小鼠中诱导了很高滴度的抗 HBs抗体产生并诱生了HBsAgS2 8 3 9特异性的CTL ,而铝佐剂组和仅包裹HBsAg的Np组诱生的抗体滴度较低 ,未检测到CTL活力。抗体亚类分析结果表明包裹pdCpGODN和HBsAg的Np诱生的免疫应答类型与pdCpGODN剂量有关 ,较低剂量 (2 4 μgpdCpGODN )诱生的为IgG2a为主的Th1型应答 ,而较高剂量(4 7μgpdCpGODN )诱生的是Th1/Th2混合型应答。铝佐剂和仅包裹HBsAg的Np组诱生的是以IgG1为主的Th2型应答。此外 ,包裹pdCpGODN和HBsAg的Np免疫小鼠脾淋巴细胞在体外HBsAg刺激培养后特异性增殖并分泌高水平的IFN γ。这些结果表明包裹pdCpGODN和HBsAg的Np能增强HBsAg的免疫原性 ,诱生体液 /细胞免疫均衡应答 ,有可能发展为慢性乙肝的治疗性疫苗     

Immunization against hepatitis B virus by mucosal administration of antigen-antibody complexes

Antigen-antibody complexes have been shown to enhance immune responses against several antigens given by parenteral immunization. Herein, we have evaluated the potential of administering such immunostimulatory complexes by a mucosal route. Hepatitis B surface antigen (HBsAg) complexed with antibodies against HBsAg (anti-HBs) (HBsAg/Ab) was administered to BALB/c mice by intranasal inhalation. HBsAg by itself did not induce immune responses, whereas with HBsAg/Ab complexes, both systemic and mucosal immune responses were observed and these could be modulated by adjuvants. With HBsAg/Ab (1 or 10 microg), anti-HBs antibodies induced were predominantly of the IgG1 isotype (Th2-like). In contrast, anti-HBs induced by HBsAg/Ab plus cholera toxin (CT) or oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG) (1 microg each) were predominantly IgG2a (Th1-like). Results from this study indicate that HBsAg/Ab complexes can induce strong humoral immune responses when delivered by a noninvasive route, whether used alone or in combination with other mucosal adjuvants.     

Immunomodulation of Japanese encephalitis vaccine through CpG oligodeoxynucleotides in mice

Synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine guanine (CpG) dinucleotides motifs act as immune adjuvant and provide means of modulation to immune responses when co-delivered with antigens. They stimulate both innate and adaptive immune responses and induce T helper 1 (Th1) immune responses. We investigated the immunomodulation of Japanese encephalitis (JE) vaccine using CpG ODN as an adjuvant. Mice were immunized with one dose of JE vaccine 0.1 ml with different concentrations (10, 25 and 100 microg) of CpG ODN. The serum antibody level and cytokines were evaluated and compared with mice immunized with two doses of JE vaccine alone. Our studies revealed that anti-JE antibody level in mice immunized with single dose of 0.1 ml JE vaccine and 100 microg CpG ODN were almost equal to mice immunized with two doses of JE vaccine alone. Furthermore, CpG ODN enhanced the production of TNF-alpha and Th1-mediated cytokines, including IFN-gamma and IL-2 compared with JE vaccine alone. In addition, absence of any significant changes in biochemical, haematological and histological studies suggest that CpG ODN are safe adjuvants for JE vaccine. Therefore, it is inferred that CpG ODN are effective and improve the efficacy of JE vaccine.     

IL-2的真核表达载体对乙肝病毒基因疫苗的免疫佐剂效应

单以乙肝病毒S区基因疫苗 (pCR3 1 S)或联合IL 2真核表达载体 (pDOR IL 2 )注射BALB/c小鼠 (H 2 d)股四头肌 ,ELISA法检测小鼠血清抗HBs,4h51Cr释放法检测小鼠脾细胞CTL活性。免疫 8周后 ,单注射pCR3 1 S及共注射IL 2真核表达载体的小鼠血清 45 0nmA值分别为 1 2 4± 0 1及 1 98± 0 17。CTL细胞杀伤活性分别为 (5 0 5± 6 4) %、 (6 1 9± 7 1) % ,两组均有明显差异 (P <0 0 1)。脾细胞悬液经抗CD4+ 单克隆抗体处理后CTL细胞杀伤活性分别为 (4 8 3± 5 9) %、 (5 6 2±6 1) % ,抗CD8+ 单克隆抗体处理后分别为 (10 6± 1 4) %、 (13 6± 1 3) %。结果表明 ,IL 2的真核表达载体能够提高小鼠对DNA疫苗的免疫应答 ,CTL细胞杀伤活性主要由CD8+ 执行。基因疫苗可能用于预防及治疗HBV感染。     

HBV与HCV融合DNA疫苗的构建及其体液免疫应答

目的 构建含乙型肝炎病毒（HBV）表面抗原基因（S区基因）与丙型肝炎病毒（HCV）核心抗原基因（C区基因）的嵌合真核表达载体,观察preS1和preS2基因对HBV表面抗原及HCV核心抗原体液免疫的影响。方法 用PCR方法,分别扩增HBV S区基因和HCV C区基因。将S区基因克隆入真核表达载体pcDNA3.1,酶切鉴定后,大量提取质粒并免疫Balb/c小鼠,用ELISA法检测抗HBs和抗HCV抗体。结果 成功地扩增出目的基因片段,克隆后酶切鉴定结果正确,序列分析与文献报告相一致。免疫后检测到抗HBs和抗HCV抗体。preS1与preS2基因对构建的融合DNA疫苗的体液免疫应答有一定的抑制作用。抗HBs抗体的产生低于只含S基因的真核表达载体;preS1基因对抗HCV抗体的产生具有抑制作用,而preS2无影响。结论 不同长度的HBV S区基因可影响抗HBs和抗HCV抗体的产生。     

CpG DNA中ISS数量与免疫刺激效果间的关系初探

研究CpG免疫刺激DNA中免疫刺激序列（ISS）的数量与免疫刺激效果间的关系。方法：将3种CpGODN（分别含2,4,8个ISS）与甲型肝炎灭活疫苗混合,通过腹腔注射途径免疫小鼠,于免疫后4,6,8w采血,用全自动微粒子酶联免疫测定分析检测抗HAVIgG水平.结果：含2个ISS的CpG ODN具有较好的佐剂效应,能增强甲型肝炎灭活疫苗的免疫效果,含4个ISS的CpG ODN不具有免疫刺激作用,而含8个ISS的CpG ODN则抑制了甲型肝炎灭活疫苗所诱导的免疫反应,结论：适当长度的CpG ODN（含2个ISS）可作为有效的佐剂来增强疫苗的免疫效果,而当其中的ISS含量过多,则反而具有免疫抑制作用。     

Inhibition of the hepatitis B virus replication in vitro by an oligodeoxynucleotide containing cytidine-guanosine motifs

Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. CpG ODNs show promise as vaccine adjuvants and immunoprotective agents in animals. Here, we investigated the inhibitory effects of D type CpG ODN on hepatitis B virus (HBV) replication in vitro. The experiments were performed in HepG2 2.2.15 cells, which contain an integrated tandem dimer of the HBV genome and are routinely used for anti-HBV study. HepG2 2.2.15 cells co-cultured with peripheral blood mononuclear cells (PBMCs) plus CpG ODN for 3 days, remarkably reduced the secretion of HBsAg and HBeAg, when compared to cells treated with PBMCs plus non-CpG ODN. The levels of intracellular HBV DNA and HBV mRNA were also decreased. Treatment of HepG2 2.2.15 cells with the culture supernatants of PBMCs activated by CpG ODN can remarkably suppress the secretion of HBsAg and HBeAg as compared with that of PBMCs without CpG ODN activation under the same conditions. There were no inhibitory effects on the replication of HBV to be found for CpG ODN treatment alone. These results suggest that CpG ODN can inhibit indirectly HBV replication in vitro via activating the immune cells, and could contribute to the development of an immunoregulator against HBV infection.