Evaluation of Hepatitis B Reactivation Among Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors.

Hepatitis B reactivation (HBVr) in cancer patients is a well-established complication due to chemotherapy-induced immunosuppression. Studies have reported HBVr associated with immunosuppressive medications, such as rituximab, methotrexate, and high dose steroids. There are different risks for different types of chemotherapy with rituximab carrying one of the highest risks for hepatitis B reactivation. Tyrosine kinase inhibitors (TKIs) are the standard of care in patients with chronic myeloid leukemia (CML). The risk of HBVr in chronic myeloid leukemia has been reported in many studies, but to this date, there are no clear guidelines or recommendations regarding screening and monitoring of HBV in CML patients receiving TKIs. We conducted this review to identify the risk of HBVr in patients with CML who are treated with tyrosine kinase inhibitors. We recommend testing for HBV status in patients who are to be treated with TKIs and to consider giving prophylaxis in those who are positive for HBsAg at baseline. More studies are needed to assess the risk of reactivation in patients with Hepatitis B core antibody positive receiving TKIs. Currently, monitoring such patients for reactivation may be the best strategy.

Expression and characterization of hepatitis B surface antigen in transgenic potato plants.

Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 microg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.

Mechanism of adsorption of hepatitis B surface antigen by aluminum hydroxide adjuvant.

Hepatitis B surface antigen (HBsAg) differs from many antigens because of its associated lipid bilayer that is largely composed of phospholipids. In general, phosphate groups adsorb strongly to hydroxylated mineral surfaces by ligand exchange. The purpose of this study was to investigate the mechanism of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant with emphasis on the role of phospholipids in this adsorption. The adsorption of HBsAg by aluminum hydroxide adjuvant exhibits a high affinity adsorption isotherm. The Langmuir equation was used to calculate the adsorptive capacity (1.7 microg/microg Al), which is the amount of HBsAg adsorbed at monolayer coverage and the adsorptive coefficient (6.0 ml/microg), which is a measure of the strength of the adsorption force. The relatively high value of the adsorptive coefficient indicates that adsorption is due to a strong attractive force. Ligand exchange between a phosphate of the antigen and a surface hydroxyl of the adjuvant provides the strongest adsorption mechanism. The adsorption capacity of HBsAg was not affected by increased ionic strength indicating that electrostatic attraction is not the predominant adsorption force. Adsorption was also not affected by the addition of ethylene glycol indicating that hydrophobic interactions were not the predominant adsorption force. The strength of the adsorption force was indicated by the resistance of HBsAg to elution when exposed to interstitial fluid. Less than 5% of the HBsAg adsorbed to aluminum hydroxide adjuvant in a model vaccine was eluted during a 12 h in vitro exposure to interstitial fluid at 37 degrees C. Less than 1% of the adsorbed HBsAg in two commercial vaccines was eluted by in vitro exposure to interstitial fluid for 48 h at 37 degrees C. Thus, it was concluded that adsorption of HBsAg by aluminum hydroxide adjuvant is predominantly due to ligand exchange between the phospholipids in HBsAg and surface hydroxyls in aluminum hydroxide adjuvant.

Factors important in the extraction, stability and in vitro assembly of the hepatitis B surface antigen derived from recombinant plant systems.

The expression of vaccine antigens in edible plant material together with their delivery by the oral route constitutes a powerful paradigm, with the potential to dramatically reduce the cost of vaccine production and administration, in addition to improving distribution and patient compliance. These products will be subject to many of the same regulations applied to current injectable vaccines, so reliable methods to quantify antigen and ensure stability in crude plant extracts are required. As a model system the hepatitis B surface antigen (HBsAg) was expressed in soybean and tobacco cell cultures. This complex antigen consists of membrane-associated small surface antigen proteins (p24(s)), disulfide cross-linked to yield dimers and higher multimers. Although the total p24(s) extracted from plant cells was relatively unaffected by detergent concentration, the quantification of antigenically reactive product depended strongly on the ratio of detergent to cell concentration. Furthermore, 1-20% w/v sodium ascorbate improved the measured levels of monoclonal-reactive antigen 4- to 12-fold. Detergent also influenced antigen stability in cell lysates stored at 4 degrees C; under optimum conditions stability was maintained for at least 1 month, whereas excess detergent rendered the antigen susceptible to proteolytic degradation. This proteolysis could be counteracted by the addition of skim milk or its protein component, which stabilized antigenically reactive p24(s) for up to 2 months. The immunologically relevant epitopes of HBsAg are critically dependent on disulfide bonding. By altering the sodium ascorbate concentration or buffer pH the proportion of HBsAg displaying the monoclonal reactive epitopes was increased between 8- and 20-fold. In addition, under certain conditions the dimerized p24(s) could be converted to oligomeric aggregates, resembling the form of the serum-derived antigen. These simple in vitro manipulations, compatible with the goal of a minimally processed oral vaccine, may prove valuable in increasing the immunogenicity of the plant-derived antigen.

Process options in hepatitis B surface antigen extraction from transgenic potato.

The process conditions for recombinant hepatitis B surface antigen (HBsAg) extraction from transgenic potato were examined. The effects of temperature, the reducing agent beta-mercaptoethanol (BME), and proteinase inhibitors on the level of antigenic activity of recovered HBsAg were determined. Sedimentation profiles were performed to characterize HBsAg assembly into virus-like particles. Increasing the temperature of the sample for about 1 min increased the measured HBsAg antigenic activity. The optimum temperature was around 50 degrees C. A 3-fold enhancement of the antigenic activity was obtained in extract from transgenic potato expressing HBsAg, when monoclonal antibodies were used to assay for HBsAg. When antigenic activity was determined by polyclonal antibodies, no enhancement in the antigenic activity was obtained. Temperature may affect the conformation of the a epitope to which the monoclonal antibodies bind or alter the fluidity of surface lipid regions. BME increased the antigenic activity of HBsAg up to 4-fold when monoclonal antibodies directed against the a determinant were used, but there was no increase with polyclonal antibodies. This observation suggests that BME affects the structure or presentation of the a epitope. In the presence of BME and leupeptin, a proteinase inhibitor, higher antigenic activity was obtained. Leupeptin might protect the antigen, which might become more susceptible to proteolytic degradation after reduction, as a result of stimulation of sulfhydryl proteases. Although both temperature and BME increased the antigenic activity of HBsAg individually, when combined their interaction was antagonistic, resulting in reduced antigenic activity. Different proteinase inhibitors, including leupeptin, aprotinin, E-64, pefabloc, and pepstatin, had no significant effect on HBsAg from potato extract in a 2 h period in the absence of BME. The sedimentation profile of potato-produced HBsAg was determined in 5-30% sucrose gradients. Yeast-derived recombinant HBsAg was used as a positive control. The HBsAg from transgenic potato showed sedimentation and density properties that are very similar to the yeast-produced antigen, indicating assembly into virus-like particles. BME treatment did not change the sedimentation profile.

Hepatitis B surface antigen. Role of lipids in maintaining the structural and antigenic properties of protein components.

Most of the lipid components of hepatitis B surface antigen (HBsAg) can be removed by treatment with the non-ionic non-denaturing detergent beta-D-octyl glucoside (OG) followed by centrifugation through caesium chloride linear density gradients (density 1.15-1.32 g/ml). The conformational changes induced by the elimination of lipids decreased the helical content of HBsAg proteins from 52 to 28% as indicated by c.d. techniques. Measurements of the extent of quenching of protein fluorescence by iodide showed that half of the tryptophan residues which are buried in the native structure of HBsAg particles are brought close to the surface of the molecule by such conformational changes. The antigenic activity, as measured by binding to polyclonal antibodies, was decreased upon removal of lipids. Moreover, the six different antigenic sites recognized by our panel of monoclonal antibodies decreased their capacity to bind to the corresponding antibody when lipids were removed. However, the extent of this decrease differed for the different antibodies. Thus the apparent dependence of antibody binding on the lipid content seemed to indicate a greater involvement of the lipid-protein interaction for some of the epitopes than for others.

[Clinical and immunological studies of an experimental series of vaccines against hepatitis B]. / Klinicheskie i immunologicheskie issledovaniia éksperimental'nykh serii vaktsiny protiv gepatita B.

Two experimental lots of hepatitis B vaccine were prepared by purification of HBsAg from human plasma, inactivation at 100 degrees C for 2 min and at 37 degrees C for 72 hours with formalin in a concentration of 1:4000. The former lot comprises purified HBsAg (40 mg/ml) adsorbed with 0.32 mg % Al(OH)3, the latter consists of purified HBsAg stabilized with 0.1% human albumin and adsorbed with 1 mg% Al(OH)3. The immune response after vaccination with the first lot was observed in 54.54% of the vaccinees. The second vaccine after 3 administrations of 80 micrograms/ml each produced a very good primary and a very good secondary immune response. Both vaccines are nonreactogenic and well tolerated by the vaccinees.

[New approaches to obtaining vaccines for preventing serum hepatitis type B]. / Novye podkhody k polucheniiu vaktsin dlia profilaktiki syvorotochnogo gepatita tipa B.

A comparative study of the antigenic and immunogenic properties of hepatitis B virus surface antigen (HBsAg) and a glycopeptide containing protective antigenic determinant "a" (aAg) isolated from purified HBsAg structures was carried out. In contrast to HBsAg structures, aAg preparations contained no antigens of normal human serum. Anti-aAg antisera unlike immune sera against HBsAg contained no antibody against normal serum proteins. The aAg preparations were at least as immunogenic as HBsAg for laboratory animals. Inoculation of aAg-immunized mice with 125I-HBsAg resulted in no accumulation of the label in the kidneys whereas it was demonstrated in the animals immunized with HBsAg. The prospects of using minimal antigenic structures carrying protective antigenic determinants as split-HBV vaccines are discussed.