乙肝病毒疫苗的类型及其免疫原性和安全性

乙型肝炎(HB)病毒(HBV)疫苗主要包括血源疫苗、基因工程酵母疫苗、地鼠及仓鼠卵细胞(CHO)疫苗．血源疫苗已被基因重组HBV(rHBV)疫苗所代替,后者现已发展到第3代及第4代,如含Pre-S的CHO疫苗及添加佐剂如寡核苷酸、3抗原(含S,Pre-S1和Pre-S2)的Hepacare疫苗和3’-单磷酸脂A(AS04)疫苗等．近年来又研制成功了肺炎球菌多糖苷蛋白结合rHBV疫苗及可口服和涂抹的DNA疫苗等．目前所应用的rHBV疫苗均具有很好的免疫原性已得到世界公认和肯定,且国内外无明显差别．不良反应普通rHBV疫苗小于2．5％,主要为针刺部位痛、红、肿、胀、痒,一般轻微,日余即消．小于0．5％的人有发热、嗜睡、食欲下降等反应,多见于HB-AS04疫苗,无需处理.

乙肝免疫球蛋白与乙肝病毒疫苗联合应用阻断乙肝母婴传播的研究

目的:评价乙肝免疫球蛋白(HBIG)联合乙型肝炎病毒疫苗(HBVac)免疫预防措施阻断乙肝病毒(HBV)母婴传播的临床效果。方法:选择2002年1月至2010年1月在本院分娩的198例HBs Ag阳性母亲及其198例新生儿为研究对象,同时依据HBIG接种方式的不同将孕妇分为研究组和对照组。研究组126例,于孕期的第28、32、36周分别肌内注射HBIG 200 U;新生儿出生后分别于出生后12小时和30天肌内注射HBIG 200 U,出生当天、第1个月和第6个月各接种5μg HBVac。对照组72例,在孕期不注射HBIG,其新生儿接种方法同研究组。结果:研究组和对照组分娩新生儿的HBV感染率分别为0.79%和2.78%,差异无统计学意义(校正χ2=0.245,P>0.05);且HBe Ag(+)母亲所生孩子比HBe Ag(-)母亲所生孩子HBV感染率有增高的趋势,但无差异无统计学意义(4.65%vs.0.65%,校正χ2=1.433,P>0.05)。结论:孕妇孕晚期使用HBIG对HBV母婴传播和新生儿对HBVac的抗体应答无明显影响,其中HBs Ag单阳性孕妇的阻断率有高于HBs Ag和HBe Ag双阳性孕妇的趋势。

乙肝免疫球蛋白与乙肝病毒疫苗联合应用阻断乙肝母婴传播的Meta分析

[目的]综合评价孕晚期注射乙肝免疫球蛋白联合新生儿注射乙肝免疫球蛋白及乙肝病毒疫苗阻断乙肝病毒母婴传播的有效性,为制定重点人群的乙肝免疫策略提供循证依据。[方法]应用计算机检索相关文献,实验组干预措施为孕晚期注射乙肝免疫球蛋白,新生儿注射乙肝免疫球蛋白和乙肝病毒疫苗;对照组干预措施仅为新生儿注射乙肝病毒疫苗。对符合纳入标准的8项随机对照研究,采用固定和随机效应模型进行Meta分析。[结果]各纳入文献的发表偏倚较小。Meta分析显示实验组母婴传播率（6.01%）低于对照组（16.37%）,χ^2=7.02,P〈0.001;合并RR为0.37,95%可信区间为0.28-0.48,敏感性分析提示模型稳定性较好。亚组分析显示无论对于单纯HBsAg阳性还是HBsAg和HBeAg双阳性的孕妇;或者不管是以新生儿出生24h内乙肝感染标志阳性或是以出生后12个月乙肝感染标志阳性为标准,实验组的母婴传播率均低于对照组（P〈0.05）。[结论]孕晚期注射乙肝免疫球蛋白联合新生儿注射乙肝免疫球蛋白及乙肝病毒疫苗能有效地阻断乙肝病毒的母婴传播。

安徽地区42 509名未成年人乙肝病毒血清标志物的调查

目的分析0~17岁不同年龄区间的未成年人体内乙肝病毒血清标志物,探讨乙肝疫苗的免疫效果和加强免疫的必要性.方法收集2018年就诊于安徽省儿童医院42509位患儿的血清,采用化学发光方法对乙型肝炎HBsAg和HBsAb定量检测,分析未成年人乙肝病毒携带情况及疫苗接种效果分析.结果经统计42509位0~17岁未成年人血清检测结果,发现HBsAg阳性者37例,占0.087%.HBsAb总阳性率71.33%,其中1~2岁的群体抗体阳性率最高,为93.41%;3岁以后HBsAb阳性率逐渐下降;儿童从出生至7个月后,抗体浓度≥100 mIU/mL的人数比例从34.21%显著提高到88.05%;从4~6岁人群开始至18岁人群抗体浓度≥100 mIU/mL的人数比例基本保持在80%左右.结论安徽地区未成年人乙肝病毒携带率较低;安徽地区的乙肝疫苗接种的效果较好,学龄前儿童可再次检测抗体浓度,必要时可加强免疫.

重点、高危和特殊人群之乙肝免疫

重点、高危和特殊人群除自身更易感染乙肝病毒(HBV)外,还包括当他们感染HBV后易形成慢性肝炎或携带并造成HBV传播,他们是乙肝免疫的重点对象或需要尽快接种HBV疫苗的特殊对象．该人群乙肝免疫覆盖率较低,而HBV感染率和携带率较高;其中一些人对HBV疫苗反应低下,常规接种程序和接种剂量免疫后抗-HBs出现晚．阳转率低,几何平均滴度低,有效保护时间短,宜用快速免疫方案和大剂量HBV疫苗或接种4针,必要时应用佐剂或免疫激动剂．

乙肝免疫过程中要注意的几个问题

乙型肝炎病毒(HBV)疫苗问世近30 a以来,成效巨大,但因各种因素影响,不同群体和不同个体乙肝(HB)免疫的重点或迫切性及免疫效果始终存在差异,即存在HBV疫苗接种群体化甚至个体化问题．因此,在实际操作中需注意根据不同年龄、不同人群甚至不同个体选择不同接种方案和不同剂量;对高危等人群应采用快速接种方案,并监测其抗-HBs水平,必要时加强免疫或应用佐剂．对HBsAg阳性母亲的子女等应加大接种剂量和／或接种4针．HBV疫苗与某些其他疫苗联合或同时接种为一省时简便的方法,效果肯定．

疫苗创新之世纪回眸

过去百年的疫苗学史研究成就斐然，但进步并非一帆风顺，预防医学创造的生物制品总有是被迫投入激烈的斗争中，以争取所需的资源和支持，尽管总有一批先驱矢志于疫苗的发明、开发和销售，其成就的荣誉多半不属于个人，而归功于由公有的、私立的非营利性机构组成的复杂而结构松散的网络，这些机构经年累月地推动该领域的创新，哪里网络运行良好，社会就获益；哪里网络运作不善，不管因何原因，社会终是输家。

低出生体重儿对乙肝疫苗接种免疫应答的反应

有研究表明，用乙肝疫苗主被动联合免疫能阻断母婴间乙肝病毒传播，保护效果达到70％～90％。母亲为乙肝病毒携带者，或乙肝患者，婴儿出生后立即肌注高价乙肝免疫球蛋白（HBIg）0．5ml，同时换部位注射重组乙肝病毒疫苗10μg。对早产儿在生后短时间内接种乙肝疫苗的免疫反应性我国已有报道。对低出生体重儿出生时是否接种乙肝疫苗尚无定论。现行政策规定：早产儿体重〈2500g时禁止接种乙肝疫苗。2002年10月至2005年10月，我们对因各种原因转入我院儿科或产科分娩的低出生体重儿（早产儿）106例，在生后1周内接种首剂乙肝疫苗，第2、第3支在1，6个月时接种。完成随访者：组Ⅰ（25例），组Ⅱ（35例），组Ⅲ（20例），组Ⅳ（39例）。

生物技术及产品

新生物药物是ACR2007会议的亮点,Aveo与Peptech和Osi公司签署了技术协议,Genzyme／Bayer开始Campath的Ⅲ期试验,Lilly公司扩展了I型糖尿病候选药的后期开发管线,Genmab从Medarex得到了HuMax—Inflam的所有权利,卫材转让BioArctic的AD治疗药,研究表明HPV6和HPV11疫苗有价值,Tripep和Inovio公司将联合开发乙肝病毒疫苗。

A mimotope of Pre-S_2 region of surface antigen of viral hepatitis B screened by phage display

To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino asids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design.

Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins

AIM: The incorporation of hepatitis B virus (HBV) preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial. Therefore our purpose was to further investigate its immunogenic domains for the epitopebased hepatitis B vaccine design.METHODS: Eight GST fusion proteins containing overlapping preS1 fragments in preS1 (21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA.RESULTS: The results in mice showed that the immunogenic domains mainly existed in preS1 (21-59) and preS1 (95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1 (60-70) also had some immunogenicity.More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1 (34-59), were speculated to be virus-neutralizing.CONCLUSION: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic domains in preS1 (21-119) region. The preS1 (34-59) fragments were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore, the incorporation of preS1 (34-59) fragments into epitopebased HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.

Novel DNA vaccine based on hepatitis B virus core gene induces specific immune responses in Balb/c mice

AIM： To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS： A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes （CTLs） of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS： HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1：97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector：target ratio of 20：1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION： pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.

Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

Objective:To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines.Methods:BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA;splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro.Results:The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone,but there was not significantly different (P>0.05).Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P<0.05) and the level of IFN-γ in supernatant of splenocytes cultured with HBsAg in vitro was significantly elevated (P<0.05) while the level of IL-4 had no significant difference (P>0.05).Conclusion:The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.

Immunogenicity of recombinant hepatitis B virus vaccine in patients with and without chronic hepatitis C virus infection:A case-control study

AIM： To compare the response of standard hepatitis B virus （HBV） vaccination between patients with chronic hepatitis C virus （HCV） infection and healthy individuals. METHODS： This is a prospective case-control study. A total of 38 patients with chronic HCV infection and 40 healthy controls were included. Vaccination was performed by injection of 20μg recombinant HBsAg into the deltoid muscle at mo 0,1 and 6. Anti-HBs concentration was determined 3 mo after the last dose and compared between the two groups. The response pattern was characterized as （1） high-response when the anti-HBs antibody titer was 〉 100 IU/L, （2） low-response when the titer was 10-100 IU/L. and （3） no-response when the titer was 〈 10 IU/L. RESULTS： In the patient group, there were 10/38 （26.3%） non-responders, 8/38 （21.1%） Iow-responders and 20/38 （52.6%） high-responders. The corresponding values in the control group were 2/40 （5.0%）, 7/40 （17.5%） and 31/40 （77.5%）, respectively. The response pattern was statistically different between the two groups. In multivariate analysis, smoking was a significant confounder, while HCV infection lost its significant correlation with lower antibody response. CONCLUSION： Patients with chronic HCV infection tend to respond weakly to HBV vaccination compared to healthy individuals, though this correlation is not independent according to multivariate analysis.

Screening of a High Growth Influenza B Virus Strain in Vero Cells

Due to the insufficient supply of embryonated chicken eggs,the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus.The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines.However,most of the influenza viruses can not grow well in Vero cells.To develop a new influenza vaccine with Vero cells as a substrate,the virus needs to adapt to this cell substrate to maintain high growth characteristics.By serial passages in Vero cells,the B/Yunnan/2/2005va(B)strain was successfully adapted to Vero cells,with the hemagglutination titer(HAT)of the virus reaching 1:512.The high growth characteristic of this strain is stable up to 21 passages.The strain was identified by hemagglutination inhibition (HAI)test and sequencing respectively;the HA1 gene sequence of the virus was cloned and analyzed.The screening and establishment of high growth B virus provides an important tool for influenza vaccine production in Vero cells.

Immune response to an indigenously developed r-Hepatitis B vaccine in mixed population: Study of an accelerated vaccination schedule

AIM: To establish the safety and efficacy of an indigenously developed r-hepatitis B vaccine using an accelerated schedule and to highlight the social awareness and commitment in preventing the spreading of hepatitis B virus infection. METHODS: The study was a multicentric, double blind, randomized (3:1) study using three doses of vaccine immunization schedule (20μg for those above 10 years old and 10 μg for those below 10 years old) on d 0, 30 and 60. One hundred and sixty-six subjects were enrolled (87 males and 76 females aged 5-35 years). The main outcome measure was assessment of immunogenicity and safety. RESULTS: A 100% seroconversion response was observed on the 30th d after the 1st injection in both the experimental groups. The sero-protection data reported a 41.2-65.6% response on the 30th d after the 1st injection and reached 100% on the 60th d. Descriptive statistical analysis showed a geometric mean titer value of 13.77 mIU/mL in the test (BEVAC) group and 10.95 mlU/mL in the commercial control (ENGERIX-B) group on the 30th d after the 1st injection. The response on the 60th d showed a geometric mean titre value (GMT) of 519.84 mlU/mL in the BEVAC group and 475.46 mlU/mL in the ENGERIX-B group. On the 90th d, the antibody titer response was observed to be 2627.58 mlU/mL in the BEVAC group and 2272.72 mlU/mL in the ENGERIX-B group. Two subjects in each group experienced pains at injection site after the first vaccination. A total of six subjects in both groups experienced a solicited adverse reaction, which included pains, swelling and redness at the injection site, three subjects in the group-B had a pain at the injection site after the third dose. No other serious adverse events occurred and no dose-related local or general symptoms were observed during the study. CONCLUSION: The vaccine is safe, efficacious and immunogenic in comparison with the well documented ENGERIX-B.

Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency

AIM： To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein （HSP） as a strategy to enhance DNA vaccine potency.METHODS： A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was Performed in BALB/c mice to monitor anti-tumor immune responses.RESULTS： In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg （44% ± 5% vs 30% ± 6% in E： T 〉 50：1, P 〈 0,05）, ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe- HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC class I or class Ⅱ epitopes derived from HBeAg （74% ± 9% vs 31% ± 6%, P 〈 0.01）. ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV- HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV- HBe-HSP when challenged with CT26-HBeAg.CONCLUSION： The results of this study demonstrate a broad enhancement of antigen-specific CD4^＋ helper,CD8^＋ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.

Four-year follow up of hepatitis C patients vaccinated against hepatitis B virus

AIM: Patients with chronic hepatitis C have been recommended to receive vaccinations against hepatitis B. Our study aimed at evaluating the hepatitis B immunogenicity and efficacy against hepatitis B virus infection 4 years after primary immunization series in a group of patients with chronic hepatitis C.METHODS: We recruited 36 out of 48 hepatitis C virus (HCV) infected individuals who were vaccinated against hepatitis B virus (20 μg of recombinant HBsAg at 0-1-6mo schedule) in 1998. Here we measured anti-HBs titers and anti-HBc 4 years after delivery of the third dose of primary immunization series.RESULTS: After 4 years a total of 13/36 (36%) HCV infected patients had seroprotective titers of anti-HBs compared with 9/10 (90%) in the control group, (P＜0.05).Similarly the mean concentration of anti-HBs found in hepatitis C patients was significantly lower than that found in healthy subjects (18.3 and 156.0 mIU/mL respectively (P＜0.05). None of the HCV infected patients or controls became infected with HBV during the study period as confirmed by anti-HBc negativity.CONCLUSION: We demonstrated that 4 years after HBV immunizations' more than 60% of vaccinated HCV patients did not maintain seroprotective levels of anti-HBs, which might put them at risk of clinically significant breakthrough infections. Further follow-up studies are required to clarify whether memory B and T lymphocytes can provideprotection in chronic hepatitis C patients in the absence or inadequate titers of anti-HBs.

疫苗与癌症预防

中国人群肿瘤死亡的29.7%由慢性感染引起,其中肝炎病毒(HBV)、幽门螺杆菌(HP)和人乳头瘤病毒(HPV)是最常见的感染致癌因子。目前已研发成功HBV预防性疫苗、HP预防性疫苗和HPV预防性疫苗,可有效预防上述感染因子引起的肝癌、胃癌和宫颈癌。2012年5月颁布的《中国慢性病防治工作规划(2012—2015年)》明确指出:我国在开发癌症高发地区重点癌症筛查适宜技术的同时,应结合国家免疫规划政策,加强对癌症高风险人群乙型肝炎、人乳头瘤病毒等疫苗的预防接种。因此,本文简要介绍上述三种癌症疫苗的最新研究进展,以期借鉴HBV疫苗在中国的推广历程和免疫成果,进一步推进和完善我国HBV疫苗的接种,加速HPV疫苗和HP疫苗的引入推广,让肿瘤一级预防的科研成果尽早惠及于民。

The Experimental Study on Treating Transgenic HBV Mice with Recombined IL-2-PreS DNA Vaccine

The aim of this study is to investigate the feasibility and mechanism of hIL-2-preS DNA vaccine as prevention and therapeutic approach against Hepatitis B. Eukaryon expression vector involving hIL-2 and preS gene was constructed with recombinant technique and transferred into normal BALB/c mice and HBV transgenic mice (Tg-Mice) respectively. Then a series of detection were performed: detection of anti-preS2, HBs antibody and HBsAg in BALB/c mice and Tg-mice with ELISA, quantification of HBV DNA copies in HBV Tg-mice serum with real-time PCR, determination of hepatitis degree with immunopathological HE staining and detection of liver function. Anti-preS1 can be detected at 4 th , 6 th and 10 th week in inoculated BALB/c mice. Injection with gene gun gained an advantage over muscular and subcutaneous injection since it acquired just 1/10 inoculation quantity (10 μg/mouse). Highest expression of IgG2a at 4 th week suggested Th1-mediated immune response, which facilitated HBV cleaning. Of all inoculated HBV Tg-mice, 80% of them showed anti-preS2, HBs antibody positive and HBV DNA decreased, and 20% showed negative for HBsAg. HE staining to hepatic tissue showed obvious infiltration of inflammatory cells, swelling and granular degeneration of hepatocytes. In our study, IL-2-preS DNA vaccine which can provoke the humoral and cellular immune response and break the immune tolerance supports the designation and construction of new vaccine against HBV and specific immune remedy for HBV continuous infection.