Recombinant Hepatitis B Virus Surface Antigen Research Articles

Decreased expression of HBV surface antigen (HBsAg) with sK122R and sV96A co-mutation is associated with an ineffective antibody response in a chronic hepatitis B patient.

Emergence and predominance of hepatitis B virus (HBV) variants carrying S gene mutations frequently occur in HBV-infected individuals. Here, coexistent serum anti-HBsAg antibody (HBsAb) and HBV surface antigen (HBsAg) were detected in a chronic HBV patient. The patient's HBsAg proteins possessed amino acid substitutions sK122R and sV96A. We reported this case and conducted relevant studies to investigate differences in expression levels and antibody neutralization of HBsAg proteins bearing sK122R and sV96A amino acid substitutions to explore causes of antigen-antibody coexistence in a chronic hepatitis B patient. We first sequenced the S gene from HBV present within the patient's serum. Based on the S gene sequence, we cloned wild-type and mutated S gene sequences via site-directed mutagenesis to construct expression plasmids pJW4303-WT (wild-type), pJW4303-sV96A, pJW4303-sK122R, and pJW4303-sV96A-sK122R. Plasmids were transfected into HEK 293T cells then culture supernatants and cells were collected. Collected cells and supernatants were next subjected to a series of quantitative and functional tests to assess expression and neutralization characteristics of wild-type and mutant HBsAg proteins. Based on quantification of HBsAg expression in cells transfected with the four plasmids, HBsAg-sK122R-sV96A was more intracellularly retained and less secreted than HBsAg-sV96A single-mutant protein and WT. Neutralization ability of serum from chronic HBV patient against culture supernatants containing recombinant HBsAg proteins were ranked from highest to lowest as HBsAg-sV96A, HBsAg-sV96A-sK122R, and HBsAg-sK122R. However, no significant differences of neutralization efficiency by high-potency antibodies from HBV-vaccinees against these three mutant proteins were observed. The levels of HBsAg proteins with amino acid substitutions sV96A-sK122R were greatly reduced in culture supernatants but were apparently increased in the intracellular fraction. This may account for the higher levels of HBV replication in patients. HBsAg neutralization by HBsAb in this patient may have been compromised by the HBsAg sK122R amino acid substitution, suggesting that antibodies produced by the patient had lost their HBV-neutralizing effect.

Protective immunity in a sample of healthy adults following vaccination with a more cost effective recombinant HBsAg vaccine

BackgroundVaccines against hepatitis B virus (HBV) infection became available from 1982, however, the HBV infection remains as a public health issue due to infection among susceptible individuals. Many HBV vaccines are available and until 2012, Sri Lanka has been using vaccines produced by different Pharmaceutical companies. Since 2012, Sri Lanka imported HBV vaccine from Serum Institute of India as the product was more cost effective. However, the immune response to this vaccine has never been studied in the country. ObjectiveThe current study was carried out to assess the immune response (anti-HBs) to the recombinant HBV surface antigen (HBsAg) vaccine (Serum Institute, India), which is currently used in the state healthcare sector of Sri Lanka. Study designA sample of vaccinated healthy adults (n=529), age ranged between 20-29 years were enrolled in this study after completing the standard 3-dose regimen of HBV vaccination from early 2015 to 2016. Sociodemographic data were collected using a self-administrated questionnaire. Serum samples were tested to detect the presence of anti-HBs using an enzyme linked immunosorbent assay (ELISA) and the results were analyzed using MS-Excel 2010. ResultsBased on the results, 96.8% of the healthy adults had protective immune response with anti-HBs levels > 10 mIU/mL. Gender did not show an association with levels of anti-HBs. All the ethnic groups in the study sample exhibited >90% of protective immune response. ConclusionThe more cost effective recombinant HBsAg vaccine taken by the healthy adults in the present study was effective in inducing the protective immunity.

Interaction of S17 Antibody with the Functional Binding Region of the Hepatitis B Virus Pre-S2 Epitope.

To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability. Flow cytometry analysis and immunocytochemical staining revealed that one scFv, S17, could recognize endogenous HBsAg expressed on the HepG2215 cell membrane. Moreover, the binding affinity of scFv S17 to the pre-S2 epitope was determined to be 4.2 × 10-8 M. Two ion interactions were observed as the major driving forces for scFv S17 interacting with pre-S2 by performing a rational molecular docking analysis. This study provides insights into the structural basis to understand the interactions between an antibody and the pre-S2 epitope. The functional scFv format can potentially be used in future immunotherapeutic applications.

Cellular uptake of hepatitis B virus envelope L particles is independent of sodium taurocholate cotransporting polypeptide, but dependent on heparan sulfate proteoglycan

Sodium taurocholate cotransporting polypeptide (NTCP) was recently discovered as a hepatitis B virus (HBV) receptor, however, the detailed mechanism of HBV entry is not yet fully understood. We investigated the cellular entry pathway of HBV using recombinant HBV surface antigen L protein particles (bio-nanocapsules, BNCs). After the modification of L protein in BNCs with myristoyl group, myristoylated BNCs (Myr-BNCs) were found to bind to NTCP in vitro, and inhibit in vitro HBV infection competitively, suggesting that Myr-BNCs share NTCP-dependent infection machinery with HBV. Nevertheless, the cellular entry rates of Myr-BNCs and plasma-derived HBV surface antigen (HBsAg) particles were the same as those of BNCs in NTCP-overexpressing HepG2 cells. Moreover, the cellular entry of these particles was mainly driven by heparan sulfate proteoglycan-mediated endocytosis regardless of NTCP expression. Taken together, cell-surface NTCP may not be involved in the cellular uptake of HBV, while presumably intracellular NTCP plays a critical role.

Hepatitis B surface antibodies in medical students from a public university in Puebla, Mexico

ABSTRACTAlthough preventable with vaccination, Hepatitis B virus (HBV) infection is a major health concern, with ∼400 million people at risk of developing the chronic form of the disease worldwide. The anti-HBV vaccine consists of a recombinant HBV surface antigen (HBsAg), which induces specific anti-HBs antibodies and confers 95% protection for >20 y. The aim of the present study was to analyze the response to HBV vaccination by measuring anti-HBs antibodies in serum samples from medical students of a public university in Puebla, Mexico. HBV infection markers HBsAg and anti-HBs, were also determined. A total of 201 students were included and vaccination coverage was found at 54%. Overall seropositivity for HBsAg, anti-HBc and anti-HBs determined by ELISA was 0.5%, 1.0% and 47%, respectively. Protective levels of anti-HBs >10 mIU/mL were found in 93.2% of subjects vaccinated with 2 or 3 doses and in 40% of those vaccinated with a single dose; while only 4.8% of unvaccinated subjects were anti-HBs positive. The response to the HBV vaccine was different in each participant, despite similar vaccination scheme. A history of blood transfusion/organ transplant or more than 2 sexual partners was significantly associated with anti-HBc positivity, OR = 399 (p = 0.010) and OR = 19.9 (p = 0.044), respectively. HBV immunization coverage was low in our sample compared with reports from countries with similar HBV prevalence, but anti-HBs in vaccinated individuals were in the expected range. It is important to promote HBV vaccination and awareness among medical students, due to their exposure risk.

Toward the development of monoclonal antibody-based assays to probe virion-like epitopes in hepatitis B vaccine antigen

Prophylactic vaccines against hepatitis B Virus (HBV) infection were produced in different expression systems under different processing conditions. Since the recombinant HBV surface antigen (HBsAg) in these vaccines is a cysteine-rich protein with 14 cysteines among a total of 226 amino acids, the epitopes are dependent on the formation of intra- and intermolecular disulfide bonds. A panel of 22 monoclonal antibodies (mAbs) were developed and evaluated with respect to their sensitivity to disulfide reduction treatment of recombinant HBsAg. Not surprisingly, different mAbs showed different degree of sensitivity to controlled HBsAg disulfide reduction. With a view to exploring the functionality of anti-HBsAg mAbs to be used in HBsAg quality analysis, in vitro neutralization activity for the mAbs was assessed. One of the mAbs tested, 5F11, which showed high sensitivity to the disulfide integrity in HBsAg, was shown also to be highly effective in neutralizing HBV in vitro. Conversely, 42B6, while exhibiting similar neutralization activity, showed comparable binding HBsAg with or without reduction treatment. Based on these mAb characteristics, a sandwich ELISA with 42B6 being the capture Ab and detection Ab was developed to quantify HBsAg (like a “mass” assay) during antigen bioprocessing or in vaccine products. In parallel, when 5F11 was used as the detection Ab (with the same capture Ab), the assay can be used to probe disulfide-dependent and virion-like epitopes in intermediates or final products of hepatitis B vaccine, serving as a surrogate marker for vaccine efficacy to elicit neutralizing antibodies. This approach enables the comparative epitope specific antigenicity analysis of HBsAg antigen preparations from different sources.

Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals.

Recombinant Hepatitis B Virus Surface Antigen Formulated with B-Type CpG Oligodeoxynucleotide Induces Therapeutic Immunity Against Hepatitis B Virus Surface Antigen-Expressing Liver Cancer Cells in Mice

To develop a therapeutic vaccine against hepatitis B virus surface antigen (HBsAg)-expressing liver cancer, we tried to prepare a vaccine by formulating recombinant HBsAg with BW006, a B type CpG oligodeoxynucleotide (ODN) with Th1-biasing activity, and examined its potency of inducing therapeutic immunity against HBsAg-expressing liver cancer cells in mice. When applied therapeutically, BW006 could assist HBsAg to induce vigorous immune responses capable of inhibiting the growth of HBsAg-expressing liver cancer cells and prolonging the survival of mice bearing HBsAg-expressing liver cancer cells. In vivo and in vitro experiments showed that the BW006-adjuvanted HBsAg enhanced the production of IgG2a antibodies, interferon-γ, and interleukin-12 and facilitated the generation of specific cytotoxic T lymphocyte that killed the HBsAg-expressing liver cancer cells. These results suggest that the BW006-adjuvanted HBsAg might be developed into a candidate tumor vaccine for the treatment of HBsAg-expressing liver cancer.

Development of multiplex serological assays to detect oncoviral infections

Serological markers of infection (antibodies or antigens) of viruses that cause cancer are most often detected using ELISA-based methodologies. In many cases, multiple markers of infection must be assessed to determine a final sero-status. Volume requirements and costs of reagents for single analyte ELISAs are high and studies which include multiple viruses can require milliliters of plasma, often not available from archived cohorts. Thus, we sought to develop a Luminex® bead-based customizable panel initially including Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Epstein-Barr Virus (EBV) and often the concomitant infection, Human Immunodeficiency Virus (HIV) to reduce sample volume requirements, overall cost and increase flexibility. Peptides, antigens and antibodies were sourced from multiple manufacturers and tested for suitability in this platform. Where necessary, suitable reagents were designed and produced in-house. The HCV assay multiplexes four HCV peptides designed to detect antibodies raised to HCV Core (2), HCV NS4, HCV NS5 gene regions. The HBV assay multiplexes a HBV early antigen peptide, a recombinant HBV core protein and either a recombinant HBV surface antigen or an antibody specific for HBV surface antigen to assess HBV infection. The EBV assay multiplexes peptides specific to viral capsid antigen, EBNA-1 and early antigen (Cyto-Barr, Zuidhorn, The Netherlands). HIV-1 assay development is ongoing and the list of antigens to be included in the assay has not been finalized. These assays can be run singly or with any combination (multi-plex) of the above listed targets. Each target has been independently validated using samples of known molecular and serological status to determine specificity (false positive versus false negative) and re-evaluated under multiplex conditions to confirm assay performance. In addition, where possible, samples were assayed on commercial testing platforms as well as our multiplex assay to assess concordance (94-99%). Dependent on the panel selected and the expected antibody titers in a particular population, plasma or serum volumes in the range of 10 µL to 125 µL per subject would be required to determine the HBV, HCV, EBV and/or HIV serostatus of a subject. This assay platform is inherently flexible and the benefits include amenability to expansion to include other oncogenic viruses as well as screening large epidemiological cohorts or smaller subsets of samples in an economical and high throughput manner.

European collaborative evaluation of the enzygnost HBsAg 6.0 assay: Performance on hepatitis B virus surface antigen variants

Amino acid changes within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may modify eventually the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its ability to detect HBV mutants. One hundred forty-seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant HBsAg mutants were tested by two generations of a commercial HBsAg test (Enzygnost® HBsAg 5.0 and 6.0, Siemens Healthcare Diagnostics Products, Marburg, Germany), and the results were compared. A significant improvement was demonstrated for the second test by comparing the mean and individual sample/cut-off values, as well as by the detection of several samples displaying amino acid changes in residues 120 and 145 of the HBsAg which were recorded as negative by the former test. The results showed that modifications in design of the assay improved considerably the ability of the test to detect HBsAg mutants, and that difficulties in detecting such HBV variants should not be expected with the routine use of the test in diagnostic laboratories and in blood transfusion centers.

Lasting immune memory against hepatitis B in children after primary immunization with 4 doses of DTPa-HBV-IPV/Hib in the first and 2nd year of life.

BackgroundFew studies have assessed long term persisting immunity against hepatitis B virus (HBV) in children vaccinated during infancy with combined vaccines containing recombinant HBV surface antigen (HBs). We assessed antibody persistence and immune memory in children 4-5 years of age, previously vaccinated with four doses of combined hexavalent DTPa-HBV-IPV/Hib vaccine (Infanrix hexa™).MethodsImmune memory was assessed in 301 children through administration of a challenge dose of monovalent HBV vaccine.ResultsAt 4-5 years of age, 85.3% of subjects had persisting anti-HBs antibody concentrations ≥ 10 mIU/mL, rising to 98.6% after the HBV challenge dose. All but 12 subjects (95.8%) achieved post-challenge anti-HBs concentrations ≥ 100 mIU/mL. The post-challenge anti-HBs GMC rose by 100-fold compared to pre-challenge concentrations. An anamnestic response to the HBV vaccine challenge was observed in 96.8% of subjects, including 17/21 (81.0%) of children with initially undetectable antibodies (<3.3 mIU/mL). All but 4 of 42 subjects (90.5%) with anti-HBs antibodies <10 mIU/mL prior to the challenge dose, achieved seroprotective levels afterwards. A 4-fold rise in antibody concentration after the challenge dose was observed in 259/264 (98.1%) of initially seropositive subjects. The magnitude of the post-challenge responses was proportional to pre-challenge anti-HBs levels. No serious adverse events were reported during the study.ConclusionThe combined DTPa-HBV-IPV/Hib vaccine induced lasting immune memory against hepatitis B. Long term protection afforded by DTPa-HBV-IPV/Hib is likely to be similar to that observed following priming with monovalent HBV vaccines.Trial registrationhttp://www.clinicaltrials.gov 106789 NCT00411697

Evaluation of 10 commercial diagnostic kits for in vitro expressed hepatitis B virus (HBV) surface antigens encoded by HBV of genotypes A to H

Genetic variability of the hepatitis B virus (HBV) constitutes one of the major challenges for diagnosis of HBV infection. It is plausible that amino acid substitutions in the “a” determinant of the HBV surface antigen (HBsAg) that affect antigenic sites, whether originating from genetic diversity or from mutations in the HBV strain itself, will affect the sensitivity of some diagnostic kits. In fact, recent studies have indicated that some diagnostic kits had false negative results with particular HBsAg mutants. There have been, however, few substantial studies evaluating sensitivities of diagnostic kits to the HBsAg encoded by different HBV genotypes. Our recent study found that 10 diagnostic kits available in Japan were able to detect HBsAg irrespective of whether it originated from HBV genotypes A, B or C, with the latter two genotypes being the dominant species in East Asia. In this study, we extended our previous efforts by assessing the ability of diagnostic kits to detect recombinant HBsAg derived from HBV genotypes A to H. Our results demonstrated that 9 out of 10 diagnostic kits evaluated were able to detect as low as 0.2 International Units (IU)/ml HBsAg, irrespective of HBV genotype. The genotypic differences in the HBV family thus appear to have little impact on the sensitivity of currently available HBsAg diagnostic kits.

The influence of glycosylation on secretion, stability, and immunogenicity of recombinant HBV pre-S antigen synthesized in Saccharomyces cerevisiae

Three types of recombinant pre-S antigens (i.e., pre-S1S2) of hepatitis B virus (HBV) were synthesized in Saccharomyces cerevisiae and secreted into extracellular medium: wild type (pre-S1S2) and two mutant antigens, pre-S1°S2 (Asn15Gln) and pre-S1°S2° (Asn15Gln and Asn123Gln). An N-terminus sequence (Ser5–Ala28) of human interleukin 1β (hIL-1β) was used as synthetic prosequence of recombinant HBV surface antigen (pre-S), secreted from S. cerevisiae. The expression cassette comprised the signal peptide of the killer toxin of Kluyveromyces lactis, the synthetic prosequence above, KEX2 dibasic endopeptidase cleavage site (–Lys–Arg–), and the surface antigen. The recombinant pre-S1S2 and pre-S1°S2 were secreted in the hyper-mannosylated form, while the recombinant pre-S1°S2° was produced without N-glycosylation. It has been demonstrated that the two particular N-linked glycans at Asn15 and Asn123 interfered with the B-cell response to the HBV-derived pre-S1S2, resulting in low titers of pre-S1S2-neutralizing antibodies. This problem was overcome by eliminating both of the N-glycosylation signals. Despite enhanced immunogenicity, the recombinant pre-S1°S2° showed two major problems: (1) inefficient Kex2 cleavage process in the secretory pathway and (2) the severe proteolytic degradation by yeast proteases. The efficiency of Kex2 cleavage increased dramatically by removing N-glycosylation signal in the synthetic prosequence, but the proteolysis of pre-S1°S2° was somewhat inevitable. Further systematic approaches including modulation of degree of N-glycosylation or relocation of N-glycosylation sites in the recombinant pre-S1S2 may make it possible to achieve both enhanced immunogenicity and resistance towards proteolytic degradation of the secreted pre-S antigen.

Hepatitis B virus surface antigen suppresses the activation of monocytes through interaction with a serum protein and a monocyte-specific receptor.

During hepatitis B virus (HBV) infection, high numbers of non-infectious HBV surface antigen (HBsAg) particles are present in circulation. It is shown here that recombinant HBsAg (rHBsAg) particles, which contain the S protein only, bind almost exclusively to monocytes. Attachment of rHBsAg to the THP-1 pre-monocytic cell line occurs upon 1,25-dihydroxyvitamin D3-induced differentiation. Binding to monocytes is enhanced by a heat-labile serum protein and is inhibited by Ca(2+)/Mg(2+), low pH and an HBsAg-specific monoclonal antibody. Furthermore, it is shown that rHBsAg suppresses lipopolysaccharide- and IL-2-induced production of cytokines. These results suggest the existence of a monocyte-specific receptor, the engagement of which by HBsAg suppresses the activity of these cells.

Efficient Haemodialysis Improves the Response to Hepatitis B Virus Vaccination

Background: As patients on chronic haemodialysis (PCHD) elicit a weaker response to vaccination with recombinant hepatitis B virus surface antigen (HBsAg), we conducted this study to see how dialysis efficacy affects response to hepatitis B virus (HBV) vaccination. Methods: Study subjects consisted of 30 PCHD. All subjects were vaccinated with 4 × 40 µg HBsAg i.m. at 0, 1, 2, and 6 months. If a subject had an HBsAg antibody (HBsAb) level <10 IU/l after vaccination, he or she received a booster dose. Subjects were divided into groups according to the level of HBsAb: non-responders (<10 IU/l), weak responders (10–100 IU/l), and good responders (>100 IU/l). Results: The group of responders had a significantly more efficient dialysis (Kt/V) than the group of non-responders (p = 0.027). This difference was not observed between groups of non-responders and weak responders. The group of good responders had a significantly better Kt/V than the group of non-responders (p = 0.012). Good responders had a significantly better Kt/V than weak responders (p = 0.019). Kt/V values showed a significantly positive correlation with the HBsAb level (r = 0.47; p = 0.006). Conclusions: The HBV vaccination reaction was weaker in PCHD with inefficient dialysis. Efficient haemodialysis significantly improves the response to vaccination with recombinant HBsAg.

Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepatitis B virus surface antigen.

In a search for monoclonal antibodies (MAbs) that can bind hepatitis B virus surface antigen (HBsAg) with amino acid substitutions in the immune dominant 'a' region (escape mutants) we investigated the epitope recognition site of the human MAb 4-7B. Pepscan analysis and experiments with alanine substitution as well as substitutions known from nature pointed to residues 178-186 in the small S protein with the amino acid sequence PFVQWFVGL (key amino acids in bold) as the minimal epitope. Single amino acid substitutions at positions 122(R/K)(d/y), 134(Y/F), 145(G/R), 148(T/A) and 160(K/R)(w/r), representing 'a' region variants in recombinant HBsAg COS-I cells, did not influence binding of MAb 4-7B. Synthetic peptides (residues 175-189) including the 4-7B epitope sequence were able to evoke an anti-HBs response in rabbits. According to established polypeptide models, the 4-7B epitope region is located in the lipid layer of 20 nm HBsAg particles. The present results, however, suggest that residues 178-186 are exposed on the surface of the 20 nm particle. This may change our view of the structure of HBsAg.

Epitope specificity of Th0/Th2 CD4+ T-lymphocyte clones induced by vaccination with rHBsAg vaccine

BACKGROUND & AIMS: Different amino acid sequences of hepatitis B virus surface antigen (HBsAg) are involved in the activation of CD4+ lymphocytes needed to induce an optimal antiviral function. The aim of this study was to characterize the CD4-mediated response to immunodominant HBsAg epitopes in hepatitis B virus (HBV) vaccine recipients by defining minimal sequences recognized by T cells, cytokine profiles, and HLA restriction of peptide recognition. METHODS: T-lymphocyte lines and clones specific for HBsAg were isolated from the peripheral blood of subjects immunized with recombinant HBsAg and stimulated in vitro with synthetic peptides spanning the whole HBsAg sequence. RESULTS: Four immunodominant epitopes (sequences 21-40, 136- 155, 156-175, and 211-226) were identified. Using panels of truncated peptides of different length, sequences 21-28, 165-172, and 215-223 were shown to correspond to the minimal epitopes recognized by T cells. The antigen-specific T-lymphocyte proliferation was HLA class II restricted, and each peptide could be presented in association with different HLA class II determinants. Th0/Th2 cytokine patterns were induced on peptide stimulation. CONCLUSIONS: These results indicate the presence of at least four immunodominant epitopes within HBsAg that represent potential candidates for the design of anti-HBV synthetic vaccines. (Gastroenterology 1997 Jun;112(6):2017-27)

Epitope specificity of Th 0/Th 2 CD4+ T lymphocyte clones induced by vaccination with rHBsAg vaccine

Abstract BACKGROUND & AIMS: Different amino acid sequences of hepatitis B virus surface antigen (HBsAg) are involved in the activation of CD4+ lymphocytes needed to induce an optimal antiviral function. The aim of this study was to characterize the CD4-mediated response to immunodominant HBsAg epitopes in hepatitis B virus (HBV) vaccine recipients by defining minimal sequences recognized by T cells, cytokine profiles, and HLA restriction of peptide recognition. METHODS: T-lymphocyte lines and clones specific for HBsAg were isolated from the peripheral blood of subjects immunized with recombinant HBsAg and stimulated in vitro with synthetic peptides spanning the whole HBsAg sequence. RESULTS: Four immunodominant epitopes (sequences 21-40, 136- 155, 156-175, and 211-226) were identified. Using panels of truncated peptides of different length, sequences 21-28, 165-172, and 215-223 were shown to correspond to the minimal epitopes recognized by T cells. The antigen-specific T-lymphocyte proliferation was HLA class II restricted, and each peptide could be presented in association with different HLA class II determinants. Th0/Th2 cytokine patterns were induced on peptide stimulation. CONCLUSIONS: These results indicate the presence of at least four immunodominant epitopes within HBsAg that represent potential candidates for the design of anti-HBV synthetic vaccines. (Gastroenterology 1997 Jun;112(6):2017-27)

DNA-mediated immunization to hepatitis B surface antigen: longevity of primary response and effect of boost

Intramuscular (i.m.) injection of mice with plasmid DNA expression vectors containing all or part of the hepatitis B virus (HBV) gene encoding the envelope proteins induces a strong humoral response to the HBV surface antigen (HBsAg) which is sustained for up to 74 weeks without boost. After a single i.m. injection of 100 μg DNA, antibodies to HBsAg (anti-HBs) reach ELISA titers of 4 × 10 4 in C57BL/6 mice and 10 4 in BALB/c mice, or somewhat less in older mice. Although antibody levels induced by a single injection of DNA do not diminish significantly over time, they can be further increased 10–200-fold by boosting with a second injection of DNA or an injection of recombinant HBsAg protein. Prior injection of DNA does not affect the strength or timing of the boosting effect, suggesting that there is no immune response against the vector itself. Boosting with a second injection of DNA is possible even in BALB/c mice, which are known to have a strong cytotoxic T-lymphocyte response against an epitope on the major HBV envelope protein, indicating that possible destruction of newly transfected muscle fibers is not so quick and efficient as to abort the boosting effect. A single injection of DNA results in a stronger and longer lasting humoral response than does a single injection of recombinant protein.

Investigation of korean plant extracts for potential phytotherapeutic agents against B‐virus hepatitis

AbstractThe present study was undertaken to identify potential phytotherapeutic agents among the aqueous extracts of 151 herbal medicinal plants of Korea. Extracts were assayed for (1) binding potency to hepatitis B‐virus surface antigen (HBsAg), and (2) inhibition of serum hepatitis B‐virus DNA polymerase activity. Of the 151 aqueous plant extracts tested, 33 demonstrated a positive precipitation reaction with HBsAg+ serum, and 15 of these specifically bound HBsAg in serum as well as recombinant HBsAg (Hepavax®). Nine extracts competitively inhibited &gt;80% of anti‐HBsAg antibody binding to Hepavax® HBsAg. Furthermore, nine of these 15 plant extracts also exhibited greater than 25%—50% inhibition of hepatitis B‐virus (HBV) DNA polymerase activity, and also significantly stimulated production of the potent cytokine, tumour necrosis factor (TNF). None of the extract concentrations used altered liver function parameters of normal rats.