HBsAg/GM-CSF融合蛋白在毕赤酵母中的表达及其免疫原性研究

为探索一种提高乙肝病毒表面抗原免疫原性的新方法,用PCR和基因重组技术构建HBsAg与GM-CSF的融合基因,并在毕赤酵母中分泌表达HBsAg/GM-CSF(S-GM)融合蛋白。表达产物用SDS-PAGE检测,W estern b lot分析,离子交换柱纯化后免疫昆明鼠,ELISA检测免疫小鼠血清中抗HBsAg的抗体水平。结果显示S-GM融合蛋白在毕赤酵母中获得了表达,离子交换柱一步纯化即可得到纯度达90%以上的S-GM。W estern b lot分析S-GM可分别与抗HBsAg及抗GM-CSF的抗体特异结合。ELISA检测发现第一次免疫后4w出现抗HBsAg的抗体,加强免疫后融合蛋白组几乎全部阳转,且抗体水平较HBsAg组(P=0.009<0.05)及HBsAg和GM-CSF的混合物组(P=0.032<0.05)高。HBsAg/GM-CSF融合蛋白能够在毕赤酵母中表达,且可增强HBsAg的免疫原性,为提高乙肝疫苗的免疫效果提供了新的思路与方法。     

Cationic Lipid-Formulated DNA Vaccine against Hepatitis B Virus: Immunogenicity of MIDGE-Th1 Vectors Encoding Small and Large Surface Antigen in Comparison to a Licensed Protein Vaccine

Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals.     

Improved immunogenicity of HIV-1 epitopes in HBsAg chimeric DNA vaccine plasmids by structural mutations of HBsAg

To improve the immunogenicity of epitopes from the envelope protein of HIV-1, we have developed gene gun-delivered subunit DNA vaccines by inserting the sequences encoding the V3 region into the hepatitis B virus (HBV) envelope gene, often called the surface antigen (HBsAg). We have examined the possibility of modifying the immune response to V3 by introducing modifications into the carrier HBsAg in gene gun DNA immunization of mice. In some plasmid constructions, the V3 sequence was introduced into the preS2 region of the HBsAg. Although this region is not present in all protein subunits of the HBsAg particles produced, abolishing the internal translational initiation site for the S protein had no effect on the immune response to V3. Expression of V3 at the N-terminal or C-terminal part of the HBsAg protein resulted in equal anti-V3 antibody and cytotoxic T-lymphocyte (CTL) responses. However, elimination of secretion by single amino-acid mutations in the HBsAg decreased the anti-HBsAg antibody response but enhanced the anti-V3 antibody response. In contrast, the CTL response to V3 was independent of the structural mutations but could be improved by a total deletion of the HBsAg sequence part. Thus, the immune response to heterologous epitopes can be altered by modifications in the carrier HBsAg protein. Modifications of the HBsAg carrier might interfere with the dominant immune response to the HBsAg epitopes, allowing better antibody induction to less immunogenic foreign epitopes. However, for induction of CTL responses, the expression of minimal epitopes may be advantageous.     

Immune response to a hepatitis B DNA vaccine in Aotus monkeys: a comparison of vaccine formulation, route, and method of administration.

BACKGROUND: Attempts to optimize DNA vaccines in mice include using different routes of administration and different formulations. It may be more relevant to human use to carry such studies out in nonhuman primates. Here we compare different approaches to delivery of a DNA vaccine against the hepatitis B virus (HBV) in Aotus monkeys. MATERIALS AND METHODS: Thirty-two adult Aotus l. lemurinus monkeys divided into 8 groups of four were immunized with 400 microg of a DNA vaccine which encoded hepatitis B surface antigen (HBsAg). DNA in saline was administered by intradermal (ID) or intramuscular (IM) injection with needle and syringe, IM injection with the Biojector needleless injection system or combined ID (needle) and IM (Biojector). DNA formulated with cationic liposomes (CellFECTIN) was injected IM with needle or Biojector. DNA with added E. coli DNA (100 microg) was injected IM with the Biojector or ID. A ninth group of 4 monkeys was injected IM (needle) with Engerix-B, a commercial vaccine containing recombinant HBsAg (10 microg) adsorbed onto alum. Monkeys were boosted in an identical fashion to their prime at 8 weeks, but all received the protein vaccine (Engerix-B) at 16 weeks. Sera was assessed for antibodies against HBsAg (anti-HBs) by enzyme-linked imunosorbent assay (ELISA). RESULTS: The primary humoral response induced by IM delivery of the DNA vaccine was very poor. In most cases there was no detectable anti-HBs even after 2 DNA doses but the kinetics of the response to subsequent protein indicated that a memory B cell response had been induced. In contrast, following IM-administration of DNA using the Biojector, detectable anti-HBs were observed in 3 of 8 animals and evidence for immunological priming was apparent in an additional 4 of the 8 monkeys. ID injection of DNA vaccine in saline induced a potent antibody response which was augmented 6-fold by the addition of E. coli DNA. Combining ID and IM administration did not improve humoral immunity over ID injection alone. CONCLUSIONS: For immunization of primates with DNA vaccines, ID may be a preferable route to IM, although it is not clear whether the Aotus monkey is a relevant model for humans in this respect. Nevertheless, the use of the Biojector needleless injection system may improve responses with IM delivery of DNA vaccines. As well, the immunostimulatory action of E. coli DNA may be used to augment the humoral response induced by a DNA vaccine.     

The effect of human gamma globulin on tbe production of antibodies to the surface antigen of the hepatitis B virus in an experiment on animals

The biological assay for the determination of HBsAg in gamma globulin is proposed. The assay is based on the immunizing action of immune complexes and some specific features of secondary immune response. For this purpose, the highly purified preparation of HBsAg is injected into guinea pigs. Subsequently the animals receive human gamma globulin. An increase in the titer of anti-HBsAg antibodies in the animals preimmunized with the antigen is indicative of the presence HBsAg in human gamma globulin. In accordance with another variant of this assay, human gamma globulin is introduced into mice and 21 days later the animals are immunized with HBsAg. The appearance of anti-HBsAg antibodies in the animals, previously treated with gamma globulin, on day 4 after the injection of the antigen indicate that the animals experience the second penetration of HBsAg, i. e. its presence in previously injected human gamma globulin.     

A vectored measles virus induces hepatitis B surface antigen antibodies while protecting macaques against measles virus challenge

Hepatitis B virus (HBV) acute and chronic infections remain a major worldwide health problem. Towards developing an anti-HBV vaccine with single-dose scheme potential, we engineered infectious measles virus (MV) genomic cDNAs with a vaccine strain background and expression vector properties. Hepatitis B surface antigen (HBsAg) expression cassettes were inserted into this cDNA and three MVs expressing HBsAg at different levels generated. All vectored MVs, which secrete HBsAg as subviral particles, elicited humoral responses in MV-susceptible genetically modified mice. However, small differences in HBsAg expression elicited vastly different HBsAg antibody levels. The two vectors inducing the highest HBsAg antibody levels were inoculated into rhesus monkeys (Macaca mulatta). After challenge with a pathogenic MV strain (Davis87), control naive monkeys showed a classic measles rash and high viral loads. In contrast, all monkeys immunized with vaccine or a control nonvectored recombinant vaccine or HBsAg-expressing vectored MV remained healthy, with low or undetectable viral loads. After a single vaccine dose, only the vector expressing HBsAg at the highest levels elicited protective levels of HBsAg antibodies in two of four animals. These observations reveal an expression threshold for efficient induction of HBsAg humoral immune responses. This threshold is lower in mice than in macaques. Implications for the development of divalent vaccines based on live attenuated viruses are discussed.     

Immunogenicity and safety assessment of the Cuban recombinant hepatitis B vaccine in healthy adults.

Manufactures of biotechnological/biological products (including vaccines) frequently make changes to manufacturing processes of products both during development and after approval. In our case, a non-inferiority bridging study was carried out to demonstrate that changes in the production plant facilities of Cuban recombinant hepatitis B vaccine, Heberbiovac HB, did not affect the safety and immunogenicity of the vaccine. This controlled, randomized, doubled-blinded trial included 501 volunteers, aged between 20 and 64, who were given three doses of vaccine (20 microg HBsAg/mL) at month 0, 1, and 2. Four lots were evaluated (three corresponding to the new production facilities and a control one produced in the older facilities). One month after the third dose, were observed protective levels of anti-HBsAg in 97% of the subjects that concluded the study with a geometric mean antibody titer (GMT) of 931.18 IU/L. Normal values of body mass index (BMI), the younger ages, and being a female, were significantly related to a good antibody response. The vaccine was well tolerated. Pain at the injection site was the most commonly reported symptom. We conclude that Heberbiovac HB vaccine maintains its characteristics after the modifications carried out in the production plant facilities and both, lot obtained in previous facilities and in the new ones, are comparable in terms of safety and immunogenicity.     

Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.

Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d.     

Structural and Functional Characterization of Liposomal Recombinant Hepatitis B Vaccine

Abstract

Liposomal hepatitis B vaccine was prepared by encapsulating recombinant 22-nm hepatitis B surface antigen (HBsAg) particles derived from a Chinese hamster ovary (CHO) cell line in multilammelar lipid vesicles (MLV) composed of 9:1 dimyristoyl phosphatidyl-choline/dimyristoyl phosphatidylglycerol. The CHO-derived HBsAg particles reveal 6 bands in polyacrylamide gel electrophoresis related to the presence of 3 peptides (S, M, & L). Four different methods were used to prepare the MLV vaccine, each resulting in freeze-dried powder which upon hydration gave MLV of a similar mean size, 4.5 μm. The humoral response to these 4 liposomal vaccines in mice was dependent on the method of preparation, but for all of them it was better than the response to alum-based vaccine (especially at a low dose of antigen). Comparison of vaccination using “naked” HBsAg particles, particles adsorbed to alum, and particles encapsulated in liposomes demonstrated that at low dose of antigen the liposomal vaccine was superior in eliciting humoral response. Encapsulation in liposomes did not improve specific cytotoxic T lymphocyte (CTL) response. The alum in the vaccine completely eliminates CTL response, though it improved the humoral response by increasing the linear range in the antigen dose-response curve (increasing the antibody titer at high antigen dose). A similar response profile was obtained with recombinant yeast (Hansenula) 22-nm particles composed of a single non-glycosylated (p24) peptide and lipids. The similarity in the response to the mammalian cell and yeast derived vaccine suggests that the physical nature of the vaccine, more than the exact composition, determines the balance between humoral and CTL responses.     

Construction and characterization of hybrid hepatitis B antigen particles carrying a poliovirus immunogen

The hepatitis B surface antigen (HBsAg) has the unique property of assembling with cellular lipids into spherical or elongated particles of 22 nm diameter which are secreted by mammalian cells expressing HBsAg. We have studied the structural requirements for particle formation and secretion by creating in-phase insertions into different regions of the S gene of the hepatitis B virus, coding for HBsAg. Modified genes were integrated into an appropriate vector and expressed in mouse L cells. Various single and double inserts in the two major hydrophilic domains of HBsAg were compatible with particle synthesis and secretion. The level of secretion was influenced by the length of the insert, its primary structure, and the site of insertion into the HBsAg molecule. One of the inserted sequences was a synthetic DNA fragment encoding a continuous type 1 poliovirus neutralization epitope (the C3 epitope). Mammalian cells expressing the modified hepatitis B virus S gene secreted hybrid particles carrying the poliovirus antigen. The hybrid polio-HBsAg particles reacted with a monoclonal antibody specific for the C3 epitope and induced poliovirus neutralizing antibodies at low, but significant, titers in mice and at high titers in rabbits. However, the immune response to HBsAg was weaker to hybrid particles than to unmodified HBsAg particles. By cotransfection with two different plasmids carrying either modified or unmodified genes, we obtained phenotypically mixed particles containing both polio-HBsAg and HBsAg molecules. Inoculated into rabbits, the mixed particles induced high antibody titers against both poliovirus and HBsAg.     

Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120-146) and preS1(21-47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.     

胸腺素α原作为乙型肝炎表面抗原佐剂的研究

目的：研究胸腺素α原（ProTα）作为佐剂对重组乙型肝炎表面抗原（HBsAg）诱导小鼠产生乙型肝炎表面抗体（抗-HBs）的影响。方法：以纯系BALB/c小鼠为免疫对象,分组情况为：①HBsAg组（1μgHBsAg）;②0.1μgProTα＋1μgHBsAg组;③0.5μgProTα＋1μgHBsAg组;④1μgProTα＋1μgHBsAg组;⑤铝佐剂组（1μgVacon疫苗）;⑥1μgPro-Tα＋1μgVacon疫苗组;每组10只小鼠,分别于0、2周各肌肉注射免疫1次。采用ELISA法检测血清抗-HBs滴度（即IgG亚类IgG1和IgG2a）。结果：与单独注射HBsAg组相比,1μgProTα＋1μgHBsAg组抗-HBs总抗体滴度明显增高（P〈0.05）,抗体持续时间更长,且ProTα可以平衡Th1和Th2免疫反应;2组间IgG1/IgG2a差异显著（P〈0.05）。与铝佐剂相比,ProTα增强了小鼠对HBsAg的反应性,提高抗-HBs阳转率。结论：ProTα在增强小鼠抗-HBs产生的同时,提高细胞免疫反应,提示ProTα是一种很有潜力的HBsAg佐剂。     

Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice

The smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have produced protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax). H3L, an intracellular mature virion envelope protein, was consistently recognized by high-titer antibodies in the majority of human donors, particularly after secondary immunization. We then focused on examining H3L as a valuable human antibody target. Purified human anti-H3L antibodies exhibited substantial vaccinia virus-neutralizing activity in vitro (50% plaque reduction neutralization test [PRNT50] = 44 microg/ml). Mice also make an immunodominant antibody response to H3L after vaccination with vaccinia virus, as determined by vaccinia virus protein microarray. Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed high-titer vaccinia virus-neutralizing antibodies (mean PRNT50 = 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD50) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02). Together, these data show that H3L is a major target of the human anti-poxvirus antibody response and is likely to be a key contributor to protection against poxvirus infection and disease.     

用rAAV8-1.3HBV制备两种品系小鼠乙型肝炎病毒感染模型的比较研究

我们先前用rAAV8-1.3HBV静脉注射C57BL/6小鼠成功地制备了慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染模型。为了探讨不同品系的小鼠对rAAV8-1.3HBV静脉注射是否具有不同反应,本研究比较了C57BL/6和BALB/c小鼠静脉注射重组病毒后外周血中HBV抗原和抗体水平、病毒载量和肝脏组织HBcAg表达情况,以及不同剂量重组病毒注射与这些指标的关系。将低(4×109 Viral genome,vg)、中(4×1010vg)和高(4×1011vg)三种剂量的rAAV8-1.3HBV通过尾静脉注射至C57BL/6和BALB/c小鼠,分别利用ELISA和荧光定量PCR方法检测血清中的HBV抗原、抗体水平以及HBV DNA,利用免疫组化检测肝脏组织HBcAg的表达。结果发现,对于C57BL/6小鼠,三种不同剂量rAAV8-1.3HBV注射均可造成100%小鼠出现HBV持续感染;血清HBsAg、HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,其表达水平随重组病毒注射剂量的增加而升高,高剂量注射时可造成超过40%的肝细胞感染HBV,血清中HBV DNA可达105 IU/mL以上;未检测到针对HBV的抗体。对于BALB/c小鼠,三种不同剂量rAAV8-1.3HBV注射也可造成100%小鼠出现HBV持续感染;血清HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,但是血清HBsAg在重组病毒注射2周之后显著下降甚至消失;在中剂量注射组的BALB/c小鼠血清中检测到低水平的Anti-HBs;血清HBeAg和肝组织HBcAg的表达水平随重组病毒注射剂量的增加而增高,并且各剂量组表达水平均高于C57BL/6小鼠,高剂量注射时可造成超过50%的肝细胞感染HBV。本研究表明,低至4×109 vg剂量的rAAV8-1.3HBV注射即可造成C57BL/6和BALB/c两种品系小鼠出现HBV持续感染,并且HBV复制水平随重组病毒注射剂量增加而增高;BALB/c小鼠对HBV的免疫反应强于C57BL/6小鼠,可以产生针对HBsAg的体液免疫反应而使血清HBsAg转阴,但无法清除携带HBV的肝细胞。     

Immunogenicity of recombinant hepatitis B virus surface antigen fused with preS1 epitopes expressed in rice seeds

To test the possibility of producing a novel hepatitis B vaccine in plants, the modified hepatitis B virus (HBV) surface antigen (HBsAg) gene SS1 was expressed in rice under the control of the seed-specific Glub-4 promoter. The SS1 gene encodes a fusion protein consisting of amino acids 21-47 of the hepatocyte receptor-binding presurface 1 region (preS1) fused to the truncated C-terminus of the major HBV surface (S) protein. The production of antibodies against the preS1 region acts to protect humans against HBV infection by preventing HBV from binding to hepatocytes. The presence of SS1 in the genome of transgenic rice was confirmed by PCR and Southern blot analysis, and RNA dot blot analysis indicated that the fused SS1 gene was specifically expressed in rice seeds, with the highest expression level being about 31.5 ng/g dry weight grain. Western blot analysis revealed that the recombinant SS1 protein could be specifically recognized by both an anti-S protein antibody and an anti-preS1 antibody. The recombinant SS1 protein was also observed to form virus-like particles with a diameter of about 22 nm and a density of 1.25 g cm(-3). Furthermore, immunological responses against both the S and preS1 epitopes were induced in BALB/c mice immunized with the recombinant SS1 protein, indicating that this rice-derived SS1 protein could be a promising candidate as an alternative HBV vaccine for preventing hepatitis B.     

Use of baculovector for the expression of HBsAg gene in insect cells

Based on the information of molecular biology of Autographa californica Nuclear Polyhedrosis virus (AcNPV), a recombinant transfer plasmid pAcMV was constructed by molecular procedures included using two synthetic localized probes, which provided an inserted position linked with BamHI sequences nearly at polyhedrin initiating ATG codon. Then an expression vector pAcMV-HBsAg was reconstructed, it contained HBsAg gene from subclone pYPSS-1 derived from adwserotype of HBV. The recombinant virus containing HBsAg gene was isolated and purified through 3 cycles plaques and hybridization experiment after cotransfection of Spodoptera frugiperda cells with DNA of pAcMV-HBsAg and AcNPV. The expression of HBsAg gene in S. frugiperda cells infected with recombinant virus AcRV-HBsAg was identified by ELISA as haemagglutination tests. The yield of HBsAg excreted from S. frugiperda cells (an appropriate density usually between 1-2 X 10(6) cells/ml) after 48-72 h infected with AcRV-HBsAg was 4-8 mg/L. HBsAg harvested from the infected culture medium was shown immunoelectromicroscopy to be composed of spherical particles of about 22 nm diameter. Using this purified HBsAg, Bal b/c mice was immunized, the titer of anti-HBsAg serum measured measured by RIA was similar to that of purified HBsAg from human blood. Stable recombinant virus was isolated and could be shown to replicate in corn borer (Ostrinia nubilalis) larvae. All of these results can be expected that this expression vector system will be commercially developed to its fullest potential for diagnosis and vaccine HBsAg.     

Monoclonal antibodies against hepatitis B virus surface antigen: preparation and characterization

Hybridomas secreting HBsAg antibodies were obtained by fusing murine myeloma cell line P3-X63-Ag8 to spleen cells of BALB/c mice sensitized with HBsAg. The surface antigen used for immunization of mice was prepared by purification from pooled human plasma specimens. Resulting monoclonal antibodies were detected by the SPRIA method. Clones producing highest anti-HBs titres were used to prepare mouse ascitic fluids. Monoclonal antibodies in ascitic fluid reached a titre of 10(6) to 10(7) at a protein concentration of 1 mg per ml. Two of the prepared monoclonal antibodies, HBS-01 and HBS-02, both belonging to IgG1 subclass of immunoglobulins, were selected for further study in order to assess their potential useability in the commercial ELISA kit. The pI values for HBS-01 ranged from 6.60 to 6.85, for HBS-02 from 5.6 to 6.1. In solid phase ELISA test the use of HBS-01 antibody improved accuracy of the assay by increasing its detection sensitivity for HBsAg subtypes adw and ayw in the reference serum; this sensitivity was evidently much better than that seen with the commercially available rabbit polyclonal anti-HBsAg antibody. The monoclonal antibody HBS-01 is specific to the determinant "a", which makes it suitable for use in ELISA test aimed at HBsAg detection. The antibody HBS-02 showed a markedly better reaction with HBsAg subtype adw than subtype ayw and can thus be used with advantage for their discrimination.     

DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV) core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic

Background

Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4) primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV) clearance.

Principal Findings

Plasmids expressing HBV core protein (HBcAg) or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc), CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI) of pAAV/HBV1.2. HBV surface antigen (HBsAg) and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance.

Conclusion

Viral clearance could be efficiently achieved by Th1/Th2-balanced immune response, with a small but significant shift in T-cell and B-cell immune responses.