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.     

Liposome/DNA complexes coated with biodegradable PLA improve immune responses to plasmid encoding hepatitis B surface antigen

We hypothesized that the addition of polymer to the surface of liposome/DNA complexes may potentially enhance in vivo delivery of plasmid DNA to antigen-presenting cells and thereby facilitate enhanced immune responses to encoded protein. BALB/c mice were immunized subcutaneously or intramuscularly three times with a total of 50 microg of the plasmid pRc/CMV-HBs(S) (ayw subtype) encoding for the hepatitis B surface antigen. We measured transgene-specific total immunoglobulin G (IgG), IgG2a, IgG2b and IgG1 antibody responses as well as splenocyte and T-cell proliferation and cytokine production upon re-stimulation following immunization. Modification of lipid/DNA complexes by the polymer precipitation method used here for the addition of poly(d,l-lactic acid) was found to be consistently and significantly more effective than either unmodified liposomal DNA or naked DNA in eliciting transgene-specific immune responses to plasmid-encoded antigen when administered by the subcutaneous route. In addition, the polymer-modified formulations delivered by this route were more effective than naked DNA delivered by the intramuscular route in inducing antibody responses (n=5, P<0.03). Our observations provide 'proof of principle' for the use of these multicomponent formulations, which offer potential for manipulation and increased transfection efficiency in vivo for the purposes of genetic immunization.     

Comparing the immunogenicity of hepatitis B virus S gene variants by DNA immunization.

DNA immunization was used to compare the immunogenicity of hepatitis B virus S gene variants. Four recombinant plasmid DNAs containing the full-length virus genome with different S gene inserts were used to immunize BALB/c and C57/BL/6 mice. These inserts were cloned from 129L (residue 129, glutamine to leucine), 129H (residue 129, glutamine to histidine) 145R (residue 145, glycine to arginine) variants and the wild-type virus. The titer of hepatitis B virus core antibodies (anti-HBc) in immunized mice was used as the control for the efficiency of DNA immunization. Serum hepatitis B surface antibody (anti-HBs) titer and cytokines induced in splenocytes stimulated with hepatitis B surface antigen (HBsAg) were monitored as specific immune responses induced by different plasmid DNAs. 129L DNA induced significantly lower anti-HBs antibodies (IgG, IgG1, and IgG2a) and less interferon-gamma, compared to those in mice immunized with the 129H variant and the wild-type HBV DNA (p < 0.05). Computer modeling showed that a change from glutamine to leucine at 129 residue led to higher hydrophobicity and could result in decreased immunogenicity. Results indicate that DNA immunization can be used to compare the humoral and cellular immunogenicity among different HBV S variants.     

骨骼肌注射乙型肝炎表面抗原基因在小鼠体内诱发乙肝表面抗原特异的免疫反应

将含有乙型肝炎表面抗原（ＨＢｓＡｇ）基因的重组质粒ｐＡｃｔ－ＭＳ直接注入小鼠骨骼肌中，２周后加强免疫１次。免疫后４～９周间，每周检测小鼠血清中ＨＢｓ。结果表明实验组６／８只小鼠ＨＢｓ为阳性，对照组８只小鼠均未检测到ＨＢｓ。本实验证实直接注射质粒ＤＮＡ外源基因能获得表达，并能引起免疫反应。     

IL-12对结构优化的HBV核心抗原DNA疫苗诱导免疫应答的影响

目的 探讨IL-12对一种结构优化的HBV核心抗原(HBcAg)DNA疫苗免疫效果的影响。方法 将小鼠IL-12基因插入结构优化的DKA疫苗pST-HBc，构成pST-HBc/IL12，将pST-HBc／IL12转染COS7细胞，ELISA检测培养上清中的HBcAg和小鼠IL-12。分别将pST-HBc／IL12和pST-HBc肌肉注射接种BALB／c小鼠，检测小鼠的体液和细胞免疫应答。结果 ELISA检测到培养上清液中的HBcAg和小鼠IL-12，pST-HBc／IL12诱导的抗-HBc总IgG阳转率和抗体水平不及pST-HBc，但其诱导的脾细胞增殖反应和细胞毒性T淋巴细胞反应均强于pST-HBc。结论 IL-12可进一步增强这种HBcAgDNA疫苗诱导的细胞免疫应答。     

Vaccination with a fusion DNA vaccine encoding hepatitis B surface antigen fused to the extracellular domain of CTLA4 enhances HBV-specific immune responses in mice: Implication of its potential use as a therapeutic vaccine

Fusion of specific antigens to extracellular domain of cytotoxic-T-lymphocyte-associated antigen 4 (CTLA4) represents a promising approach to increase the immunogenicity of DNA vaccines. We evaluated this interesting approach for its enhancement on HBV-specific immune responses and its antiviral effects in HBV transgenic mice. A fusion plasmid encoding the extracellular domain of CTLA4 linked with HBsAg was constructed. Mice were immunized by this fusion plasmid. Vaccination with the CTLA4-fused DNA not only induced much higher level of anti-HBs antibody, but also increased HBsAg-specific CD8+ response as well as CTL response in BALB/c mice. Furthermore, both Th1 and Th2 responses were augmented. In HBV transgenic mice, the levels of circulating HBsAg and HBV DNA replication were down-regulated by induction of higher anti-HBs antibody and HBsAg-specific CD8+ response after vaccination with the fusion plasmid. Thus, the CTLA4-fused DNA vaccine led to breakdown of immune tolerance to viral infection in HBV transgenic mice, which might be used as a therapeutic vaccine in HBV infection.     

DNA-mediated immunization of mice with plasmid encoding HBs antigen.

In order to develop an experimental DNA vaccine for the prevention and treatment of hepatitis B virus infection, hepatitis B virus surface antigen (HBsAg) DNA was subcloned into an E. coli-eukaryotic cell shuttle vector and was expressed in the Baculovirus expression system. Intramuscular, intradermal, and intraperitoneal injections of 30 microg of the plasmid DNA expressing HBsAg induced humoral and cellular immune responses in ICR mice. The first IgG antibodies were detected after ten days and specific IgG antibody titers peaked after two months of a single intramuscular DNA injection. Anti-HBs antibody titers gradually increased and peaked at four months following intradermal DNA injection, and in case of intraperitoneal injection they peaked at seven months. Generation of HBs-specific helper T lymphocytes was also investigated through the production of interleukin-2 by T helper cells. Boosting effects of HBs DNA were investigated without much results. In general, DNA-mediated HBs immunization induced humoral and cellular immune responses in mice that appears to simulate immune responses in human during the course of HBV vaccination.     

IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠(H-2d)特异性免疫应答的影响

目的：观察IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠（H-2d）特异性体液免疫和细胞免疫应答的影响.方法：用肌肉注射法将HBV核心区DNA疫苗、IL-12质粒和IL-18 质粒接种BALB/c小鼠;ELISA法检测小鼠血清抗-HBc（IgG）及IgG亚类（IgG1、IgG2a）;LDH释放法检测小鼠脾细胞HBcAg特异性CTL活性.结果：免疫6周后,HBcAg DNA疫苗联合IL-12质粒、IL-18质粒和IL-12＋IL-18质粒组小鼠的血清抗HBc终点滴度均明显高于单纯注射HBcAg DNA疫苗组小鼠（P＜0.05）,抗HBc IgG亚类以IgG2a占优.DNA疫苗免疫的各组小鼠,HBcAg特异性细胞毒性T淋巴细胞杀伤率均高于对照组（P组）,其中C＋IL-18组和C＋IL-12＋IL-18组中CTL值明显高于C组,尤以C＋IL-12＋IL-18组中的CTL杀伤率最高.结论：IL-12和IL-18基因与HBcAg DNA疫苗联合免疫,不仅能增强HBcAg特异性体液免疫应答,而且能增强HBcAg特异性CTL的杀伤活性.     

Regulation of the neutralizing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vaccine recipients and patients with acute or chronic hepatitis B virus (HBV) infection

Antibodies directed to the HBs antigen indicate viral clearance and the development of life-long immunity in patients that recovered from HBV infection. In HBs antigen vaccine recipients anti-HBs antibodies provide protective immunity. However, little is known about the regulation of this HBs-specific antibody response. The existence of anti-HBs-secreting B cells was demonstrated using the highly sensitive ELISPOT technique compared with conventional ELISA in serum and cell culture supernatants. In the peripheral blood of patients with acute self-limited hepatitis B, HBs-specific B cells were demonstrated with a high frequency despite undetectable anti-HBs serum antibodies. HBV-immunized patients that had recovered from infection and vaccine recipients had significantly lower frequencies, whereas chronic HBV carriers and negative controls showed no anti-HBs-secreting B cells. Coculture experiments of isolated B and T cells revealed that the anti-HBs antibody response was restricted to the presence of T helper cells, but not to identical HLA class II molecules. Allogeneic T cells derived from vaccine recipients or chronic HBV carriers stimulated the HBs-specific B cell response in HBs vaccine recipients. Otherwise, isolated T helper cells could never provide sufficient help to induce the HBs-specific B cell response in chronic HBV carriers. Furthermore, peripheral blood mononuclear cells (PBMC) of six out of 10 vaccine recipients, one out of five HBV-immunized patients, but of no chronic HBV carrier showed a proliferative response to different HBs antigen preparations. This study demonstrated a high frequency of circulating anti-HBs-producing B cells in the early phase of acute HBV infection, but a lower frequency of HBs-specific B cells years after resolution of HBV infection. In chronic HBV carriers, however, deficient HBs-specific T and B cell responses were observed.     

Antigen-antibody complex as therapeutic vaccine for viral hepatitis B

In a previous study, hepatitis B surface antigen (HBsAg) complexed to human anti-HBs immunoglobulins (HBIG) in excess of HBsAg was used as therapeutic vaccine to treat chronic hepatitis B patients and promising results were obtained. To study the mechanisms of this approach, mice were immunized with HBsAg or IC (immunogenic complex, i.e. HBsAg complexed with mouse polyclonal anti-HBs). Studies indicate that IC induced enhanced immune responses by increasing uptake of HBsAg through Fc receptors on antigen presenting cells and modulated HBsAg processing and presentation. This modulation led to stimulation of T cell responses, and increased production of IL-2 and IFN-gamma. Assay for antibody subclasses showed that higher ratio of IgG 2a was observed in the IC immunized group, which correlated with the production of lymphokine pattern. When alum was used as the adjuvant, though antibody response was enhanced, production of cytokines decreased. When DNA from a recombinant plasmid was added to IC as an adjuvant, the titer of anti-HBs was significantly higher than those in mice immunized only with the DNA or the IC. Since DNA immunization can induce both cellular and humoral immune responses, combined immunization using IC and DNA might serve as another type of therapeutic vaccine for viral hepatitis B.     

B7-2表达质粒对HBV DNA疫苗诱导的特异性免疫应答的影响

目的：探讨B7－2分子是否能够增强乙型肝炎病毒（HBV）DNA疫苗诱导的特异性免疫应答。方法：将B7－2表达质粒与HBV DNA疫苗共接种于小鼠腓肠肌内，检测细胞毒性T淋巴细胞（CTL）活性，迟发性超敏反应（DTH）及抗－HBs滴度。结果：B7－2表达质粒与HBV DNA疫苗共接种组的DTH反应和CTL活性，明显强于单独接种HBV DNA疫苗组（P＜0．01）。两组的抗－HBs滴度差异无显著性（P＞0．05）。结论：B7－2表达质粒与HBV DNA疫苗共接种可显著增强抗－HBV特异性细胞免疫应答（CMI）。     

不同剂量乙肝疫苗与HBIG联合免疫阻断HBV母婴传播的效果对比

目的 探讨10 μg和20 μg乙肝疫苗与HBIG联合免疫阻断HBV母婴传播的效果.方法 124例HBsAg阳性孕妇所生的婴儿随机分为两组,即10 μg乙肝疫苗组和20 μg乙肝疫苗组.婴儿于出生6h内及30 d分别注射200 IU HBIG,同时分别于出生24 h内、1个月及6个月注射3次10 μg或20 μg重组酵母乙肝疫苗.检测婴儿出生时以及1岁时血清HBV标志物.结果 两组新生儿血清HBsAg、HBeAg及抗-HBe阳性率与滴度之间差别均无统计学意义（P＞0.05）.所有新生儿血清HBV DNA水平均小于检测下限（500 U/ml）.出生12个月时,所有124例婴儿血清HBsAg和HBeAg检测结果均为阴性;血清HBV DNA水平均在检测下限以下;10 μg和20 μg乙肝疫苗组血清抗-HBs阳性率分别为90.3％和96.8％,差异无统计学意义（P＞0.05）;抗-HBs水平分别为325.5±342.2 mIU/ml和463.7±353.3 mIU/ml,后者显著高于前者（P=0.01）.而且,20 μg乙肝疫苗组产生高应答抗-HBs（＞ 100 mIU/ml）的比例显著高于10μg乙肝疫苗组（P =0.035）.结论 20 μg乙肝疫苗联合HBIG方案阻断HBV母婴传播的效果优于10 μg乙肝疫苗联合HBIG方案.     

Long-term persistence of hepatitis B surface antigen and antibody induced by DNA-mediated immunization results in liver and kidney lesions in mice

DNA-mediated immunization has been recognized as a new approach for prevention and treatment of hepatitis B virus (HBV) infection. However, the side effects of this approach have not been well described. Here we report that DNA-mediated immunization by intramuscular injection of plasmid DNA encoding HBV surface antigen (HBsAg) induced long-term persistence of HBsAg and HBsAg-specific antibody (anti-HBs) in the sera of the immunized BALB/c mice and resulted in liver and kidney lesions. The lesions persisted for 6 months after injection. Lesions were also found in normal mice injected with the sera from immunized mice, and in HBV-transgenic mice injected with anti-HBs antibody, or sera from immunized mice. Furthermore, lesions were accompanied by deposition of circulating immune complex (CIC) of HBsAg and anti-HBs antibody in the damaged organs. These results indicate that long-term persistence of HBsAg and anti-HBs in the immunized mice can result in deposited CIC in liver and kidney, and in development of lesions. The use of DNA containing mammalian replication origins, such as the plasmids used in this study, is not appropriate for human vaccines due to safety concerns relating to persistence of DNA; nevertheless, the safety of DNA-mediated immunization protocols still needs to be carefully evaluated before practical application.     

A novel HBV DNA vaccine based on T cell epitopes and its potential therapeutic effect in HBV transgenic mice

DNA vaccination represents a novel therapeutic strategy for chronic hepatitis B virus (HBV) infection. Recently, some HBV DNA vaccines have been used in the preliminary clinical trials and exhibited exciting results in chronic HBV carriers. But these vaccines only encoded the single viral antigen, the S or the PreS2/S antigen. In this study, we designed a polytope DNA vaccine encoding multiple T cell epitopes. We found that it induced stronger CTL responses than the vaccine encoding the single antigen in H-2d and H-2b mice, although the CTL response to Ld-restricted epitope suppressed the CTLs to other epitopes in H-2d-restricted mice. Interestingly, heat shock protein 70 as an adjuvant not only enhanced CTL response to the viral antigen but also overcame this epitope suppression. Furthermore, the polytope DNA vaccine resulted in a long-term down-regulation of hepatitis B virus surface antigen and inhibition of HBV DNA replication in a HBV transgenic mouse model. Therefore, our research indicates that it is practicable and feasible to design a polytope DNA vaccine for chronic hepatitis B immunotherapy.     

乙型肝炎病毒多表位抗原DNA疫苗的免疫原性

目的 :探讨HBV复合多表位DNA疫苗诱导体液及细胞免疫的可行性。方法 :将HBV多表位抗原基因BPT克隆到真核表达载体pcDNA3.1中 ,构建重组真核表达载体pcDNA3.1/BPT。将其通过肌肉注射免疫BALB/c小鼠 ,用间接免疫ELISA法、CTL杀伤功能检测和淋巴细胞增殖试验 ,检测特异性体液免疫和细胞免疫的水平 ,并观察其对免疫小鼠的毒副作用。结果 :用重组质粒pcDNA3.1/BPT免疫BALB/c小鼠后 ,在效靶比为 10 0∶1时 ,可诱导显著地特异性CTL应答 (P <0 .0 5 )。ELISA检测免疫小鼠血清特异性IgG的水平明显升高 (P <0 .0 5 )。在BPT基因原核表达蛋白的刺激下 ,免疫小鼠脾淋巴细胞增殖显著 (P <0 .0 5 )。RT PCR分析表明 ,IL 12mRNA的水平亦明显升高。结论 :HBV多表位基因疫苗可诱发特异性免疫应答 ,为预防、治疗性HBV疫苗的研制提供了一定的实验依据     

Vaccination against hepatitis B virus at the Lyon Pasteur Institute. A seven-year evaluation]

Results of immunization against hepatitis B among Pasteur Institute staff members are reported. Prior to immunization, 439 subjects were tested for hepatitis B virus (HBV) markers, including HBs antigen, anti-HBs antibody, and anti-HBc antibody (Ausria, Ausab, Corab assays; Abbott). Forty-seven subjects tested positive for anti-HBs antibody. 317 subjects negative for all the HBs markers studied were given three intramuscular doses of Hevac B (Pasteur vaccins) at one-month intervals. Anti-HBs antibodies were assayed after the third injection with the following results: mean titer, 1,454 mIU/ml, standard deviation, 5,349 mIU/ml, and range, 4 to 41,100 mIU/ml. Anti-HBs titers above 10 mIU/ml were found in 879.4% of subjects. Non-responders and weak responders (anti-HBs titer under 10 mIU/ml) were given a fourth dose of vaccine. Ultimately, after the last (third of fourth) injection 97.6% of subjects had protective antibody titers. No case of HBV infection was seen during the seven-year follow-up period.     

High rate of seroconversion following administration of a single supplementary dose of recombinant hepatitis B vaccine in Iranian healthy nonresponder neonates

Forty-nine Iranian neonates who failed to develop a protective anti hepatitis B surface antigen (HBs) response following primary vaccination with triple doses of recombinant hepatitis B vaccine, were classified as hypo-responders (anti-HBs > 1 < 10 IU/l) or non-responders (anti-HBs < 1 IU/l) and subsequently challenged with a single supplementary vaccine dose. A protective and anamnestic type of response was observed in 90% (44/49) of the neonates. The mean titer of anti-HBs antibody was significantly higher following supplementary vaccination, compared to that achieved after primary vaccination. This was more evident in the primary hypo-responder neonates (P < 0.00001) than the non-responder group (P < 0.002). The results indicate that a significant proportion of the non-responder neonates to HBs antigen can be induced to develop a protective and long-lasting antibody response by administration of a single additional vaccine dose. Received: 24 September 1996     

Immune responses to recombinant Mycobacterium smegmatis expressing fused core protein and preS1 peptide of hepatitis B virus in mice

Attenuated strains of bacteria have been developed as potential live vectors to express homologous or heterologous antigens of many pathogens for inducing protective immune responses. The non-pathogenic and rapidly growing Mycobacterium smegmatis can be transformed effectively by genes for pathogenic antigens, and has been used as a valuable vector for the development of live vaccines. However, little is known on whether M. smegmatis could be transformed with the genes for HBV antigens and could express those genes, and whether vaccination with recombinant M. smegmatis could induce humoral and cellular immune responses in vivo. Both the core protein and preS1 peptide of the hepatitis B virus (HBV) are immunogenic and can induce cellular and humoral immune responses. This made them ideal platform for the development of new vaccines. In the present study, both recombinant M. smegmatis and DNA vaccines were generated to express the CS1 antigen, a fusion protein that comprises truncated core protein (amino acids 1-155) and preS1 peptide (amino acids 1-55) of HBV. Following vaccination of BALB/c mice with the live recombinant M. smegmatis, the CS1-based DNA vaccine, or controls, antigen-specific humoral and cellular immune responses were characterized. Vaccination with live recombinant M. smegmatis induced a stronger cellular immune response and a longer period of humoral immune response than with the DNA vaccination. These results indicate that the recombinant M. smegmatis can express efficiently immunogenic CS1 antigen of HBV in vivo, and may be used for the prevention of HBV infection.     

Passively acquired antibody to hepatitis B surface antigen. Pitfall in evaluating immunity to hepatitis B viral infections

Antibody to hepatitis B surface antigen (anti-HBs) has been used clinically to indicate an immune response to hepatitis B virus (HBV) and a protection against reinfection with the virus. We describe a child with hemophilia who had high-titer IgG anti-HBs in his serum and who subsequently developed viral B hepatitis. The child had received a unit of fresh frozen plasma 17 days prior to the determination of anti-HBs. The fresh frozen plasma donor was later found to be anti-HBs positive. The patient's anti-HBs was most likely passively acquired and therefore did not signify immunity to HBV. Various tests, including hepatitis B surface antigen group-specific and subtype determinants, ratio units of anti-HBs, and antibody class, have been used to determine whether or not anti-HBs will confer immunity. Although these tests have been thought to accurately predict immune status against infection with HBV, our case shows this may not be true, especially in patients who have been recently transfused. Anti-HBs testing may be predictive of immunity to HBV in the absence of a source of passively acquired anti-HBs.     

The isolated presence of anti-HBc antibodies: prevalence and interpretation based on the results of viral DNA research and anti-HBs antibodies measurement after vaccination

In order to study the significance of the isolated presence of anti-HBc antibodies, we have looked for the 3 classic serological markers of the hepatitis B (HBs antigen, anti-HBs and anti-HBc antibodies) in 1,586 hospital agents who are to vaccinate in the framework of a campaign of systematic vaccination of the hospital personnel of university hospitals of Sfax for a period of 18 months. We identified subjects who presented isolated anti-HBc antibodies (33 individuals = 2.08%). In these subjects'serum, we performed a research of the DNA of hepatitis B virus (HBV) with a PCR hybridization technique using a couple of primers. One week after administration of a vaccine dose, we also measured anti-HBs antibodies in their sera. Among the tested 18 personnel with anti-HBc isolated antibodies, 11.1% had low rates of anti-HBs antibodies indicating that there is presumably primary antibody response and therefore a false positivity of anti-HBc antibodies in pre-vaccinal serology; while 11.1% others had higher rates of anti-HBs antibodies corresponding to a secondary antibody response, which witness a previous HBV immunisation. The research of the HBV-DNA was positive in 11.1% of tested personnel, testifying a presumably chronic portage of the virus with low rates of the HBs antigen undetectable with the serological techniques. For the remainder subjects with isolated anti-HBc antibodies (66.7%), the interpretation remains ambiguous.