pre-S Antigens Research Articles

Prophylactic vaccination against hepatitis B: achievements, challenges and perspectives.

Large-scale vaccination against hepatitis B virus (HBV) infection started in 1984 with first-generation vaccines made from plasma of chronic carriers containing HBV surface antigen (HBsAg). Thereafter, it was replaced in most countries by second-generation vaccines manufactured in yeast cells transformed with gene S encoding HBsAg. Both generations of vaccines have been applied for universal neonate and early childhood vaccination worldwide and have led to a 70-90 % decrease in chronic HBV carrier rates. However, 10-30% of newborns from HBsAg/HBeAg-positive mothers cannot be protected by passive/active vaccination alone and become chronic HBV carriers themselves. Asymptomatic occult HBV infections are frequent even in those who have protective levels of anti-HBs. Suboptimal protection may be due to heterologous HBsAg subtypes that are present in 99% of HBV carriers worldwide. Second-generation vaccines contain partially misfolded HBsAg and lack preS1 antigen that carries the major HBV attachment site and neutralizing epitopes. Third-generation vaccines produced in mammalian cells contain correctly folded HBsAg and neutralizing epitopes of the preS antigens, induce more rapid protection, overcome nonresponse to second-generation vaccines and, most importantly, may provide better protection for newborns of HBV-positive mothers. PreS/S vaccines expressed in mammalian cells are more expensive to manufacture, but introduction of more potent HBV vaccines should be considered in regions with a high rate of vertical transmission pending assessment of health economics and healthcare priorities. With optimal vaccines and vaccination coverage, eradication of HBV would be possible.

Induction of a robust T- and B-cell immune response in non- and low-responders to conventional vaccination against hepatitis B by using a third generation PreS/S vaccine

Non-responsiveness to conventional hepatitis B vaccines in individuals at high risk of exposure to hepatitis B virus (HBV) is an important public health problem and of particular relevance in health care providers. Yeast-derived conventional HBsAg vaccines fail to induce protective antibody titers in up to 10% of immune competent vaccinees. Therefore, a third generation HBV vaccine, Sci-B-Vac™, was developed which contains in addition to the small S antigen the PreS1 and PreS2 antigens. This vaccine proved to induce a highly potent cellular and humoral immune response in healthy individuals as well as protective antibody levels in non- and low-responders to conventional HBV vaccines. The aim of the study was to examine whether Sci-B-Vac™ triggers cellular and humoral immunity in individuals who failed immunization with conventional vaccines. We immunized 21 volunteers (15 non- and 6 low-responders) according to the standard vaccination schedule (0, 4 and 24 weeks), determined the cellular immunity by proliferation assay and interferon (IFN)-γ ELISpot and measured the anti-HBs antibody titers prior to each vaccination and four weeks after the third vaccine dose. Following three vaccinations, PreS/S-specific T-cell proliferation was detected in 8 out of 15 non-responders and 5 out of 6 low-responders. Specific IFN-γ responses were measured in 2 out of 15 non-responders and 4 out of 6 low-responders. All but one (20/21) study participants developed anti-HBs titers ≥10IU/l after three vaccinations. Anti-HBs ≥100IU/L were detected in 12 out of 15 non-responders and in 6 out of 6 low-responders. Anti-HBs ≥10IU/l and <100IU/l were found in 2 non-responders. These results indicate that Sci-B-Vac™ induces cellular immunity as well as protective anti-HBs antibody titers in non- and low-responders. In conclusion, these results confirm that Sci-B-Vac™ should be administered to non-responders to conventional HBV vaccines and patients with impaired immune function.

Atypical serological profiles in hepatitis B virus infection

During hepatitis B virus (HBV) infection, at least four antigen-antibody systems are observed: HBsAg and anti-HBs; preS antigen and anti-preS antibody; HBcAg and anti-HBc; and HBeAg and anti-HBe. Through the examination of these antigen-antibody systems, hepatitis B infection is diagnosed and the course of the disorder may be observed. Although the serologic findings that allow both the diagnosis of HBV infection as well as assessing of its clinical course are already well established, the dynamics of viral proteins expression and of the antibodies production may vary during the infection natural course. This causes the HBV infection to be occasionally associated with the presence of uncommon serological profiles, which could lead to doubts in the interpretation of results or suspicion of a serological result being incorrect. This paper is dedicated to the discussion of some of these profiles and their significance.

Analysis of relationship of HBV PreS1 antigen, anti-HBc IgM, DNA load and genotypes in hepatitis B patients

To explore the relationship of HBV PreS1 antigen, anti-HBc IgM, DNA load and genotypes, and the significance for clinical diagnosis and prognostic. Enzyme linked immune-sorbent assay was used to test the HBV serum markers of HB patients; HBV-DNA copies was detected by time fluorescence quantitative PCR; using nested PCR to amplify the S fragment of HBV genome, then sequence and make blast with HBV standard sequences to ascertain genotypes. Make comprehensive analysis of these indexes. 355 serum specimens of acute or chronic HB patients were collected. The positive rates of HBV PreS1-Ag and HBV-DNA in model I (positive for HBeAg) were 80.2% and 73.7% respectively, which both higher than other models. The abnormal rate of ALT and AST were higher in PreS1-Ag positive group than negative, as well as in anti-HBc IgM positive group. There are 4 samples is genotype B (2.9%), 76 genotype C (55.9%) and 56 genotype D (41.2%). Positive rate of HBeAg and HBV-DNA of genotype C samples were both higher than which of genotype B and D. PreS1-Ag and Anti-HBe-IgM indexes are of great value to viral hepatitis B early diagnosis, HBV replication surveillance and prognostic evaluation; the major HBV genotypes in Henan province are C and D, and the positive rate of HBeAg and HBV-DNA were both higher in genotype C HBV infection population than genotype B and D.

Association between polymorphisms of the E-selectin gene, hepatitis B virus DNA copies and preS1 antigen in patients with chronic hepatitis B infection

The aim of this study was to investigate the relationship between E-selectin +G98T, +A561C polymorphisms and the levels of hepatitis B virus (HBV) DNA and preS1 antigen (preS1Ag) in patients with chronic hepatitisB (CHB) infection. Polymorphisms of the E-selectin gene in 150CHB patients and 150healthy controls of two different nationalities were detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time quantitative PCR was used to detect the levels of HBV DNA. preS1Ag and five items of hepatitisB were detected by enzyme-linked immunosorbent assay. Two genotypes, GG (94%, 96%) and GT (6%, 4%) of the E-selectin +G98T polymorphism, and AA (78.67%, 80.67%) and AC (21.33%, 19.33%) of the +A561C polymorphism, were found in these patients. There were also significant differences in the two nationalities in the genotypic frequencies in +A561C polymorphisms between patients and healthy subjects (χ2=5.489, χ2=5.653; P<0.05). In the patients studied, the relative risk of suffering from CHB in genotype AC was 2.122 and 2.313-fold higher for the two nationalities, respectively, than that of the AA genotype (OR=2.122, 95%CI 1.121-4.019; OR=2.313, 95%CI 1.002-5.360). There was also significant over-representation in the Callele frequency between the two groups (χ2=5.000, χ2=5.30; P<0.05), and the levels of HBV DNA and preS1Ag in the AC genotype patients were higher than those in the AA genotype (P<0.01 and P<0.05). E-selectin +A561C and +G98T polymorphisms were present in the populations studied. Therefore, there is a correlation between E-selectin +A561C polymorphisms and CHB. Allele C may be one of the predisposing factors, and mutation of this locus may impact the virus copy number.

DETECTION OF HEPATITIS B VIRUS PRES1 ANTIGEN USING A TIME-RESOLVED FLUOROIMMUNOASSAY

The hepatitis B virus (HBV) PreS1 antigen is expressed at the distal most region of the envelope protein and contains the hepatocyte receptor-binding site. The presence of the HBV PreS1 antigen in serum and liver of HBsAg-positive patients is a new marker used for diagnosing HBV infection, and is indicative of viral replication. Our objective is to establish a method of time-resolved fluoroimmunoassay (TRFIA) with higher sensitivity and broader detection range for detecting serum HBV PreS1 antigen. Eu3+ labeling of antibodies was performed with respective labeling kits, and Eu3+ fluorescence intensity was measured with an auto DELFIA1235 TRFIA analyzer. The established method was evaluated for its performance. Serum specimens (574 in total) from Wuxi People's Hospital were analyzed for PreS1 antigen using the TRFIA and ELISA. The precision, specificity, and sensitivity of the TRFIA were clearly better than ELISA. The detection limit was 0.01 ng/mL. The average recovery rate for PreS1 antigens was 103.3%. There was significant correlation between the PreS1 antigen results obtained by TRFIA and ELISA in 374 serum samples with HBV >103 IU/mL (χ2 = 25.04, p < 0.01) and 183 HbeAg-positive serum samples (χ2 = 12.07, p < 0.01). Normal reference ranges were established at 0–0.32 ng/mL based on the values obtained from 100 healthy controls. TRFIA is a significantly effective method for clinical detection of serum HBV PreS1 antigens.

The clinical value of hepatitis B virus Pre-S1 antigen detection

Objectives To explore the clinical significance of detection of HBV Pre-S1 antigen in the diagnosis of Hepatitis B. Methods 254 blood samples with positive HBV markers detected by ELISA were divided into three groups based on different modes. Pre-S 1 antigen, HBV DNA, and ALT were detected in each group and then were comparatively analyzed. Results The detection rates for PreS1 antigen and HBV-DNA were 77.5％ and 93.1％ in the group with HbsAg ( + ), HBeAg ( + ), and HBcAb ( + ), 45.7％ and 60.1％ in the group with HBsAg ( + ), HBeAb ( + ), and HBcAb ( + ); and 0％ and 14.3％ in the group with HBsAg (-), HBeAb ( + ), and HBcAb ( + ). The positive rate of PreSl was 64.4％ ( 116/180 ) and A LT abnormality rate was 61.1％ ( 110/180 ) in 180 samples with a HBVDNA copy number of greater than 500IU/ml, and they were 35.1％ and 33.8％ in 74 samples with a copy number of less than 500IU/ml, respectively. The positive of Pre-S1 antigen was increased with an elevation in HBV DNA viral load, but the ALT abnormality rate was not linearly associated with the increase in HBV DNA viral load. Conclusions HBV Pre-Sl antigen has a higher consistency with HBV-DNA, which has an important clinical value in the diagnosis of type B hepatitis and can provide more reliable referrence for judging viral replication in vivo and efficacy of treatment. Key words: Hepatitis B virus; Pre-Sl antigen; HBV DNA viral load; ALT

The influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and antiviral proteins of IFN alpha

To investigate the possible influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and the antiviral proteins of IFN alpha. The HepG2 cells were transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids which express HBV whole particles or S-antigen, Pre-S antigen and core antigens. The infectious supernatant from HepG2.2.15 cells and the pured HBV proteins which contained the S, Pre-S antigens were used to treat the HepG2 cells. Northern blot and RT-PCR were applied to analyse the expressions of the antiviral proteins MxA, 2' -5' OAS, 9-27 and the JAK-STAT signal transduction pathway molecules STAT1 in HepG2 cells responded to the IFN alpha treatment. The HepG2 cells transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids could express whole HBV particles and HBsAg, Pre-S antigen and HBcAg. The quantitation of expressed HBV particles and antigens increased significantly during the course of transfection. Northern blot hybridization analysis indicated that the HepG2 cells expressed IFN alpha antiviral proteins MxA, 2' -5' OAS and 9-27. When transfected with pHBV-dimer, pHBS2-S, pHBc-EGFP plasmids, the IFN/A antiviral proteins MxA, 2' -5' OAS and 9-27 in transfected cells were reduced greatly as compared to the un-transfected HepG2 cells, and the expressed antiviral proteins decreased sharply with the development of transfection time. Furthermore, the expression of IFN alpha JAK-STAT signal transduction pathway molecule STAT1 was also inhibited with the expression of HBV particles and HBV antigens in transfected HepG2 cells. The HBV and its antigens influence the expressions of IFN alpha JAK-STAT signal transduction pathway molecules and antiviral proteins in the hepatocellular models in vitro. It is indicated that HBV might possess the activity to antagonise or counteract the IFN alpha antiviral action.

The correlation and clinical significance of HBV infection makers

Objective To explore the internal relations of HBV markers and its clinical significance.Methods HBV Pre S1-Ag、HBV-M(HBsAg、HBsAb、HBeAg、HBeAb、HBcAb)and HBV DNA of 106 patients were detected by ELISA and FQ-PCR respectively.Results The total positive rates of Pre-S1 antigen and HBV DNA in 106 cases were 81.1%and 84.9%respectively.In41 HBeAg-positive cases.Pre S1-Ag and HBV DNA detection rate was 95.1%.90.2%.And HBeAg-negative 2 groups Pre S1-Ag and HBV DNA detection rate was also higher.Conclusion HBV Per S1-Ag Was a very valuable serum marker in terms of direct HBV replication alone can not HBeAg-positive to determine whether the replication of the virus. Key words: Biological narkers; DNA virus Infections

Functional expression of anti-hepatitis B virus (HBV) preS2 antigen scFv by cspA promoter system in Escherichia coli and application as a recognition molecule for single-walled carbon nanotube (SWNT) field effect transistor (FET)

The preS2 antigens of hepatitis B virus (HBV), which causes a serious health problem in the world, have been implicated in hepatocyte cell binding and viral penetration. Therefore, the importance of antibody production against preS2 antigen for early diagnosis of HBV has been well established. In this study, the recombinant HBV preS2 single chain variable fragment (scFv) antibody was successfully expressed in E. coli with the novel cold shock vector (pCold) under the cspA promoter, and its expression level was compared with the pET vector under the T7 promoter. Additionally, a host with an oxidizing cytoplasm, E. coli trxB/gor double mutant, was used to improve the soluble expression. The anti-HBV preS2 scFv using pCold vector was successfully expressed in a soluble and functional form in both wild type and double mutant E. coli, while the scFv using the pET vector was expressed in an insoluble form in spite of using a double mutant providing an oxidizing environment. The induction with 0.05 mM IPTG showed a 2-fold higher functional expression compared to induction with 1 mM IPTG, and the functional expression at the induction temperature (15°C), which is optimal temperature for pCold vector, was improved 2-fold and 3- fold at 4 and 25°C, respectively. The efficacy of anti-HBV preS2 scFv for detecting HBV preS2 antigen was tested and verified by using Ni-decorated single-walled carbon nanotube (SWNT) field effect transistors.

The underlying mechanisms for the “isolated positivity for the hepatitis B surface antigen (HBsAg)” serological profile

During HBV infection, four structural antigen/antibody systems are observed: hepatitis B surface antigen (HBsAg) and its antibody (anti-HBs); the pre-S antigens associated with HBsAg particles and their antibodies; the particulate nucleocapsid antigen (HBcAg) and anti-HBc; and an antigen structurally related to HBcAg, namely HBeAg and its antibody (anti-HBe). Through the examination of this antigen-antibodies system, hepatitis B infection is diagnosed and the course of the disorder may be observed. Isolated HBsAg seropositivity is a peculiar serological pattern in HBV infection observed some times in routine laboratory. In most cases is not clear how this profile should be interpreted neither its significance. This pattern, however, may be associated with some clinical and laboratorial situations of great relevance, some of which will be addressed in this article.

A novel immunoassay for PreS1 and/or core-related antigens for detection of HBsAg variants

A novel immunoassay that detects simultaneously hepatitis B virus (HBV) PreS1 and/or core-related antigens was developed and evaluated for its potential for detecting hepatitis B surface antigen (HBsAg) variants. The detection limits of the assay was 10 2.9±0.5 copies/mL (mean ± SD) for HBsAg-positive sera with different genotypes, and 10 3.5±1.2 copies/mL for HBsAg variants sera. The specificity of the assay was 99.9% (95% CI: 99.7–99.9%, 4551 healthy individuals). The sensitivities were 93.9% (95% CI: 92.8–94.9%), 59.3% (95% CI: 38.7–77.6%) and 80% (95% CI: 44.4–97.5%) in three independent groups which include: 2065 hepatitis patients, 27 patients with occult hepatitis B and 10 HBsAg variants, respectively. In addition, a novel premature stop code mutation at position 112 of HBsAg was observed in two patients with chronic hepatitis B with different genotypes.

Review article: Hepatitis B and dialysis

The incidence of hepatitis B virus (HBV) infection in dialysis populations has declined over recent decades, largely because of improvements in infection control and widespread implementation of HBV vaccination. Regardless, outbreaks of infection continue to occur in dialysis units, and prevalence rates remain unacceptably high. For a variety of reasons, dialysis patients are at increased risk of acquiring HBV. They also demonstrate different disease manifestations compared with healthy individuals and are more likely to progress to chronic carriage. This paper will review the epidemiology, modes of transmission and diagnosis of HBV in this population. Prevention and treatment will be discussed, with a specific focus on strategies to improve vaccination response, new therapeutic options and selection of patients for therapy.

Selection of peptides bind to PreS1 antigen from a phage-displayed peptide library

Objective:To screen for peptides that specifically bind to PreS1 antigen from a phage-displayed peptide library.Methods: The PreS1 antigen was used as the target molecule to screen the binding peptide from the Ph.D.-7 peptide library with phage display technique,and the positive clones were identified by ELISA.Results: After three rounds of biopanning,the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA.Sequencing result showed that the binding peptides had high affinity and specificity.Conclusion: A peptide binding PreS1 antigen has been successfully obtained by screening the phage display library,which paves a way for the diagnosis and treatment of hepatitis B infection.

Prevalence of the precore G1896A mutation in Chinese patients with e antigen negative hepatitis B virus infection and its relationship to pre-S1 antigen

This study investigated the prevalence of the precore G1896A mutation in Chinese patients with hepatitis B e antigen (HBeAg) negative HBV infection and its relation to serum HBV pre-S1 antigen. The overall prevalence of the precore G1896A mutation was 72.6% in HBeAg-negative Chinese patients with detectable serum HBV DNA. The prevalence of the precore G1896A is significantly higher in Chinese HBeAg-negative patients with chronic hepatitis B than that in inactive HBV carriers with detectable serum HBV DNA. Serum pre-S1 and the precore G1896A mutation were simultaneously detected in most of Chinese HBeAg-negative patients.

Transient occult hepatitis B virus infection in a blood donor with high viremia

Screening of blood donors for viral nucleic acids has recently been introduced in several countries. With the use of transcription-mediated amplification, a blood donor was detected who had 90,000 copies of hepatitis B virus (HBV) DNA/mL but no hepatitis B surface antigen (HBsAg) or antibody to hepatitis B core antigen (anti-HBc). One month later, anti-HBc and hepatitis B surface antibody (anti-HBs) appeared; HBV DNA disappeared after 2 months. This study asked why HBsAg was undetectable in this rare case of transient occult HBV infection. The HBV DNA in the first sample was cloned and sequenced to identify mutations. The physical nature of the virus was examined by polyethylene glycol precipitation, DNase digestion, density gradient centrifugation, and immunoprecipitation. Several mutations were found all over the genome, but the HBs antigen loop was unchanged. A stop mutation in the precore region led to loss of hepatitis B e antigen (HBeAg) expression. No HBV DNA–containing immune complexes were present. The plasma did not contain nonencapsidated HBV DNA that could explain the absence of HBsAg. The virus was immune precipitated by antibodies against HBsAg or preS1 antigen. The ratio of HBV to HBsAg subviral particles was estimated to be 1 in less than 20 whereas in overt cases the ratio is 1 in more than 1000. The acute resolving occult HBV infection was caused by an HBeAg-negative variant, which otherwise was almost normal. The negative HBsAg result was probably due to an unusually low production of surplus HBsAg. The absence of the viral immunomodulator HBeAg and the early appearance of anti-HBs suggested a rapid noncytolytic HBsAg-specific T-cell response leading to low expression of HBsAg.

The Study on Relation of Hepatitis B virus Serum Markers and Pre-S1 Antigen

Objective To discuss relationship of hepatitis B virus gerum markers and Pre-S1 antigen.Methods Analyze the markers of HBV in blood serum and pre-S1 of HBV in 610 patients with ELISA.Results Pre-S1 antigen positive detection rate was 85.84% in 226 blood serum samples of HBsAg(+),HBeAg(+) and anti-HBcAg(+),Pre-S1 antigen positive detection rate was 93.33% in 45 blood serum samples of HBsAg(+) and anti-HBeAg(+).Pre-S1 antigen positive detection rate was 44.97% in 298 blood serum samples of HBsAg (+),anti-HBeAg(+) and anti-HBcAg(+).Pre-S1 antigen positive detection rate was 70.73% in 41 blood serum samples of HBsAg(+) and anti-HBcAg(+).The detection rate of Pre-S1 Ag was 87.08% in HBeAg(+) group and 35.80% in HBeAg(-) group,and there was significant difference between two groups(P＜0.05).The detection rate of Pre-S1Ag was 64.50% in anti-HBcAg(+) group and 82.55% in anti-HBcAg(-) group,and there was significant difference between two groups(P＜0.05).Conclusion Pre-S1Ag is a credible index of HBV replication,and may be helpful for HBV markers in detecting. Key words: Pre-S1; HBV marker; ELISA

Analysis of the correlation between the pre-S1 antigen, pre-S2 antigen and DNA of hepatitis B virus in the serum of chronic hepatitis B patients undergoing nucleoside analogue therapy.

To investigate the dynamic correlation between pre-S1 antigen, pre-S2 antigen and HBV DNA in the serum of chronic hepatitis B (CHB) patients undergoing nucleoside analogue therapy. 12 CHB patients with transient virological response after lamivudine treatment, and 20 patients treated with adefovir for 5 years were recruited in this study. Serum samples were collected at four time points when HBV DNA fluctuated sharply during lamivudine treatment, and at 0, 8, 12, 28, 52, 104, 156, 208, 260 weeks following adefovir treatment. HBV DNA was quantified by real-time PCR, pre-S1 and pre-S2 antigens were detected by ELISA. The titers of pre-S1 and pre-S2 antigens were not correlated with the HBV DNA level in the serum of lamivudine treated patients. Only in one case of the adfovir treated patients, the decrease of pre-S1 and pre-S2 antigens was in parallel with the decrease of HBV DNA. Linear regression analysis indicated that neither pre-S1 antigen nor pre-S2 antigen was correlated with HBV DNA in the serum of lamivudine or adfovir treated patients (P more than 0.05). Our results indicate that the titers of pre-S1 and pre-S2 antigens are not correlated with the serum HBV DNA in CHB patients undergoing nucleoside analogue therapy. Neither pre-S1 nor pre-S2 is a good predictor for the outcome of nucleoside analogue treatment.

The value of detection of Pre S1 antigen of hepatitis B virus

Objective To discuss the value of detection of Pre S1 antigen （Pre-S1Ag） of hepatitis B virus. Methods ELISA was used to detect Pre-S1Ag and HBeAg in serum from 854 HBsAg-positive persons in medical examination who have no symptom. In addition, Pre-S1Ag in serum from 300 random samples of HBsAg-negative persons were detected in medical examination. Results Pre-S1Ag positive rate was 41.1% （351/854） in 854 samples with HBsAg（ ＋ ） ,and HBeAg positive rate was 26. 6% （227/854） in them. Pre-S1Ag positive rate was 79. 7% （181/227） in HBeAg（ ＋ ） group but was 27.1% （170/627） in HBeAg（ - ） group. Pre-S1Ag wasn＇t detected in 300 samples with HBsAg（ - ）. Conclusions In HBsAg-positive samples who have no symptom,the detection rate of Pre-S1Ag is higher than that of HBeAg. Pre-S1Ag can reflect duplication and infectivity of HBV better than HBeAg and it has more value to epidemiology investigation and clinical diagnosis than the latter. Key words: Pre S1 antigen; Hepatitis B virus

PreS1 epitope recognition in newborns after vaccination with the third-generation Sci-B-Vac™ vaccine and their relation to the antibody response to hepatitis B surface antigen

BackgroundSci-B-Vac™ is a recombinant, hepatitis B vaccine derived from a mammalian cell line and containing hepatitis B surface antigen (HBsAg) as well as preS1 and preS2 antigens. Few studies have been performed on the antibody responses to preS1 in relation to the antibody to hepatitis B surface antigen (anti-HBs) response during immunisation of healthy children with preS-containing vaccines.ResultsIn this study 28 healthy newborns were randomly selected to receive either 2.5 ug or 5.0 ug of the Sci-B-Vac vaccine. Children received three doses of vaccine according to a 0-, 1-, 6-month scheme. Antibodies against the S-protein and three synthetic peptides mimicking three B-cell preS1 epitopes, (21–32 amino acid epitope), (32–47 amino acid epitope) and the C-terminal (amino acid epitope 94–117) were determined at 6 and 9 months. Fourteen (50%) of the 28 newborns had detectable levels of anti-preS1 (21–32) antibodies; 15 (54%) were anti-preS1 (32–47) reactive and 12 (43%) were anti-preS1 (94–117) reactive at 6 or 9 months after initiation of the vaccination. Significantly higher levels of anti-HBs were observed in the sera of patients with detectable anti-preS1 (32–47) reactivity (24 550 ± 7375 IU/L, mean ± SEM) as compared with the non-reactive sera (5991 ± 1530 IU/L, p < 0.05). The anti-HBs levels were significantly lower if none (p < 0.05) or one (p < 0.025) of the preS1 (21–32, 32–47, 94–117) peptides were recognised compared with the anti-HBs levels if two or three peptides were recognised.ConclusionRecognition of several preS1 epitopes, and in particular, the epitope contained within the second half of the hepatocyte binding site localised in the hepatitis B surface protein of the third-generation hepatitis B vaccine is accompanied by a more pronounced antibody response to the S-gene-derived protein in healthy newborns.