乙肝核酸疫苗诱导BALB/C小鼠的CTL免疫应答

目的：研究携带HBsAg基因的载体质粒pcDHBs诱导小鼠CTL应答效果。方法：将HBsAg基因连接到真核表达载体pcDNA3．1上，构建成载体质粒pcDHBs。将纯化后的质粒pcDRBs和pcDNA3．1肌肉注射免疫小鼠，眼眶采血检测血清中抗体水平。用质粒pcDHBs转染P815细胞制备乙肝疫苗诱导BALB／C小鼠CTL活性检测的靶细胞。免疫后，取脾细胞，按效靶比为10：1、25：1、50：1进行CIL杀伤检测。结果：免疫pcDHBs疫苗后，检测到小鼠血清中的RBsAb。用pcDHBs进行转染的P815细胞能够检测到HBsAg基因片段和蛋白质抗原的表达。用pcDHBs免疫组小鼠的CTL杀伤率均明显高于pcDNA3．1免疫组。结论：质粒pcDHBs作为核酸疫苗能够诱导小鼠体液免疫应答和CTL免疫应答。     

不同免疫途径对chitosan-DNA疫苗诱导CVB3特异性免疫应答的影响及其意义

目的：确定新型chitosan-DNA疫苗的有效免疫途径。方法：将chitosan-pcDN3-VPI疫分别苗以肌注、口服、滴鼻3种免疫方式免疫Balb/c小鼠；以ELISA检测免疫小鼠血清中IgG、IgM、、IgA，评估其特异性体液免疫应答；以特异性淋巴细胞增殖反应和CTL活性反映其诱导细胞免疫；以5LD50致死剂量CVB3攻击免疫小鼠，评价不同免疫途径的免疫保护效果。结果：①在诱导CVB3特异性体液免疫方面：chitosan-pcDNA3-VPI疫苗肌注组诱生了高水平IgM和IgG，但未能诱生黏膜IgA；口服免疫组仅诱生低水平的黏膜IgA，未能诱生特异性IgM和IgG；滴鼻组可诱生低水平的I埘及高水平的IgG和黏膜IgA。②在诱导CVB3特异性细胞免疫方面：仅滴鼻组诱导了较高水平的淋巴细胞特异性增殖反应和CTL活性；口服组的淋巴细胞增殖活性和CTL活性稍弱；肌注组几乎不能诱导特异性细胞免疫应答。③免疫保护作用：滴鼻组可保护33．3％小鼠长期存活；口服组仅达到16．7％的保护率；肌注组无保护作用。结论：滴鼻免疫途径可能是chitosan-pcDNA3-VPI基因疫苗最合适的诱导全面免疫应答的免疫途径。     

CCL20对HBV基因疫苗的免疫增强作用

CCL20为CC家族的趋化因子,对未成熟DC和T细胞具有较强的正向趋化作用。HBsAg是乙肝病毒中具有保护作用的结构抗原。以HBsAg为目的抗原进行基因免疫可诱导HBV特异性免疫应答。为进一步增强基因疫苗的免疫效果,研究中,应用基因重组技术,构建CCL20和HBsAg的真核表达载体,将重组体用肌肉注射方式免疫C57BL/6小鼠,通过ELISA方法检测C57BL/6小鼠的抗-HBs抗体水平、淋巴细胞增殖试验检测抗原特异性Th活性、FACS检测CTL效应。结果显示,用CCL20/HBsAg真核表达质粒共注射免疫后,100%小鼠能在第4、6周检测到抗-HBs抗体,CCL20可显著增强HBsAg基因疫苗诱导的体液免疫应答;Th活性和CTL效应检测也显示,CCL20增强了HBsAg诱导的特异性细胞免疫反应。这将为新型乙肝疫苗的分子设计和研制提供新的理论与实践依据。     

C3d-P28对基因免疫诱导的HBV特异性细胞免疫应答的调节作用

目的：观察补体C3d-P28对基因免疫诱导的HBV特异性细胞免疫应答的调节作用，为增强基因免疫效果寻求新方法。方法：将质粒pVAON33-S2／S质粒(仅含HBV-preS2／S编码基因)和pVAON33-S2／S-P28．4质粒(含HBV-prS2/S和4拷贝C3d-P28的编码基因)以肌肉注射法对BALB／C小鼠实施基因免疫(100μg/100μl只)，并以空载质粒为对照。定期采集免疫小鼠血清、ELISA法检测其中特异性抗HBs-IgG及其亚型的水平；免疫小鼠脾细胞经特异性抗原(HBsAg)刺激后，采用，H-TdR掺入法、半定量RT-PCR法、同位素释放法分别检测其特异性淋巴细胞增殖活性、IL-4和IFN-γ基因表达的水平、CTL杀伤活性。结果：pVAON33-S2／S-P28．4质粒基因免疫诱导的特异性淋巴细胞增殖活性、抗HBs-IgG水平以及特异性CTL杀伤活性均明显高于pVAON33-S2／S质粒；C3d-P28未改变基因免疫诱导的抗HBs-IgG各亚型水平的格局，同时增强IgG1、IgG2a、IgG2b的水平；pVAON33-S2／S-P28．4质粒诱导的IL-4和IFN-γ的基因表达水平均明显高于pVAON33-S2／S质粒。结论：C3d-P28可在提高体液免疫应答的同时增强基因免疫诱导的HBV特异性细胞免疫应答。     

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     

基于枯草芽孢杆菌系统制备的EV71VP1重组抗原诱导小鼠免疫应答的研究

目的观察基于枯草芽孢杆菌系统制备的EV7lVPl重组抗原诱导BALB／c小鼠产生的免疫应答反应。方法通过建立BALB／c小鼠动物模型,分别将前期构建的基于枯草芽孢杆菌系统制备的EV71重组抗原（rVPl）、灭活EV71病毒免疫组（EV71）和空白对照组（PBS）,经鼻腔接种6～8周龄BALB／c小鼠,ELISA方法检测免疫后不同时间产生的特异性IgG、IgA以及IgGl和IgG2a水平。结果EV71重组抗原可以有效诱导小鼠产生高水平的体液免疫和明显的黏膜免疫应答,且能够诱发均衡的Th1、Th2免疫调节。结论肠道病毒71型重组VPl抗原可诱发小鼠产生明显的特异性体液免疫和黏膜免疫应答。     

乙肝病毒核酸疫苗诱导H-2b小鼠特异性免疫反应的初步研究

目的：观察并证实adr和adw亚型NHBs核酸疫苗单独应用，或与HBc核酸疫苗联合免疫诱导H－2^b小鼠的特异性体液和细胞免疫反应，方法：C57BL／6（H－2^b)小鼠随机分为4组：pJW4303组（P组，5只），pJW4303/MHBs/adw组（W组，6只）；pJW4303/MHBs/adr组（R组，6只）;pJW4303/MHBs/adr pJW4303/HBc组（R＋C组，6只），免疫方法为各组小鼠每只每次注射相应的质粒DNA疫苗100μg(100μl)，免疫程序为0，2，4w，小鼠血清抗HBs和抗HBc水平以及小鼠脾淋巴细胞培养上清中的IFN－γ和IL－4浓度用ELISA法检测，小鼠淋巴细胞抗原特异性增殖试验采用3H－TdR掺入法，计算刺激指数（SI），结果：W，R，R＋C各免疫组在末次免疫后4w所有小鼠均产生抗－HBs，但各免疫组间无明显差异，抗－HBs以R＋C组最早出现，R＋C组小鼠在第1次免疫后2w抗－HBc即全部阳性，P组免疫后始终末出现抗－HBs和抗－HBc，核酸疫苗免疫的W，R，R＋C各组免疫小鼠HBsAg特异性脾淋巴细胞增殖指数（SI）均高于P组（P＜0．01－P＜0．001），R＋C（n=6)组小鼠脾淋巴细胞HBsAg特异性SI比R组显著升高（P＜0．05），R＋C免疫小鼠HBcAg特异性脾淋巴细胞增殖指数（SI）均≥2，与P组（其SI均＜2）相比差异非常显著（P＜0．001），W，R，R＋C各免疫组小鼠脾淋巴细胞与HBsAg共培养的上清中IFN－γ的浓度比P组显著升高（P＜0．001），而它们的IL－4 浓度比P组明显下降（P＜0．001），R＋C免疫组小鼠脾淋巴细胞与HBcAg共培养的上清中IFN－γ浓度也较P组明显升高（P＜0．05），但其IL－4浓度与P组无差别（P＞0．05），结论：adr亚型和adw亚型HBV外膜中蛋白（MHBs)核酸疫苗和HBcAg核酸疫苗能诱导H－2b小鼠产生特异性的体液和细胞免疫应答，具有良好的体液和细胞免疫原性，MHBs核酸疫苗与HBcAg核酸疫苗联合免疫能加强MHBs核酸疫苗诱导的体液和细胞免疫反应，所诱导出的辅助性T细胞免疫反应为Th1型。     

IL-15真核表达质粒的构建及对乙肝疫苗诱导的体液免疫应答的影响

目的 研究两种不同的IL-15真核表达质粒对乙肝蛋白疫苗诱导的免疫应答的影响。方法：构建IL-15真核表达质粒(简称pIL-15)和含有IL-12信号肽的IL-15真核表达质粒(简称pIL-2s-15)，CTLL-2细胞增殖实验验证两种质粒真核表达产物的生物学活性。将这两种质粒分别与HBsAg共免疫BALB／C小鼠，用ELISA法检测小鼠血清抗-HBs IgG及IgGl、IgG2a亚类的效价。结果：与HBsAg蛋白疫苗共免疫时，pIL-15可使HBsAg诱导的抗-HBsIgG效价升高，显著高于载体pcDNA3．1与HB—sAg共免疫对照组，pIL-2s-15对HBsAg诱导抗-HBsIgC效价没有明显影响。与HBsAg pcDNA3．1组相比，HBsAg pIL-2s-15组和HBsAg pIL-15组诱生的抗HBsIgG2a亚类均升高，但前者IgG2a／IgG1比值最高，与HBsAg pcDNA3．1组相比差别有显著性；HBsAg pIL-15组IgG2a／IgG1比值与HBsAg pcDNA3．1组相比差别无显著性。结论 pIL-15真核表达质粒可增强蛋白疫苗诱导的体液免疫应答，pIL-2s-15真核表达质粒则主要使免疫应答趋向Th1型。     

不同HBsAg疫苗联合免疫后诱导小鼠细胞和体液免疫应答的研究

目的：了解HBsAg的蛋白疫苗（P）、痘苗病毒疫苗（V）、DNA疫苗（D）联合免疫小鼠诱导的特异性体液和细胞免疫应答。方法：以P、V或D疫苗中的一种疫苗初次免疫BALB／c小鼠后，于第2、5、8、11周再用另一种疫苗加强，共产生9种免疫组合：即PP、PV、PD、VP、VV、VD、DP、DV及DD。于初免后第2、5、8、11周采血检测血清中抗HBsAgIgG的总滴度及其IgG1和IgG2a亚类，并于每次加强免疫后第7天，检Nd,鼠脾脏的CTL对P815S细胞的特异性杀伤率。结果：在P、V、D3种疫苗中，V疫苗诱导产生抗HBsAg抗体的速度最快，P疫苗诱导的体液免疫回忆反应最强，D疫苗诱导产生的抗体最弱。除PP疫苗组合诱导的抗体明显倾向于IgG1外，其他均无明显的倾向性。各种免疫组合中，VD和DV疫苗组诱导的CTL应答最强，对P815S的特异性杀伤率分别为71％和64％。结论：在各种联合免疫组合中，PV、PD、VP和VD疫苗组的抗体应答较好；而DV和VD疫苗组诱导的CTL杀伤效应最强。     

炭疽PA和芽孢双组分候选疫苗免疫豚鼠的免疫学初步评价

目的 对豚鼠免疫重组PA抗原和甲醛灭活芽孢组成的炭疽候选疫苗,评价其免疫效果.方法 将豚鼠随机分成5个实验组,分别免疫高、中、低剂量候选疫苗、炭疽活疫苗或生理盐水;免疫后不同时间点采血进行抗体检测、XTT法淋巴细胞增殖检测以及皮肤迟发型超敏反应检测.结果 抗体检测结果显示,双组分炭疽候选疫苗能诱导较强的体液免疫应答;XTT细胞增殖结果显示,外周血淋巴细胞特异性增殖不明显,但所有候选疫苗组针对rPA DTH阳转率24 h后均为100％.结论 双组分炭疽候选疫苗能诱导豚鼠产生体液免疫和细胞免疫应答.     

乙肝表面抗原诱生γ-干扰素的研究

对 8名抗 HBs阳性健康成人 ,12名慢性乙肝患者外周血单个核细胞 (PBMC )分别用酵母菌表达的重组HBsAg ,s抗原第 12 0～ 147位氨基酸合成肽及重组HBsAg HBIG复合物刺激 ,测定特异性诱生γ 干扰素水平。结果 2 0名中有 14名产生了比对照更高的γ 干扰素。用重组HBsAg与合成肽为抗原诱生的γ 干扰素阳性数基本相符 ,但用HBsAg HBIG复合物诱生 ,仅有 7名产生升高的γ 干扰素。结果提示今后可用酵母菌表达的重组HBsAg特异诱生PBMC产生γ 干扰素 ,作为一种筛选病人与预测治疗性疫苗疗效的指标     

Two vaccinia virus recombinants expressing HBsAg with different concentration of A- and pre-S2 antigenic determinants.

Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M-protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L-pre-S2/15 induced anti-HBsAg a-determinant antibody as well.     

Comparative studies of the immunogenic activity of hepatitis B surface antigen (HBsAg) and HBsAg polypeptides

Three hepatitis B surface antigen (HBsAg) preparations were compared: purified intact 22-nm HBsAg particles; HBsAg-derived, sodium dodecyl sulfate (SDS)-denatured P25 + GP30 polypeptide pool; and nondenatured P25 + GP30 micelles. The micelles had the same polypeptide composition as the P25 + GP30 pool. The immunogenicity in mice of each preparation, administered either in saline suspension or adsorbed to aluminum gel, was compared. The SDS-denatured polypeptides were less immunogenic than intact HBsAg particles, whereas the micelles were more immunogenic. High anti-HBs titers were observed in mice immunized with micelle preparations in either saline suspension or adsorbed to aluminum gel for as long as 200 days after a booster inoculation, administered 26 days after the primary dose.     

Hepatitis B surface antigen polypeptide micelles from antigen expressed in Saccharomyces cerevisiae

Hepatitis B micelles containing the p25 component of hepatitis B surface antigen (HBsAg) have been produced by Triton X-100 solubilization followed by ultracentrifugation in linear sucrose gradients. The product was found to resemble micelle forms prepared from plasma-derived HBsAg with the surface being composed of discrete globular and stranded sub-units. The degree of immunochemical relatedness of the micellular preparation was compared to the native 22-nm HBsAg particle present in either plasma or yeast cell extracts. The yeast micelle preparation competed for anti-HBs in a similar manner as intact HBsAg of plasma origin. Enhanced immunogenicity may be expected for micelles containing a recombinant HBsAg protein as has previously been shown for the plasma-derived antigen.     

Levamisole enhances immune response by affecting the activation and maturation of human monocyte-derived dendritic cells

The primary aim of this study was to evaluate the role of natural killer (NK) cells on antigen-specific adaptive immune responses. After analysing the mechanism of impaired adaptive immune responses of NK-depleted mice, an immune interventional approach was developed to restore adaptive immunity in NK-depleted mice. NK cells were depleted from mice by administration of anti-asialo GM1 antibody (100 mul/mouse), twice, at an interval of 48 h. Hepatitis B surface antigen (HBsAg) was administered intraperitoneally to normal C57BL/6 mice (control mice) and NK-depleted mice. The levels of antibody to HBsAg (anti-HBs) in the sera and HBsAg-specific lymphocytes in the spleen were assessed. The functions of T lymphocytes, B lymphocytes and dendritic cells (DCs) were evaluated in vitro. HBsAg-pulsed DCs were prepared by culturing spleen DCs with HBsAg for 48 h and administered once to NK-depleted mice. The levels of anti-HBs in the sera and HBsAg-specific lymphocytes were significantly lower in NK-depleted mice compared with control mice (P < 0.05). The functions of T and B lymphocytes were similar between control mice and NK-depleted mice. However, the functions of spleen DC and liver DC were significantly lower in NK-depleted mice compared with control mice (P < 0.05). Administration of HBsAg-pulsed DCs, but not HBsAg, induced HBsAg-specific humoral and cellular immune responses in NK-depleted mice. Our study suggests that cross-talk between NK cells and DCs regulates the magnitude of adaptive immunity. In addition, antigen-pulsed immunogenic DCs represent potent immune modulator even if subjects with diminished innate immunity.     

Immunoregulation of the in vitro anti-HBs antibody synthesis in chronic HBsAg carriers and in recently boosted anti-hepatitis B vaccine recipients.

We report a study on immunoregulation of in vitro antibody to hepatitis B surface antigen (anti-HBs) synthesis induced by pokeweed mitogen (PWM) from peripheral blood mononuclear cells (PBMC) in chronic hepatitis B surface antigen (HBsAg) carriers and in 'high responders', (anti-HBs RIA ratio greater than or equal to 20 in serum), recently boosted with anti-hepatitis B vaccine. Anti-HBs was detected in 11 days PBMC supernatants (SN) from 24 out of 36 'high responders', but in none from 31 chronic HBsAg carriers, despite detectable amounts of polyclonal IgG and antibody to hepatitis B core antigen (anti-HBc) were produced. The lack of anti-HBs production by chronic HBsAg carriers did not seem to be determined by suppressor influences because T lymphocytes from the majority of chronic HBsAg carriers, co-cultured with 'high responders' PBMC did not suppress anti-HBs production. Co-cultures between HBsAg carriers T4 positive (helper/inducer) cells and allogenic 'high responder' non-T cells produced anti-HBs antibody, indicating that HBsAg carrier T cells are not deficient in this allogenic helper function under PWM stimulation. Allogenic cocultures between HBsAg carrier non-T cells and 'high responder' T4 positive cells failed in anti-HBs production: a specific B lymphocyte defect might be involved in the lacking anti-HBs synthesis in chronic HBV patients. Antigen-induced specific anti-HBs synthesis experiments indicate that B cells themselves seem to be the target for HBsAg-induced suppression of anti-HBs antibody response.     

Enhanced immunogenicity of DNA fusion vaccine encoding secreted hepatitis B surface antigen and chemokine RANTES

To increase the potency of DNA vaccines, we constructed genetic fusion vaccines encoding antigen, secretion signal, and/or chemokine RANTES. The DNA vaccines encoding secreted hepatitis B surface antigen (HBsAg) were constructed by inserting HBsAg gene into an expression vector with an endoplasmic reticulum (ER)-targeting secretory signal sequence. The plasmid encoding secretory HBsAg (pER/HBs) was fused to cDNA of RANTES, generating pER/HBs/R. For comparison, HBsAg genes were cloned into pVAX1 vector with no signal sequence (pHBs), and further linked to the N-terminus of RANTES (pHBs/R). Immunofluorescence study showed the cytoplasmic localization of HBsAg protein expressed from pHBs and pHBs/R, but not from pER/HBs and pER/HBs/R at 48 h after transfection. In mice, RANTES-fused DNA vaccines more effectively elicited the levels of HBsAg-specific IgG antibodies than pHBs. All the DNA vaccines induced higher levels of IgG(2a) rather than IgG(1) antibodies. Of RANTES-fused vaccines, pER/HBs/R encoding the secreted fusion protein revealed much higher humoral and CD8(+) T cell-stimulating responses compared to pHBs/R. These results suggest that the immunogenicity of DNA vaccines could be enhanced by genetic fusion to a secretory signal peptide sequence and RANTES.     

Hemokinin-1 As an Adjuvant Molecule Enhancing Humoral and Memory Responses to HBsAg DNA Vaccination

Abstract Incorporation of molecular adjuvants into DNA vaccines is often used to improve the induction of immune responses, but few approaches aim to specifically activate B cells for an enhanced humoral response only. Hemokinin-1 (HK-1) is a factor that activates B cells for proliferation, survival, differentiation into plasma cells, and Ab production. Therefore, we investigated if it may be used as a molecular adjuvant for DNA vaccines to elicit strong humoral and memory responses. The HK-1 coding sequence was sub-cloned as single or triple copies in-frame downstream of S2 HBsAg in the proVAX/S2 construct. Compared to mice immunized with proVAX/S2 or proVAX/S2-HK-1, proVAX/S2-3HK-1 induced a higher level of IgG production, a higher percentage of differentiated antibody-secreting plasma cells, and a higher level of T-cell proliferation. Furthermore, a higher proportion of B cells had the B220(+)CD27(+) phenotype in these groups, and specific antigen re-challenge induced a higher level of total IgG production 60?d after the last immunization, suggesting that the use of HK-1 as an adjuvant promoted immunological memory. Taken together, these results suggest that using HK-1 as an adjuvant molecule could enhance the immunogenicity of HBsAg DNA vaccines, and result in stronger humoral and memory responses. Therefore, HK-1 may lead to the development of a novel humoral-biased molecular adjuvant for an HBsAg DNA vaccine against hepatitis B infection.     

HBsAg-induced interferon-gamma secretion in T cells from asymptomatic HBsAg carriers and HB-immune donors in vitro.

Peripheral T lymphocytes obtained from asymptomatic carriers of hepatitis B surface antigen (HBsAg) and donors immune to hepatitis B (HB) through natural infection or vaccination were induced by the envelope protein (HBsAg) of the hepatitis B virus (HBV) into secretion of interferon-gamma (IFN-gamma) in vitro. The kinetics of the IFN-gamma response varied between individuals, but was constantly found to be biphasic, with an early peak attained after 12 hr-4 days and a late peak after 5-8 days of antigen stimulation. The early release of IFN-gamma activity was antigen-specifically induced, as it was in T cells from HB-immune and asymptomatic carriers of HBsAg but not HB-susceptible controls. The second peak of HBsAg-induced IFN-gamma secretion was induced in all three donor groups and the kinetics of IFN-gamma release were similar to that of the mitogen phytohemagglutinin(PHA)-induced IFN-gamma production in similarly prepared T-cell cultures. Thus the late burst of IFN-gamma activity seems to result from a mitogenic property contained within the envelope material of HBV. The mitogenic response was three- to fivefold higher for 4/7 asymptomatic carriers of HBsAg compared to HB-immune donors and HB-susceptible controls, indicating that some patients with chronic asymptomatic carriership of HBsAg exhibit enhanced mitogenic responses to HBsAg.