Determinant Of Hepatitis B Surface Antigen Research Articles

Computational Design of a Novel VLP-Based Vaccine for Hepatitis B Virus.

Hepatitis B virus (HBV) is a global virus responsible for a universal disease burden for millions of people. Various vaccination strategies have been developed using viral vector, nucleic acid, protein, peptide, and virus-like particles (VLPs) to stimulate favorable immune responses against HBV. Given the pivotal role of specific immune responses of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in infection control, we designed a VLP-based vaccine by placing the antibody-binding fragments of HBsAg in the major immunodominant region (MIR) epitope of HBcAg to stimulate multilateral immunity. A computational approach was employed to predict and evaluate the conservation, antigenicity, allergenicity, and immunogenicity of the construct. Modeling and molecular dynamics (MD) demonstrated the folding stability of HBcAg as a carrier in inserting Myrcludex and “a” determinant of HBsAg. Regions 1–50 and 118–150 of HBsAg were considered to have the highest stability to be involved in the designed vaccine. Molecular docking revealed appropriate interactions between the B cell epitope of the designed vaccine and the antibodies. Totally, the final construct was promising for inducing humoral and cellular responses against HBV.

Occult Hepatitis B Virus Infection in Maintenance Hemodialysis Patients: Prevalence and Mutations in "a" Determinant.

Background: Occult hepatitis B virus infection (OBI) is defined as undetectable serum hepatitis B surface antigen (HBsAg) with detectable HBV-DNA in the serum or liver. Patients with maintenance hemodialysis (MHD) are at a high risk of OBI. The prevalence of OBI in MHD patients in China is not well evaluated. In this study, we aim to assess the prevalence of OBI in MHD patients in Sichuan Province, Southwest of China and investigate the mutations in the “a” determinant of HBsAg.Methods: A total of 330 patients undergoing MHD at Sichuan Provincial People's Hospital were enrolled. Serum samples were collected for ELISA assay to test the serological markers of HBV infection, real-time PCR assay to identify the presence of HBV-DNA, and nested PCR plus sequencing analysis to investigate the gene mutations.Results: In a total of 330 MHD patients, we found that the prevalence of OBI was 4.2% (7/165) in the test group, 2.1% (7/330) in the overall dialysis cohort. After a follow-up study of 7 MHD patients with OBI for 2 years, 2 (isolated HBcAb+) of them were still detectable for HBV-DNA. By sequencing analysis, we revealed mutations at the “a” determinant of HBsAg, including Q129R, T131N, M133S, F134L and D144E. The Q129R and M133S mutations were first reported.Conclusions: Our study clarifies the prevalence of OBI in MHD patients in Sichuan Province(4.2% in the test group, 2.1% in the overall dialysis cohort), and demonstrate the mutations of Q129R and M133S in the “a” determinant of HBsAg for the first time.

Antibody response after hepatitis B vaccine boost mapped with peptide-phage display

Recombinant hepatitis B virus vaccines confer protection by eliciting specific antibodies against the hepatitis B surface antigen (HBsAg), known as anti-HBs. However, the performance of rapid anti-HBs diagnostic tests generates concerns regarding consistency. Novel indicators of protection might be developed by monitoring changes in targeted HBsAg-epitope profile after vaccination. In this work, we test the feasibility of our peptide-phage display platform in identifying B-cell epitopes targeted at different time-points after hepatitis B vaccination. We combined this platform with a unique approach for in silico analysis of enriched sequences. Serum samples collected from one single patient who had two boosting immunizations against hepatitis B virus were used in two-rounds of selection experiments. Five epitope candidates from HBsAg were identified in silico; most of them were previously reported in the literature. Our results suggest that the number of recognized HBsAg epitopes is related to the decrease of anti-HBs over time.

A novel method for sensitive, low-cost and portable detection of hepatitis B surface antigen using a personal glucose meter

Hepatitis B virus (HBV) infection is the major public health problem leading cause of death worldwide. The most important diagnostic marker for this infection is hepatitis B surface antigen (HBsAg). In this study, a novel, inexpensive, portable and sensitive ELISA method was designed and investigated for diagnosis of HBsAg based on the functionalized Fe3O4 and Al2O3 nanoparticles, with the strategy for detecting the concentration of glucose using a cheap and accessible personal glucose meter (PGM). The ELISA system was constructed using hepatitis B antibody against HBsAg immobilized on streptavidin coated magnetic iron oxide particles (S-Fe3O4) as the capture antibody (Ab1). In addition, another hepatitis B antibody against different epitope of HBsAg (Ab2) and glucoamylase both were immobilized on Al2O3 nanoparticles. After formation of the sandwich immune complex between Ab1 and Ab2 immobilized on S-Fe3O4 and Al2O3 NPs, respectively, through HBsAg, starch was converted into glucose using glucoamylase. Then, the glucose concentration was measured using PGM. The concentration of HBsAg was calculated based on the linear relation between the concentrations of HBsAg and glucose. Under optimal conditions, this assay showed detection limit values of 0.3 to 0.4 ng ml−1 for “ay” and “ad” subtypes of HBsAg, respectively. The results indicate that the designed assay is comparable to the commercial kits in terms of sensitivity, on-site, specificity, cost, simplicity, portability and reproducibility. The presented method can be used in disadvantaged areas of the world and blood transfusion centers. To the best of our knowledge, this is the first report of using PGMs for HBSAg detection.

Prevalence and natural course of occult hepatitis B virus infection in residents of 2 communities of Wuwei City, Gansu Province, China

Occult hepatitis B infection (OBI) is characterized by serum hepatitis B surface antigen (HBsAg) negative and hepatitis B virus (HBV) DNA positive (HBsAg-/HBV DNA+). Occult hepatitis B infection in community-based populations has been scarcely investigated, and OBI outcomes remain unclear, especially in Wuwei, a region located in Northwest China. This region is one of the areas in China that has the highest prevalence of chronic HBV infection. A prospective study was performed in the general population of 2 towns of Wuwei from June 2011 to May 2014. A questionnaire was used to collect demographic and medical data, and serum samples were collected from the participants and stored until analysis. DNA was detected using quantitative PCR (qPCR) or nested PCR, the HBV DNA from HBV DNA-positive or possible positive (below the detection limit) subjects was extracted and amplified by nested PCR, and the PCR products were sequenced. Sequence analysis was performed using the Mega 6.0 program and CLC sequence viewer software. Hepatitis B virus DNA was detected in 90 of 3,080 HBsAg-negative subjects, and the prevalence of OBI in the study population was 2.92% (90/3,080, 95% CI: 2.33%-3.51%). Hepatitis B virus genomes in 51 of 80 objects (63.75%) contained mutations in the "a" determinant of HBsAg. After 2years follow-up, 42 of 90 HBV DNA of OBI subjects remained positive, and the natural clearance rate of OBI subjects was 53.3%. Occult hepatitis B infection prevalence in this cohort was much lower than chronic HBV infection in the same region. HBV DNA was cleared in most OBI subjects during the 2year period. Our data suggest that some OBI may represent a late stage of resolving the HBV infection process.

Minimization of hepatitis B infection among children in Jiangsu, China, 12 years after integration of hepatitis B vaccine into the expanded program on immunization

BackgroundChina has integrated hepatitis B vaccine into the Expanded Program on Immunization since 2002. We aimed to survey the seroprevalence of and immunity to hepatitis B virus (HBV) in children born from 2002 to 2014 in Jiangsu, China. MethodsTotally 3442 children (M:F=2072:1370) at the age of 7months to 12years (5.5±3.6), from five cities and rural areas across Jiangsu province, were enrolled. Blood samples were measured for HBV markers by ELISA and quantitative microparticle enzyme immunoassay. HBV DNA was tested by real-time PCR and S region was amplified by nested PCR. ResultsTwelve (0.35%) children were positive for hepatitis B surface antigen (HBsAg) and 34 (0.99%) were HBsAg negative and positive for antibody against hepatitis B core antigen (anti-HBc). Totally 2542 (73.85%) children had anti-HBs levels ⩾10mIU/ml and 535 (15.54%) with 2–9.9mIU/ml. All 12 HBsAg-positive children had detectable HBV DNA with a mean level of 6.1±1.7logIU/ml (3.3–8.1logIU/ml); 8 were genotype C and 4 were genotype B. No mutation was detected in the a determinant of HBsAg. HBV DNA was not detected in all the 34 children with positive anti-HBc and negative HBsAg. ConclusionHBsAg prevalence among children in Jiangsu born after the introduction of universal vaccination against hepatitis B has significantly decreased. No mutation of S gene is associated with vaccine failure in the cohort of children.

Development of an Allergy Immunotherapy Leads to a New Type of Hepatitis B Vaccine

Hepatitis B is an old disease and the possibility to successfully vaccinate against infection by hepatitis B virus (HBV) was first shown 36 years ago in a convincing trial (Szmuness et al., 1980). Thus, it may appear unspectacular when in this issue of EBioMedicine, a small clinical trial with a new type of hepatitis B vaccine is described (Cornelius et al., 2016). Do we really need this? But the significance of the paper should not be underestimated. The classical hepatitis B vaccine is produced in genetically transformed yeast cells and consists of 20-nm-large particles formed by the small (S) protein of hepatitis B surface antigen (HBsAg). The antibodies against conformational epitopes of HBsAg (anti-HBs) neutralize the infectivity of hepatitis B virus (HBV) in vitro and indicate protection in vivo. However, the classical vaccine has some shortcomings. In spite of multiple injections some persons remain unprotected, particularly those with a weakened immune response. Furthermore, asymptomatic infections by heterologous HBV genotypes with transient viremia are frequent in vaccinated subjects with low or moderate anti-HBs titers (for review see Gerlich, 2015). While the WHO, public health authorities and the main producers of hepatitis B vaccines still consider these drawbacks as insignificant, an enhanced protective capacity against a wider HBV genotype spectrum would not hurt.

Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There isan increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG).

Functional analysis of 'a' determinant mutations associated with occult HBV in HIV-positive South Africans.

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the absence of hepatitis B surface antigen (HBsAg). Occult HBV is associated with the development of hepatocellular carcinoma, reactivation during immune suppression, and virus transmission. Viral mutations contribute significantly to the occult HBV phenotype. Mutations in the 'a' determinant of HBsAg are of particular interest, as these mutations are associated with immune escape, vaccine escape and diagnostic failure. We examined the effects of selected occult HBV-associated mutations identified in a population of HIV-positive South Africans on HBsAg production in vitro. Mutations were inserted into two different chronic HBV backbones and transfected into a hepatocyte-derived cell line. HBsAg levels were quantified by enzyme-linked immunosorbent assay (ELISA), while the detectability of mutant HBsAg was determined using an HA-tagged HBsAg expression system. Of the seven mutations analysed, four (S132P, C138Y, N146D and C147Y) resulted in decreased HBsAg expression in one viral background but not in the second viral background. One mutation (N146D) led to a decrease in HBsAg detected as compared to HA-tag, indicating that this mutation compromises the ability of the ELISA to detect HBsAg. The contribution of occult-associated mutations to the HBsAg-negative phenotype of occult HBV cannot be determined adequately by testing the effect of the mutation in a single viral background, and rigorous analysis of these mutations is required.

THU-104 - Comparative Performance of Three Rapid HBsAg Assays for Detection of HBS Diagnostic Escape Mutants in Clinical Samples

The development of rapid tests (RTs) for hepatitis B surface antigen (HBsAg) detection is expanding, especially for point-of-care (POC) testing. As mutations in the a determinant of HBsAg have been associated with failures to detect HBsAg by using some traditional HBsAg assays, this study aimed to

Universal infant immunization and occult hepatitis B virus infection in children and adolescents: a population-based study.

To determine whether universal infant immunization affects occult hepatitis B virus (HBV) infection (OBI), serum samples from hepatitis B surface antigen (HBsAg)-negative subjects <18 years enrolled during six sequential seroepidemiological surveys conducted between 1984 (just before universal infant immunization) and 2009 were analyzed. Study subjects were divided into unvaccinated cohorts (born before 1984) and vaccinated cohorts (born after 1984). HBV-DNA positivity was determined by positivity of nested polymerase chain reaction in at least two of three regions (pre-S, S, and pre-core/core genes). OBI frequency was lower in vaccinated than unvaccinated antibody to hepatitis B core antigen (anti-HBc)-negative subjects (0 of 392 [0%] vs. 4 of 218 [1.8%]; P = 0.007), tended to be higher in vaccinated than unvaccinated anti-HBc-positive subjects (16 of 334 [4.8%] vs. 3 of 181 [1.7%]; P = 0.072), and was higher in vaccinated than unvaccinated subjects seropositive for both antibody to hepatitis B surface antigen (anti-HBs) and anti-HBc (13 of 233 [5.6%] vs. 3 of 170 [1.8%]; P = 0.025). By using known anti-HBc seropositivity rate in children in our serosurveys, the estimated OBI frequency per 10(4) HBsAg-negative subjects declined from 160.7 in unvaccinated cohorts to 11.5 in vaccinated cohorts. In vaccinated cohorts, OBI frequency was higher in anti-HBc-positive subjects than in anti-HBc-negative subjects (16 of 334 [4.8%] vs. 0 of 392 [0%]; P < 0.001). Subjects with OBI had much lower viral load (P < 0.001) and a trend of higher mutation rates in "a" determinant of HBsAg than age-comparable, HBsAg-positive subjects. Reduction of OBI in immunized subjects complements the well-documented universal infant immunization-related benefit of markedly reduced overt HBV infection. Breakthrough infections in immunized subjects seem to associate with more occurrence of OBI than natural infections in unvaccinated subjects. In the postvaccination era, anti-HBc seropositivity is a useful marker for OBI screening in HBsAg-negative subjects, and a very-low-level viral replication and HBsAg expression is the major mechanism underlying OBI.

Monoclonal antibodies to various epitopes of hepatitis B surface antigen inhibit hepatitis B virus infection

Antibodies against the "a" determinant of hepatitis B surface antigen (HBsAg) are able to neutralize circulating hepatitis B virus (HBV) particles and prevent HBV infection. It has been proposed that a single amino acid exchange may allow the virus to escape the immune response. We used a set of monoclonal antibodies (MAbs) to investigate whether a single mutation may account for virus escape from humoral immunity. Nine murine HBsAg-specific MAbs were raised. Reactivity of all antibodies with 14 recombinant mutants of HBsAg was assessed by ELISA. HBV infection of HepaRG cells was used to evaluate viral neutralization capacity of MAbs in vitro. All MAbs were able to inhibit the establishment of HBV infection in a dose-dependent fashion, but recognition of HBsAg variants varied. The MAbs were classified into three subgroups based on their pattern of reactivity to the HBsAg variants. Accordingly, three MAbs showed weak reactivity (< 40%) to variants with mutations within the first loop of "a" determinant, five MAbs displayed negligible binding to variants with mutations within the second loop, and one MAb lost its binding to variants having mutations in both loops of the "a" determinant. Our results indicate that antibodies against different epitopes of the "a" determinant of HBsAg are able to neutralize HBV. It seems that mutations within a single or a limited number of amino acids within this determinant can hardly result in viral escape. These results have important implications for the development of antibody-based therapies against HBV.

Construction and expression of hepatitis B surface antigen escape variants within the "a" determinant by site directed mutagenesis.

The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ''a'' determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.

Hepatitis B virus surface protein mutations clustered mainly in CTL immune epitopes in chronic carriers: results of an Iranian nationwide study

Mutations within the coding region of hepatitis B surface antigen (HBsAg) have been found naturally in chronic carriers. To characterize the mutations of HBsAg from Iranian chronic carriers who were vaccine and/or medication naive. The surface genes from 360 patients were amplified and directly sequenced. The distribution of amino acid substitutions was classified according to different immune epitopes of the surface protein. All isolates belonged to genotype D. 222 (61.6%) of 360 patients contained at least one amino acid substitution. 404 (74.5%) of 542 amino acid changes occurred in different immune epitopes of HBsAg, of which 112 (27.7%) in 32 residues of B-cell epitopes (62 in the 'a' determinant); 111 (27.4%) in 32 residues of T helper; and 197 (48.7%) in 32 residues inside cytotoxic T lymphocyte (CTL) epitopes. One Th (186-197) and two CTL (28-51 and 206-215) epitopes were found to be hotspot motifs for the occurrence of 213 (52.7%) substitutions. 20 stop codons were identified in different epitopes. There was a significant association between amino acid substitutions and anti-HBe seropositivity; however, the correlation between such changes with viral load and ALT levels was not significant. In chronic hepatitis B virus(HBV) carriers, positive selection in particular outside the 'a' determinant appeared to exert influence on the surface proteins. These changes could be immune escape mutations naturally occurring due to the host immune surveillance especially at the T-cell level.

HBsAg Variants: Common Escape Issues

Mutations have been described in all of the four open reading frames of the hepatitis B virus (HBV), however, from a clinical perspective the surface escape mutant is the most troublesome. Hepatitis B surface antigen (HBsAg) variants may impair diagnosis, or allow the virus to escape vaccine-induced immunity or passive immunoglobulin therapy. HBV mutants with amino acid substitutions, within the common “a” determinant of HBsAg have been identified, and as a result, the HBsAg cannot be detected in some assays (diagnostic-escape). In these situations, the HBsAg mutants may arise in patients with a HBV infection, but they have been diagnosed as HBsAg negative. This review deals with the latest results on the performance of HBsAg assays, as well as the reactivity of native and or recombinant mutants of HBsAg.

Analysis of Mutations in the'a'Determinant of HBsAg in Chronic Hepatitis B Patients with Coexisting HBsAg and Anti-HBs Positivity

B형 간염바이러스 표면항원에 대한 항체가 형성되면 HBsAg이 사라지고 HBV 감염으로부터 회복되는 것이 보편적이지만, HBsAg이 양성인 만성 간염 환자들 중 지속적으로 anti-HBs가 양성인 경우를 관찰할 수 있다. 이에 저자는 HBsAg과 anti-HBs가 동시에 양성인 만성 B형 간염환자에서 HBsAg S gene의?"a"?결정기 변이 존재 및 임상적 의의를 조사하였다. 53명의 환자 중 47명은 표면항원과 항체가 동시에 양성이었으며, 6명은 표면항원만 양성인 환자였다. 53명의 환자 중 17명(32.1%)이?"a"?결정기 변이가 관찰되었다. 시험군의 4명과 대조군의 1명은?"a"?결정기에 최소한 2개 이상의 변이가 관찰되었다. 가장 빈번한 변이 부위는 aa126, aa131 그리고 aa133였다. 이상의 결과로써 HBsAg과 anti-HBs가 동시에 양성인 B형 간염 환자에서 숙주의 면역체계를 벗어날 수 있는 HBsAg?"a"?결정기 부위에 다수의 변이가 있음을 확인하였다. Serologic markers are used to screen and diagnosis hepatitis B virus(HBV) infection. After an infection with HBV, hepatitis B surface antigen(HBsAg) is the first virologic marker detectable in the serum. If anti-HBs against?"a"?determinant of HBsAg appears, HBsAg will be disappeared and the patients will recover from the HBV infection in most cases. However, the coexistence cases of HBsAg and anti-HBs were frequently observed. The author investigated the clinical significance and presence of mutations of "a"?determinant in the S gene of chronic hepatitis B patients with coexisting HBsAg and anti-HBs. 47 out of the 53 patients were both positive for HBsAg and anti-HBs, 6 were positive for HBsAg only. 17(32.1%) mutations were found within the?"a"?determinant of HBV in the 53 patients. 4 patients from the test group, but only 1 from the control group, presented at least two residue changes in the?"a"?determinant. The most frequent changes in the test group were located at positions aa126, aa131, and aa133. These results demonstrated the presence of mutations at HBsAg?"a"?determinant in chronic hepatitis B patients with concurrent HBsAg and anti-HBs

Detection of hepatitis B virus DNA among accepted blood donors in Nanjing, China

BackgroundPosttransfusion hepatitis B virus (HBV) infection still occurs although its incidence has been substantially reduced since the introduction of screening of hepatitis B surface antigen (HBsAg) in blood donors. This study aimed to investigate the occult HBV infection in accepted blood donors in Nanjing, China.ResultsThe lower detection limit of the nested PCR in this study was estimated to be 20 copies/ml HBV DNA. The positive rate of occult HBV infection was 0.13% (5 of 2972) in the accepted blood donors. Sequencing data showed that the amplified HBV sequences were not identical each other and to the known sequences cloned in our laboratory, excluding the false-positive caused by cross-contamination. Phylogenetic analysis showed that the HBV in all five donors was genotype B; a single base deletion was detected in the S region of HBV DNA from one donor, and no mutation was observed in the "a" determinant of HBsAg from four other donors. All five donors were negative for anti-HBs and one was positive for anti-HBc.ConclusionsThe prevalence of occult HBV infection in the accepted blood donors in Nanjing, China is relatively high. The data would be meaningful in adapting strategy to eliminate posttransfusion HBV infection in China.

Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen

5S is a mouse monoclonal IgG1 that binds to the 'a' epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K(D) = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.

Association between genomic heterogeneity of hepatitis B virus and intrauterine infection

Hepatitis B virus (HBV) intrauterine infection remains to be an important cause for a large number of persistent hepatitis B surface antigen (HBsAg) positive carriers in areas with a high HBV prevalence, particularly in China and Southeast Asia. In this study, the possible association between the HBV genomic heterogeneity and intrauterine infection was investigated by comparing the quasi species isolated from eight pairs of HBsAg-positive mothers and their neonates, who were infected intrauterinely with HBV, with clones from eight HBsAg-positive mothers whose neonates were not infected with HBV. The proportion of clones with specific mutations was compared among different subject groups, and phylogenetic analysis was performed to evaluate the significance of specific mutations. It was observed that the core promoter with conserved major functional regions and conserved hepatitis B e antigen (HBeAg) might be beneficial to HBV maternal–fetal transmission. Particularly, A1762T/G1764A mutations seemed to be disadvantageous for fetal infection. It was also shown that amino acid substitutions located in the immune epitopes of HBsAg were strongly associated with intrauterine HBV transmission. The clones with mutations such as amino acid P110S in preS1 region, P36L in preS2 region and C107R in S region might infect fetuses more readily. In addition, positively selected site analysis confirmed the above results.

Multiple copies of a tumor epitope in a recombinant hepatitis B surface antigen (HBsAg) vaccine enhance CTL responses, but not tumor protection

We propose the replacement of endogenous epitopes with foreign epitopes to exploit the highly immunogenic hepatitis B surface antigen (HBsAg) as a vaccine vector to elicit disease-protective cytotoxic T-lymphocyte (CTL) responses. Locations were defined within the HBsAg gene where replacements of DNA encoding HBsAg epitopes may be made to generate functional recombinant (r) HBsAg DNA vaccines. We demonstrate that rHBsAg DNA vaccines encoding multiple copies of a model tumor epitope from human papillomavirus (HPV) elicit enhanced CTL responses compared to rHBsAg DNA vaccines encoding a single copy. We show that rHBsAg DNA vaccines elicit a marked prophylactic and long-lived therapeutic protection against epitope expressing tumor, although protective efficacy was not improved by increasing the number of copies of the tumor epitope DNA. These results demonstrate the efficacy of HBsAg as a vector for the delivery of foreign CTL epitopes using the epitope replacement strategy, and have implications for rHBsAg vaccine design. The results also have implications for the derivation of a therapeutic vaccine for HPV-associated squamous carcinoma.