Transgenic <Emphasis Type="Italic">Cucumis sativus</Emphasis> Expressing the Hepatitis B Surface Antigen

A DNA fragment encoding the hepatitis B virus surface antigen was amplified from a positive blood (hepatitis B) sample and introduced into the pET 32c prokaryotic expression vector. The gene encoding the HBV surface protein antigen was introduced into pCAMBIA 3300, and immobilized into Agrobacterium tumefaciens strain LBA4404. Cotyledonary leaf sections of Cucumis sativus (cucumber) cv ‘Swarnamukhi’ were cocultivated with Agrobacterium harboring the binary vector pCAMBIA 3300 carrying the HBV surface antigen gene driven by the CaMV35S promoter and the herbicide resistance gene phosphinothricin. Putative transformed shoots were induced on a Murashige and Skoog (MS) medium containing phosphinothricin, and these were then rooted on MS basal medium supplemented with 1 mg/L Indole 3-butyric acid (IBA). Integration of the T-DNA into in putative transgenic plants was confirmed by PCR and Southern blot analyses. RT-PCR and Northern blot analyses were conducted to determine RNA expression. Levels of expression in transgenic plants were confirmed by Western blot analysis, and quantification of the protein was determined by enzyme linked immuno assay (ELISA). Molecular mass of the recombinant protein was measured by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) Mass Spectrometry.     

Expression of hepatitis B virus S gene in Pichia pastoris and application of the product for detection of anti-HBs antibody

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying a hexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5 % of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100 % with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.     

Selective synthesis and secretion of particles composed of the hepatitis B virus middle surface protein directed by a recombinant vaccinia virus: induction of antibodies to pre-S and S epitopes.

Selective synthesis in mammalian cells of the hepatitis B virus middle surface (MS) protein, which is 55 amino acids longer than the major surface (S) protein, was achieved by using a recombinant vaccinia virus. The 33-kilodalton MS polypeptide was glycosylated and secreted as particles that resembled human hepatitis B surface antigen as well as particles composed solely of S protein with regard to antigenicity, buoyant density, size, and electron micrographic appearance. The MS particles differed from S particles, however, by binding to polymerized human albumin and inducing antibodies that reacted with a pre-S peptide and inhibited the binding of human plasma-derived hepatitis B surface antigen to polymerized human albumin.     

Expression of the hepatitis B surface S and preS2 antigens in tubers of <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

In an attempt to develop an edible vaccine, we transformed a recombinant hepatitis B virus (HBV) gene encoding the middle protein of HBV that contains the surface S and preS2 antigen into potato by Agrobacterium-mediated transformation. The HBV gene was under control of either the CaMV 35S promoter, the double 35S promoter with the AlMV 5 non-translated leader sequence, or the tuber-specific patatin promoter. HBV mRNA levels were higher with the 35S promoter than with the double 35S and patatin promoters; however, the levels of the S and preS2 antigen in the transformed tubers were higher with the patatin promoter than with the CaMV 35S and double promoters. The levels of preS2 antigen produced are the highest reported to date. Transgenic potato tubers were fed to mice, and the mice showed an immune response against the HBV S antigen.     

Analysis of Transgenic Tobacco Plants Carrying the Gene for the Surface Antigen of the Hepatitis B Virus

The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBs antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HBsAg gene under a single 35S promoter was 0.0001–0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002–0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F₁ plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F₀ plants.     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg)

Summary A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-assciated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 1.12-0.18 h^–1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fedbatch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product fromation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-l culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth. Offprint requests to: M. B. Gu     

Prevalence and determinants of antibodies to hepatitis C virus and markers for hepatitis B virus infection in patients with HIV infection in Aquitaine. Groupe d'Epidémiologie Clinique du SIDA en Aquitaine.

OBJECTIVE: To evaluate the prevalence of antibodies to hepatitis C virus and serological markers for hepatitis B virus infection in patients with HIV. DESIGN: Cross sectional survey. SETTING: Aquitaine, southwestern France, 1991-94. SUBJECTS: 1935 HIV positive patients seen at least once since June 1991. MAIN OUTCOME MEASURES: Presence of antibodies to hepatitis C virus were detected by second or third generation enzyme linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA) and markers for hepatitis B virus detected by ELISA. RESULTS: The prevalence was 42.5% (823) for antibodies to hepatitis C virus, 56.4 (507) for antibodies to hepatitis B core antigen, 6.9% (133) for hepatitis B surface antigen, 30.2% (584) for antibodies to hepatitis B core and surface antigen with no detectable surface antigen, 26.2% (507) for antibodies to core antigen only, and 4.8% (92) for antibodies to surface antigen only. The prevalence of antibodies to hepatitis C virus was 86.1% (726/843) in subjects who had bloodborne HIV infection and 7.3% (66/899) in those with sexually acquired infection. The prevalence of markers for hepatitis B was higher among homosexuals than in the other groups of patients, except for antibodies to surface antigen alone. The relation between markers for hepatitis B and hepatitis C virus was negative among men but positive among women. CONCLUSIONS: The results favour the hypothesis that hepatitis C virus is sexually transmitted much less commonly than either HIV or hepatitis B virus.     

Cloning and production of Xylanase B from Paenibacillus barcinonensis in Bacillus subtilis hosts

Xylanase B from Paenibacillus barcinonensis was cloned in shuttle vectors for Escherichia coli and Bacillus subtilis, and expressed in Bacillus hosts. Several recombinant strains were constructed, among which B. subtilis MW15/pRBSPOX20 showed the highest production. This recombinant strain consists of a protease double mutant host containing P. barcinonensis xynB gene under the control of a phage SPO2 strong promoter. Maximum production was found when the strain was cultured in nutrient broth supplemented with xylans. Analysis of xylanase B location in B. subtilis MW15/pRBSPOX20 showed that the enzyme remained cell-associated in young cultures, consistent with its intracellular location in its original host, P. barcinonensis, and the lack of a signal peptide. However, when cultures reached the stationary phase, xylanase B was released to the external medium as a result of cell lysis. The amount of enzyme located in the supernatants of old cultures could account for 50% of total xylanase activity. Analysis by SDS–PAGE showed that xylanase B is an abundant protein found in the culture medium in late stationary phase cultures.     

A bio‐nanocapsule containing envelope protein domain III of Japanese encephalitis virus protects mice against lethal Japanese encephalitis virus infection

An engineered bio‐nanocapsule (BNC) comprising modified hepatitis B surface antigen L protein was used as a physical scaffold for envelope protein domain III (D3) of Japanese encephalitis virus (JEV). At the N terminus, the BNC contained a two‐tandem repeat of the Z domain (ZZ) derived from Staphylococcus aureus protein A (ZZ‐BNC). The Lys‐rich ZZ moiety exposed on the surface of ZZ‐BNC was used for chemical conjugation with the JEV D3 antigen, which had been expressed and purified from Escherichia coli. Immunization of mice with D3 loaded on the surface of ZZ‐BNC (ZZ‐BNC:D3) augmented serum IgG response against JEV and increased protection against lethal JEV infection. The present study suggests that innocuous recombinant antigens, when loaded on the surface of ZZ‐BNC, can be transformed to immunogenic antigens.     

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.     

Expression of the hepatitis B virus surface antigen in Drosophila S2 cells

Drosophila melanogaster S2 cells were transfected with a plasmid vector (pAcHBsAgHy) containing the S gene, coding for the hepatitis B virus surface antigen (HBsAg), under control of the constitutive drosophila actin promoter (pAc), and the hygromycin B (Hy) selection gene. The vector was introduced into Schneider 2 (S2) Drosophila cells by DNA transfection and a cell population (S2AcHBsAgHy) was selected by its resistance to hygromycin B. The pAcHBsAgHy vector integrated in transfected S2 cell genome and approximately 1,000 copies per cell were found in a higher HBsAg producer cell subpopulation. The HBsAg production varied in different subpopulations, but did not when a given subpopulation was cultivated in different culture flasks. Higher HBsAg expression was found in S2AcHBsAgHy cells cultivated in Insect Xpress medium (13.5 μg/1E7 cells) and SFX medium (7 μg/1E7 cells) in comparison to SF900II medium (0.6 μg/1E7 cells). An increase of HBsAg was observed in culture maintained under hygromycin selection pressure. Data presented in the paper show that S2AcHBsAgHy cells produce efficiently the HBsAg which is mainly found in the cell supernatant, suggesting that HBsAg is secreted from the cells. The data also show that our approach using the Drosophila expression system is suitable for the preparation of other viral protein preparation.     

Increased Hepatitis B surface antigen production by recombinant <Emphasis Type="BoldItalic">Aspergillus niger</Emphasis> through the optimization of agitation and dissolved oxygen concentration

The capacity of the filamentous fungi Aspergillus niger to produce and assemble complex immunogenic viral proteins into virus-like particles (VLPs) in batch culture was enhanced by optimizing the bioprocessing parameters, agitation intensity and dissolved oxygen (dO₂) concentration. Response surface methodology (RSM) and a two-factor-two-level central composite rotatable design (CCRD) were employed to evaluate the interactive response pattern between parameters and their optimum combination. The recombinant hepatitis B surface antigen (HBsAg) was used as a model VLP system to determine the effect of these parameters on biomass yield, fungal morphology, HBsAg production and bioreactor kinetics. The response surface model predicted optimum cultivation conditions at an agitation of rate of 100 rpm and a dO₂ concentration of 25%, obtaining highest intracellular membrane-associated HBsAg levels of . HBsAg production levels were increased tenfold compared to yields obtained in shake flask cultivation. Although hepatitis B VLPs mostly accumulated intracellularly, optimal bioreactor conditions resulted in significant HBsAg release in culture supernatant. These results compare favourably with other recombinant VLP systems in batch culture, and therefore, indicate a substantial potential for further engineering of the A. niger production system for the high level of intracellular and extracellular VLP production.     

Expression of hepatitis B surface antigen in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> utilizing glyceraldeyhyde-3-phosphate dehydrogenase promoter of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus ‘S’ gene under the control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited ∼20–22 nm particle formation. Stability and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale production.     

Consolidation treatment needed for sustained HBsAg-negative response induced by interferon-alpha in HBeAg positive chronic hepatitis B patients

Hepatitis B surface antigen (HBsAg) clearance is considered as functional cure in patients with chronic hepatitis B (CHB). This study aimed to assess the durability of HBsAg clearance achieved by interferon-based therapies in patients with CHB who were originally positive for hepatitis B envelope antigen (HBeAg). In this prospective study, HBeAg-positive CHB patients with confirmed HBsAg loss under interferon-based therapies were enrolled within 12 weeks from end of treatment and followed up for 48 weeks. Virological markers, biochemical indicators, and liver imaging examinations were observed every 3-6 months. Sustained functional cure was analysed as primary outcome. Factor associated with sustained HBsAg loss or reversion was also investigated. The rate of HBsAg loss sustainability was 91.8% (212/231). Patients receiving consolidation treatment for 12-24 weeks or ≥ 24 weeks had higher rates of sustained HBsAg negativity than those receiving consolidation treatment for < 12 weeks (98.3% and 91.2% vs. 86.7%, P=0.068), and the former groups had significantly higher anti-HBs levels than the later (P < 0.05). The cumulative incidence of HBsAg reversion and HBV DNA reversion was 8.2% and 3.9%, respectively. Consolidation treatment of ≥ 12 weeks[odd ratio (OR) 3.318, 95% confidence interval (CI) 1.077-10.224, P=0.037) was a predictor of sustained functional cure, and HBeAg-positivity at cessation of treatment (OR 12.271, 95% CI 1.076-139.919, P=0.043) was a predictor of HBsAg reversion. Interferon-alpha induced functional cure was durable and a consolidation treatment of ≥ 12-24 weeks was needed after HBsAg loss in HBeAg-positive CHB patients.     

Biosynthesis of recombinant human hepatitis B M-HBsAg in silkworm larvae

The middle surface antigen (M-HBsAg) of human hepatitis B virus is virus envelope protein. It's used as a basis for development of vaccine and test-system for detecting of hepatitis B virus. The cDNA of M-HBsAg was inserted into transfer vector pBK273 under the polyhedron promoter with obtaining of recombinant plasmid DNA pBHep-2. As a result of cotransfection pBHep-2 with wild type BmNPV the recombinant baculovirus rBmNPVHep which included the cDNA of M-HBsAg under the polyhedron promoter was obtained. Infection of silkworm larvae Bombyx mori with recombinant virus resulted in expression of foreign gene and accumulation of middle surface antigen of human hepatitis B virus mostly (>90%) in fat bodies of silkworm larvae.     

Size-exclusion chromatographic study of the reduction of recombinant hepatitis B surface antigen

The reduction of the P. pastoris-derived hepatitis B surface antigen (HBsAg) has been investigated by size exclusion chromatography performed in a detergent solution containing 0.3% sodium dodecyl sulfate (SDS) and 0.1 M Tris–HCl, pH 7.0. The HBsAg, reduced under different conditions and passed through the TSK G4000 SW column (600×7.5 mm I.D.) at 0.9 ml min⁻¹, was resolved into two peaks corresponding to the reduced, monomeric, and non-reduced forms, respectively. Under these conditions, the antigen fraction corresponding to the HBsAg dimer can be separated and completely reduced to monomers by repeated reductive treatment with simultaneous lipid removal. The efficiency of reduction was maximal after sample treatment with an equal volume of a solution containing 417 mM dithiothreitol, 4.2% (w/v) SDS and 16% (v/v) 2-mercaptoethanol. In conclusion, complete reduction of recombinant HBsAg to monomer subunits is possible and depends on the efficiency of lipid removal during the reductive treatment.     

乙肝病毒核心蛋白病毒样颗粒表面抗原密度对抗体应答水平的影响

【目的】为了探究乙肝病毒核心蛋白(HepatitisBviruscoreprotein,HBc)病毒样颗粒(Virus-like particles,VLPs)表面抗原密度对免疫后抗体应答水平的影响,制备了不同抗原密度的HBc VLPs疫苗,并检测了其在小鼠体内的抗体应答水平。【方法】首先制备了N端带有3个甘氨酸的人巨细胞病毒重组抗原域AD-4作为模式抗原,接着通过Sortase A的介导将AD-4连接到HBc VLPs表面上。将系列浓度梯度AD-4抗原在SortaseA介导下分别与相同浓度的HBcVLPs发生反应,制备不同抗原密度的HBc-AD-4 VLPs。将其分别免疫6–8周龄BALB/c小鼠3次,每次免疫间隔2 w,间接ELISA法检测被免疫小鼠血清的抗体应答水平。【结果】结果表明,当HBc VLPs表面抗原密度为44.4%时,即HBc反应浓度∶AD-4反应浓度为1:0.5时,不足以引起高滴度的抗体产生;当HBc VLPs表面抗原密度为64.2%时,即HBc反应浓度∶AD-4反应浓度为1:1时,HBc-AD-4 VLPs诱导的AD-4特异性抗体滴度与100%抗原密度的HBc-AD-4VLPs所引起的抗体滴度相当;当HBcVLPs表面抗原密度大于64.2%时,引起的抗体应答水平不因抗原密度增加而进一步增强。【结论】发现了HBcVLPs表面抗原密度与免疫后抗体滴度呈正相关,然而免疫64.2%抗原密度的HBc VLPs所产生的抗体滴度可达峰值,抗原密度进一步增加,抗体应答水平不会进一步加强。     

Application of recurrent neural network for online prediction of cell density of recombinant Pichia pastoris producing HBsAg

Abstract

Artificial neural networking (ANN) seems to be a promising soft sensor for implementing current approaches of quality by design (QbD) and process analytical technologies (PAT) in the biopharmaceutical industry. In this study, we aimed to implement best-fitted ANN architecture for online prediction of the biomass amount of recombinant Pichia pastoris (P. pastoris) – expressing intracellular hepatitis B surface antigen (HBsAg) – during the fed-batch fermentation process using methanol as a sole carbon source. For this purpose, at the induction phase of methanol fed-batch fermentation, carbon evolution rate (CER), dissolved oxygen (DO), and methanol feed rate were selected as input vectors and total wet cell weight (WCW) was considered as output vector for the ANN. The obtained results indicated that after training recurrent ANN with data sets of four fed-batch runs, this toolbox could predict the WCW of the next fed-batch fermentation process at each specified time point with high accuracy. The R-squared and root-mean-square error between actual and predicted values were found to be 0.9985 and 13.73, respectively. This verified toolbox could have major importance in the biopharmaceutical industry since recombinant P. pastoris is widely used for the large-scale production of HBsAg.     

Oral vaccination of mice with <Emphasis Type="Italic">Tremella fuciformis</Emphasis> yeast-like conidium cells expressing HBsAg

Tremella fuciformis yeast-like conidium (YLC) cells were transformed by co-cultivation with Agrobacterium cells harboring the hepatitis B surface antigen (HBsAg) gene construct under the control of the CaMV35S promoter. Integration of HBsAg DNA into the YLC genome was confirmed by PCR and dot-blot hybridization. Immunoblotting verified expression of the recombinant protein. Oral administration of YLC cells expressing HBsAg in mice significantly increased anti-HBsAg antibody titer levels using a double prime-boost strategy that combined parenteral and oral HBsAg boosters.