pre-S Antigens Research Articles

A retrospective study to determine the correlation among HBV PreS1 antigen, HBV e antigen, alanine aminotransferase, and HBV DNA

Background and aimHepatitis B virus (HBV) infection presents with indicators of varying clinical significance. We aimed to evaluate the correlation among HBV Pre-S1 antigen (HBV PreS1-Ag), HBV e antigen (HBeAg), HBV DNA, and alanine aminotransferase (ALT) levels. MethodsWe retrospectively analyzed 6180 serum samples collected between 2020 and 2022 at the Shanghai General Hospital, China. Data regarding PreS1-Ag, HBeAg, ALT, and HBV DNA were compiled. Correlation analyses and cross-tabulations were employed to explore the diagnostic indicators. ResultsThe detection rates of both antigen indicators showed a proportional increase with HBV DNA loads. The correlation between PreS1-Ag and HBV DNA (r = 0.616) was stronger than that between HBeAg and HBV DNA (r = 0.391). The specificity of PreS1-Ag (84.30 %) was lower than that of HBeAg (97.44 %), whereas the sensitivity of HBeAg (91.13 %) significantly surpassed that of PreS1-Ag (29.56 %). Among the HBV DNA positive patients, 92.04 % tested positive for at least one indicator, which exceeded the rate of PreS1+HBeAg- and PreS1-HBeAg+ (52. 28 % and 68. 56 %, respectively). Only 1.75 % of the patients exhibited double negativity, which was lower than the percentage of patients with single negativity (1.95 % and 12.00 % for PreS1-Ag and HBeAg, respectively). The PreS1 levels correlated with ALT levels (r = 0.317); patients with PreS1-positive status had higher ALT levels than patients with PreS1-negative status. ConclusionPreS1-Ag is a more robust HBV replication indicator than HBeAg. PreS1-Ag displayed high sensitivity, whereas HBeAg demonstrated high specificity. Moreover, PreS1-Ag levels correlated with ALT levels. A combination of these indicators demonstrated dependable clinical value for detecting HBV infection and evaluating liver function.

Synthesis and evaluation of pentacyclic triterpenoids conjugates as novel HBV entry inhibitors targeting NTCP receptor

Chronic liver diseases caused by hepatitis B virus (HBV) are the accepted main cause leading to liver cirrhosis, hepatic fibrosis, and hepatic carcinoma. Sodium taurocholate cotransporting polypeptide (NTCP), a specific membrane receptor of hepatocytes for triggering HBV infection, is a promising target against HBV entry. In this study, pentacyclic triterpenoids (PTs) including glycyrrhetinic acid (GA), oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA) were modified via molecular hybridization with podophyllotoxin respectively, and resulted in thirty-two novel conjugates. The anti-HBV activities of conjugates were evaluated in HepG2.2.15 cells. The results showed that 66% of the conjugates exhibited lower toxicity to the host cells and had significant inhibitory effects on the two HBV antigens, especially HBsAg. Notably, the compounds BA-PPT1, BA-PPT3, BA-PPT4, and UA-PPT3 not only inhibited the secretion of HBsAg but also suppressed HBV DNA replication. A significant difference in the binding of active conjugates to NTCP compared to the HBV PreS1 antigen was observed by SPR assays. The mechanism of action was found to be the competitive binding of these compounds to the NTCP 157–165 epitopes, blocking HBV entry into host cells. Molecular docking results indicated that BA-PPT3 interacted with the amino acid residues of the target protein mainly through π-cation, hydrogen bond and hydrophobic interaction, suggesting its potential as a promising HBV entry inhibitor targeting the NTCP receptor.

Pre-S1 Mutations as Indicated by Serum Pre-S1 Antigen Negative is Associated with an Increased Risk of Hepatocellular Carcinoma in Chronic Hepatitis B Patients.

Pre-S1 antigen (pre-S1) is a component of hepatitis B virus large surface antigen (L-HBsAg). This study aimed to investigate the association between clinical pre-S1 antigen (pre-S1) status and adverse prognostic events in chronic hepatitis B (CHB) patients. This study retrospectively enrolled 840 CHB patients with comprehensive clinical data, including 144 patients with multiple follow-up of pre-S1 status. All patients were tested for serum pre-S1 and divided into pre-S1 positive and negative groups. Single factor and logistic multiple regression analyses were performed to explore the association between pre-S1 and other HBV biomarkers with the risk of hepatocellular carcinoma (HCC) in CHB patients. The pre-S1 region sequences of HBV DNA were obtained from one pre-S1 positive and two pre-S1 negative treatment-naïve patients using polymerase chain reaction (PCR) amplification followed by Sanger sequencing. The quantitative HBsAg level was significantly higher in the pre-S1 positive group than that in the pre-S1 negative group (Z=-15.983, P<0.001). The positive rate of pre-S1 increased significantly with the increase in HBsAg level (χ 2=317.963, P<0.001) and HBV DNA load (χ 2=15.745, P<0.001). The pre-S1 negative group had a higher HCC risk than the pre-S1 positive group (Z=-2.00, P=0.045, OR=1.61). Moreover, patients in the sustained pre-S1 negative group had a higher HCC risk (Z=-2.56, P=0.011, OR=7.12) than those in the sustained pre-S1 positive group. The sequencing results revealed mutations in the pre-S1 region from samples of pre-S1 negative patients, including frameshift and deletion mutations. Pre-S1 is a biomarker that indicates the presence and replication of HBV. Pre-S1 sustained negativity attributed to pre-S1 mutations in CHB patients may be associated with a higher risk of HCC, which has clinical significance and warrant further investigations.

PreS1 Antigen of HBV Down-Regulates MHC-Ⅰ on the Surface of Hepatocytes and Promotes Hepatocarcinogenesis

To explore the internal mechanism of hepatocellular carcinoma (HCC) induced by chronic hepatitis B virus (HBV) infection. L02, HepG2 and Huh7 cells stably overexpressing HBV preS1 antigen were analyzed by flow cytometry, qRT-PCR and tumorigenesis in nude mice to evaluate the effect of preS1 antigen in HBV-related hepatocarcinogenesis. Our results showed that the expression of cancer stem cell (CSCs) related factors and cell surface markers in preS1 overexpressing cells were up-regulated, and the tumorigenicity of these cells was enhanced in nude mice. In addition, preS1 overexpression could down-regulate the expression of major histocompatibility complex Ⅰ (MHC-Ⅰ). The expression of MHC-Ⅰ on the cell surface could be restored by adding interferon gamma (IFN-γ) in the process of cell culturing and the tumorigenicity of cells in nude mice could thus be reduced. Based on the above results, we believe that preS1 is a carcinogen of HBV and that it promotes the formation of liver cancer through down regulating MHC-Ⅰ on the surface of hepatocytes.

A Rapid Immunochromatographic Method Based on a Secondary Antibody-Labelled Magnetic Nanoprobe for the Detection of Hepatitis B preS2 Surface Antigen.

Hepatitis B is a globally prevalent viral infectious disease caused by the hepatitis B virus (HBV). In this study, an immunochromatographic assay (ICA) for the rapid detection of hepatitis B preS2 antigen (preS2Ag) was established. The magnetic nanoparticles (MNPs) indirectly labelled with goat anti-mouse (GAM) secondary antibody were applied as a nanoprobe for free preS2 antibody (preS2Ab) capturing and signal amplification. By employing sample pre-incubation processing as well, preS2Ag-preS2Ab was sufficiently caught by the GAM-MNPs probe in 5 min. A qualitative sensitivity of 625 ng/mL was obtained by naked-eye observation within 15–20 min. A standard curve (0–5000 ng/mL) was established, with a quantitative limit of detection (LOD) of 3.6 ng/mL, based on the stability and penetrability of the magnetic signal characteristics. The proposed method for preS2Ag was rapid (~25 min, cf. ELISA ~4 h) and had a good accuracy, which was verified using an ELISA kit (relative error < 15%). Large equipment and skilled technicians were not required. The sensitivity and specificity of the developed GAM-MNPs-ICA method were 93.3% and 90% in clinical serum samples (n = 25), respectively. A good detection consistency (84%) was observed between the developed ICA method and 2 types of commercial ELISA kits, indicating that the GAM-MNPs-ICA has a potential application in large-scale screening for and point-of-care diagnosis of hepatitis B or other infectious diseases.

Identification of hepatitis B virus aetiologic antigens, HBx and Pre‐S2, in diffuse large B‐cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) has been reported to have a significant association with the hepatitis B virus (HBV) infection. However, there has been no experimental evidence to determine whether the components of the hepatitis B virus are expressed in lymphoid cells. In this study, we used immunohistochemical methods to explore whether the antigens of hepatitis B virus are expressed in DLBCL lymphoma cells in HBsAg-positive DLBCL patients (HBsAg+DLBCL). HBx antigen was detected in 48.9% of HBsAg+DLBCL patients, and the expression rate of the Pre-S2 antigen was 57.2%. HBx expression was significantly associated with high-level expression of c-Myc, while the Pre-S2 antigen was not. In this study, we demonstrated that HBx antigen and Pre-S2 antigen could be detected in lymphoma cells, and HBx expression was related to c-Myc expression. Our findings provide a strong basis for further study of the HBV-infected DLBCL and molecular mechanism underlying the lymphomagenesis.

Correlation between HBV large protein and HBV replication in patients with chronic hepatitis B

Objective To investigate the correlation of serum hepatitis B virus large protein (HBV-LP), HBV-DNA, and Pre S1 antigen (Pre S1-Ag) with HBV replication. Methods Serum samples were collected from 650 patients with chronic hepatitis B (CHB) who were treated in Beijing Friendship Hospital from March 2017 to March 2019. Serum HBV-LP and Pre S1-Ag were detected by enzyme-linked immunosorbent assay (ELISA). HBV markers (HBV-M) were measured using chemiluminescent microparticle immunoassay (CMIA). Quantitative real-time PCR (qRT-PCR) was used to detect HBV-DNA. The positive detection rates of HBV-DNA, HBV-LP and Pre S1-Ag were calculated and compared, and the correlation of HBV-LP (S/CO value) and hepatitis B surface antigen (HBsAg, log10 IU/ml) with HBV-DNA(log10 IU/ml)was analyzed. Results In the 650 CHB patients, the positive rates of HBV-DNA, HBV-LP and Pre S1-Ag were 65.4% (425/650), 79.2% (515/650) and 43.1% (280/650), respectively (P<0.01). The positive rates of HBV-DNA and HBV-LP in 243 HBeAg-positive patients were 93.0% (226/243) and 94.6% (230/243), and no significant difference was found between them (P=0.45). However, there was significant difference between the positive rates of HBV-DNA and HBV-LP in 407 patients negative for HBeAg [48.9% (199/407) vs 70.0% (285/407), P<0.01]. The positive rates of HBV-DNA and HBV-LP in HBsAg-, HBeAg- and HBcAb-positive groups were 92.8% (206/222) and 94.1% (209/222), which showed no significant difference (P=0.56). In HBsAg-, HBeAb- and HBcAb-positive groups, the positive rates of HBV-DNA and HBV-LP were 45.4% (124/273) and 69.9% (191/273) (P<0.01). The detection rate of HBeAg was lower than that of HBV-LP significantly in both HBV-DNA-positive and HBV-DNA-negative groups (P<0.01). With the increasing of HBV-DNA load, the S/CO value and the positive rate of HBV-LP increased significantly (P<0.05). Conclusions HBV-LP had a good correlation with HBV-DNA load as compared with Pre S1-Ag, HBeAg and HBsAg. HBV-LP in combination with HBV-M might be used as predictive markers that could efficiently reflect the status of HBV replication. Key words: Hepatitis B virus; DNA; Large protein; Pre S1 antigen; HBV markers

LB13. Trivalent Hepatitis B (HepB) Vaccine Yields Superior Seroprotection Rates in Adults: Results from the Phase 3 Double-Blind, Randomized Study Comparing Immunogenicity and Safety of a 3-Dose Regimen of Sci-B-Vac™ and Engerix B® (PROTECT)

BackgroundMany adults fail to achieve seroprotection after receiving 3 doses of monovalent HepB vaccines such as Engerix-B® and the response decreases with age and with common co-morbidities. Sci-B-Vac™ is a trivalent HepB vaccine produced in mammalian cells, adjuvanted with aluminum hydroxide, which in addition to small S antigen, contains preS1 and preS2 antigens expressing highly immunogenic T- and B-cell epitopes that may enhance seroprotection rates (SPR) in adults.MethodsIn a multicentre study, the immunogenicity of 10 µg dose of Sci-B-Vac™ was compared with a 20-µg dose of Engerix-B® given at days 0, 28, and 168 (NCT03393754). Randomization was stratified by study center and age (18–44, 45–64, ≥65 years). Immunogenicity, including SPR (% subjects with anti-HBs levels ≥10 mIU/mL), and safety outcomes were followed to Day 336. The co-primary objectives were (1) non-inferiority in adults ≥18 years and (2) superiority in adults ≥45 years of SPR, 4 weeks after the third dose.ResultsOf 1,607 randomized subjects, 42.3% were from United States, 41.6% EU, and 16.1% Canada. Males (38.5%) and females (61.5%) were enrolled to 18–44 (18.6%), 45–64 (44.6%), and ≥ 65 year (36.8%) age groups. Both co-primary endpoints were met. In the non-inferiority analysis, SPR in Sci-B-Vac™ recipients aged ≥18 years was 91.4% vs. 76.5% for Engerix-B®; SPR difference: 14.9%; 95% confidence interval (CI) [11.2%, 18.6%]. Superiority analysis showed that SPR in Sci-B-Vac™ recipients aged ≥45 years was 89.4% vs. 73.1% for Engerix-B®—SPR difference: 16.4%; 95% CI [12.2%, 20.7%] (figure). Significantly higher SPR for Sci-B-Vac™ vs. Engerix-B® was noted in subgroups (gender, BMI, diabetes, smoking and particularly age—SPR difference for 45–64 (14.7% [9.8–19.8%]) and ≥ 65 (18.9% [11.6–26.1%]) years. No major safety signals were observed; solicited and unsolicited adverse events were consistent with the known vaccine safety profiles.ConclusionSci-B-Vac™ met immunogenicity endpoints for non-inferiority in adults aged ≥ 8 years and was superior in adults aged ≥45 years, compared with the monovalent vaccine, Engerix-B®. Sci-B-Vac™ SPR was higher compared with Engerix-B® in key subgroups. No safety signals were observed and safety and tolerability were consistent with the known profile of Sci-B-Vac™. DisclosuresTimo Vesikari, MD, VBI (Grant/Research Support, I have received funding from VBI to carry out the reported clinical trial in Finland), Joanne M. Langley, MD, VBI Vaccines (Grant/Research Support), Johanna Spaans, BSc, MSc, VBI Vaccines (Employee), Nathalie Machluf, PhD, SciVac Ltd. (Employee), Dave Anderson, PhD, VBI Vaccines, Inc. (Employee, Shareholder), Vlad Popovic, MD, VBI Vaccines (Employee), Francisco Diaz-Mitoma, MD, VBI Vaccines, Inc. (Consultant, Shareholder, Independent Contractor). Other Authors: No reported disclosures.

A novel chimpanzee adenoviral vectored HBV vaccine, encoding multiple HBV antigens with a shark invariant chain adjuvant, for use in HBV immunotherapy

Background: Chronic hepatitis B virus (HBV) infects 257 million people globally. Current therapies suppress HBV but rebound occurs on cessation of therapy; novel therapeutic strategies are urgently required. Aim: To develop a potent therapeutic HBV vaccine that will induce T-cells to all major HBV antigens and generate anti-HBs antibodies, using Chimpanzee adenoviral vectors and shark class-II invariant (sIi) chain as a genetic adjuvant. Method: We designed two HBV immunogens SIi-HBV-CPmutS and HBV-CPmutS; encoding precore (PreC), core, non-functional polymerase (Pmut), PreS1, PreS2 and surface antigens (Figure 1a). The large envelope protein of HBV (composed of PreS1, PreS2 and surface proteins) was generated separately using furin 2A (F2A) peptide cleavage protein. A 26 amino-acid sequence, derived from shark invariant chain (sIi), was inserted at the 5’ end of the SIi-HBV-CPmutS immunogen. The immunogens were encoded in chimpanzee-adenoviral vector (ChAdOx2) and tested in naive mice given i.m. HBV-specific T-cell responses were assessed using IFN-ϒ ELISpot and intracellular cytokine (ICCS) assays. Results: Vaccination generated very high magnitude HBV specific T-cell responses to all HBV antigens (Figure 1b). The mean magnitude of total HBV-specific T-cell responses in inbred BALB/c and outbred CD1 mice were 3858 and 3821 spot forming units [SFU]/106 splenocytes (shown on the left side of figure 1b) and 4514 and 2979 SFU/106 intrahepatic lymphocytes (shown on the right side of figure 1b) respectively. ICCS showed that HBV specific CD8+ T cells were poly-functional producing both IFN-ϒ and TNF-α. The inclusion of shark invariant chain (data shown in blue coloured bars in figure 1b) significantly enhances the T-cell magnitude for both splenocytes (3821 mean total SFU/106 with sIi vs. 386 mean total SFU/106 without sIi, p<0.0001) and intrahepatic lymphocytes (2979 mean total SFU/106 with sIi vs. 461 mean total SFU/106 without SIi, p=0.0002). Importantly, T cells to the non-HBV SIi peptides were not generated. Conclusion: We have generated a highly potent HBV vaccine that induces T-cells against all major HBV proteins, using chimpanzee adenoviral vector and class-II invariant chain technologies. These pre-clinical studies pave the way for new studies of HBV immunotherapy in humans with chronic HBV infection.

Molecular and serological characterization of hepatitis B vaccine breakthrough infections in serial samples from two plasma donors

BackgroundAlthough vaccines for hepatitis B virus (HBV) are highly effective, HBV infections in vaccinees occur. Index samples of breakthrough infections are typically anti-HBc negative but HBV DNA positive with protective anti-HBs levels while HBsAg detection may be delayed or absent. HBsAg mutations have been associated with some vaccine breakthrough cases.MethodsThis research characterizes the serological and molecular profiles of vaccine breakthrough infections in serial samples from two commercially available plasma donor panels. Samples were tested with commercially available assays for HBV antigens and antibodies: HBsAg, HBeAg, anti-HBc, anti-HBc IgM, anti-HBe, and anti-HBs. Different immunoassay approaches for earlier detection of breakthrough infection were explored including hepatitis B core-related antigen (HBcrAg), a research assay for preS2 antigen, and a new prototype ARCHITECT HBsAg assay with improved sensitivity. The prototype HBsAg assay is fully automated and involves no sample pre-treatment. Molecular testing included HBV DNA quantitation and sequencing of preS1, preS2, surface, and basal core promoter/core promoter genes.ResultsAlthough the research preS2 antigen assay allowed earlier detection of the breakthrough infections than current HBsAg assays and HBcrAg, the new prototype ARCHITECT HBsAg assay provided the earliest serologic detection. The ability of the new prototype HBsAg assay to detect HBsAg in the presence of anti-HBs was investigated using known concentrations of native HBsAg mixed with anti-HBs from a vaccinee. The results demonstrated that the prototype ARCHITECT assay is more sensitive in detecting HBsAg in the presence of anti-HBs than current HBsAg assays. Sequencing revealed multiple substitutions in preS1, preS2, and S regions for one panel including a rare D144N substitution associated with vaccine breakthrough that emerged with increasing frequency as the breakthrough infection developed.ConclusionsWhen compared with other immunoassay approaches, the new prototype ARCHITECT HBsAg assay allows earlier detection of vaccine breakthrough infections and more sensitive detection of HBsAg in the presence of anti-HBs. Molecular characterization of longitudinal samples demonstrated the progressive appearance of a rare HBsAg mutation associated with vaccine breakthrough.

HBV antigen and DNA loss from mouse serum is associated with novel vaccine-induced HBV surface antigen-specific cell-mediated immunity and cytokine production

Therapeutic vaccination is a promising strategy for controlling chronic hepatitis B virus (HBV). Here, we tested whether several novel vaccination strategies could be used to induce HBV-specific adaptive immune responses and control/eradicate HBV in a mouse model. Robust HBV antigen-specific antibody responses were elicited by several vaccination strategies using a novel particle vaccine (HBSS1), which expresses a fusion of the S (amino acids [aa] 1–223) and preS1 (aa 21–47) antigens, and/or a recombinant adenovirus rAdSS1 vaccine. However, antigen-specific cell-mediated immunity and high levels of production of multiple cytokines were elicited only by heterologous prime-boost immunization; i.e., priming with the HBSS1 vaccine followed by a rAdSS1 boost. Furthermore, the most rapid loss of serum HBsAg, HBeAg and DNA was achieved by the novel vaccination regimen (priming with HBSS1 formulated with adjuvants [alum plus PolyI:C]), which was strongly associated with more potent and functional HBsAg-specific CD4+ and CD8+ T-cell responses and increased production of interleukin (IL)-2, interferon (IFN)-γ, tumor necrosis factor-α, IL-12, and IFN-γ-induced protein (IP)-10. Thus, our novel heterogeneous prime-boost vaccine regimen shows promise as a therapeutic strategy against HBV.

HBV genotyping based on key epitopes of PreS1 antigen and its correlation with genotyping by full-length PreS1 sequencing

Objective: The aim was to investigate the genotype distribution of two major epitopes of large surface protein (PreS1) of hepatitis B in Chinese patients and to explore the association between the genotypes of these two epitopes, and to determine whether PreS1 full-length genotype could be revealed according to the polypeptide sequence of key epitopes. Methods: HBV DNA was extracted from the serum of patients for PCR amplification. 278 samples amplified successfully were sequenced and compared with the known HBV sequences in Genbank to determine the two key epitopes of HBV PreS1 genotype (amino acid epitope 21-47 and 94-117, abbreviated as P21 and P94) and PreS1 full-length genotypes. The correlation among three genotyping approaches was analyzed by Cohen's kappa coefficient to verify the consistency between the key-epitope genotyping and the full-length preS1 genotyping. Results: 232 samples were successfully sequenced. The genotyping based on the kind of P21 epitope protein sequence, 201 cases for genotype C, 23 cases for genotype B and 8 cases for uncertain genotypes and genotyping based on the form of P94 epitope protein sequence, 199 cases for genotype C, 25 cases for genotype B and 8 cases for indeterminate genotypes. Lastly, the genotyping based on sequence of the full-length PreS1 sequence, 207 and 25 cases for genotype C and B. P21 or P94 epitope genotyping and PreS1 full length genotyping were highly consistent, respectively, 96.55% and 96.12%, and the two epitopes (P21and P94) genotyping have parallel consistency (93.10%). Conclusion: In this study, an innovatively genotyping method based on the amino acid sequence of key epitopes was proposed. The genotypes of HBV in china were mainly B and C genotypes, and the genotypes of key conserved epitopes of HBV PreS1 were highly consistent with the full-length genotyping ( > 96%). Moreover, genotyping with one or two key epitopes can be used in place of the full-length genotyping.

The immune response of rhesus macaques to novel vaccines comprising hepatitis B virus S, PreS1, and Core antigens

Therapeutic vaccines represent a unique approach to hepatitis B virus (HBV) treatment and have the potential to induce long-term control of infection. This study explored the immune responses of rhesus macaques to novel vaccines comprising the S, PreS1, and Core antigens of the HBV that showed promise as prophylactic and therapeutic approaches in a mouse model. The tested vaccines included two DNA vaccines (pVRC-SS1, pVRC-CS1), an HBV particle subunit (HBSS1) vaccine and the recombinant vaccinia virus- (RVJ-) based vaccines (RVJSS1 and RVJCS1) in which SS1 containing S (1–223 aa) and PreS1 (21–47 aa), CS1 containing Core (1–144 aa) and PreS1 (1–42 aa). The humoral immunity and cell-mediated immunity (CMI) induced by vaccines comprising the S, PreS1, and Core antigens of HBV were investigated in a longitudinal study that continued up to 98 weeks after the firstvaccination. In rhesus macaques, anti-PreS1 antibody was induced more rapidly than anti-S or anti-Core antibody after DNA vaccination. The antibody and cell-mediated immune responses against S, PreS1, and C were significantly enhanced in macaques boosted with RVJSS1 and RVJCS1, whereas the cell-mediated response to C was most robust and durable. The immune response to S, PreS1, and C was restored by HBSS1 boosting and detected in macaques until weeks 74 and 98 after the first vaccination. Additionally, robust neutralizing activity was detected at week 52. In conclusion, novel HBV vaccine candidates, especially those used for therapeutic applications should incorporate the PreS1 and Core antigens.

SAT-407 - A novel chimpanzee adenoviral vectored HBV vaccine, encoding multiple HBV antigens with a shark invariant chain adjuvant, for use in HBV immunotherapy

Background: Chronic hepatitis B virus (HBV) infects 257 million people globally. Current therapies suppress HBV but rebound occurs on cessation of therapy; novel therapeutic strategies are urgently required. Aim: To develop a potent therapeutic HBV vaccine that will induce T-cells to all major HBV antigens and generate anti-HBs antibodies, using Chimpanzee adenoviral vectors and shark class-II invariant (sIi) chain as a genetic adjuvant. Method: We designed two HBV immunogens SIi-HBV-CPmutS and HBV-CPmutS; encoding precore (PreC), core, non-functional polymerase (Pmut), PreS1, PreS2 and surface antigens (Figure 1a). The large envelope protein of HBV (composed of PreS1, PreS2 and surface proteins) was generated separately using furin 2A (F2A) peptide cleavage protein. A 26 amino-acid sequence, derived from shark invariant chain (sIi), was inserted at the 5’ end of the SIi-HBV-CPmutS immunogen. The immunogens were encoded in chimpanzee-adenoviral vector (ChAdOx2) and tested in naive mice given i.m. HBV-specific T-cell responses were assessed using IFN-ϒ ELISpot and intracellular cytokine (ICCS) assays. Results: Vaccination generated very high magnitude HBV specific T-cell responses to all HBV antigens (Figure 1b). The mean magnitude of total HBV-specific T-cell responses in inbred BALB/c and outbred CD1 mice were 3858 and 3821 spot forming units [SFU]/106 splenocytes (shown on the left side of figure 1b) and 4514 and 2979 SFU/106 intrahepatic lymphocytes (shown on the right side of figure 1b) respectively. ICCS showed that HBV specific CD8+ T cells were poly-functional producing both IFN-ϒ and TNF-α. The inclusion of shark invariant chain (data shown in blue coloured bars in figure 1b) significantly enhances the T-cell magnitude for both splenocytes (3821 mean total SFU/106 with sIi vs. 386 mean total SFU/106 without sIi, p<0.0001) and intrahepatic lymphocytes (2979 mean total SFU/106 with sIi vs. 461 mean total SFU/106 without SIi, p=0.0002). Importantly, T cells to the non-HBV SIi peptides were not generated. Conclusion: We have generated a highly potent HBV vaccine that induces T-cells against all major HBV proteins, using chimpanzee adenoviral vector and class-II invariant chain technologies. These pre-clinical studies pave the way for new studies of HBV immunotherapy in humans with chronic HBV infection.

A Highly Sensitive Detection System based on Proximity-dependent Hybridization with Computer-aided Affinity Maturation of a scFv Antibody

The hepatitis B virus (HBV) infection is a critical health problem worldwide, and HBV preS1 is an important biomarker for monitoring HBV infection. Previously, we found that a murine monoclonal antibody, mAb-D8, targets the preS1 (aa91-107) fragment of HBV. To improve its performance, we prepared the single-chain variable region of mAb-D8 (scFvD8) and constructed the three-dimensional structure of the scFvD8-preS1 (aa91-107) complex by computer modelling. The affinity of scFvD8 was markedly increased by the introduction of mutations L96Tyr to Ser and H98Asp to Ser. Furthermore, a highly sensitive immunosensor was designed based on a proximity-dependent hybridization strategy in which the preS1 antigen competitively reacts with an antibody labelled with DNA, resulting in decreased proximity-dependent hybridization and increased electrochemical signal from the Fc fragment, which can be used for the quantisation of preS1. The results showed a wide detection range from 1 pM to 50 pM with a detection limit of 0.1 pM. The sensitivity and specificity of this immunosensor in clinical serum samples were 100% and 96%, respectively. This study provides a novel system based on proximity-dependent hybridization and the scFv antibody fragment for the rapid quantisation of antigens of interest with a high sensitivity.

A virus-like particle of the hepatitis B virus preS antigen elicits robust neutralizing antibodies and T cell responses in mice

The preS antigen of hepatitis B virus (HBV) corresponds to the N-terminal polypeptide in the large (L) antigen in addition to the small (S) antigen. The virus-like particle (VLP) of the S antigen is widely used as a vaccine to protect the population from HBV infection. The presence of the S antigen and its antibodies in patient blood has been used as markers to monitor hepatitis B. However, there is very limited knowledge about the preS antigen. We generated a preS VLP that is formed by a chimeric protein between preS and hemagglutinin (HA), and the matrix protein M1 of influenza virus. The HBV preS antigen is displayed on the surface of preS VLP. Asn112 and Ser98 of preS in VLP were found to be glycosylated and O-glycosylation of Ser98 has not been reported previously. The preS VLP shows a significantly higher immunogenicity than recombinant preS, eliciting robust anti-preS neutralizing antibodies. In addition, preS VLP is also capable of stimulating preS-specific CD8+ and CD4+ T cell responses in Balb/c mice and HBV transgenic mice. Furthermore, preS VLP immunization provided protection against hydrodynamic transfection of HBV DNA in mice. The data clearly suggest that this novel preS VLP could elicit robust immune responses to the HBV antigen, and can be potentially developed into prophylactic and therapeutic vaccines.

Construction and Characterization of an Anti-Hepatitis B Virus preS1 Humanized Antibody that Binds to the Essential Receptor Binding Site.

Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma. With recent identification of HBV receptor, inhibition of virus entry has become a promising concept in the development of new antiviral drugs. To date, 10 HBV genotypes (A-J) have been defined. We previously generated two murine anti-preS1 monoclonal antibodies (mAbs), KR359 and KR127, that recognize amino acids (aa) 19-26 and 37-45, respectively, in the receptor binding site (aa 13-58, genotype C). Each mAb exhibited virus neutralizing activity in vitro, and a humanized version of KR127 effectively neutralized HBV infection in chimpanzees. In the present study, we constructed a humanized version (HzKR359-1) of KR359 whose antigen binding activity is 4.4-fold higher than that of KR359, as assessed by competitive ELISA, and produced recombinant preS1 antigens (aa 1-60) of different genotypes to investigate the binding capacities of HzKR359-1 and a humanized version (HzKR127-3.2) of KR127 to the 10 HBV genotypes. The results indicate that HzKR359-1 can bind to five genotypes (A, B, C, H, and J), and HzKR127-3.2 can also bind to five genotypes (A, C, D, G, and I). The combination of these two antibodies can bind to eight genotypes (A-D, G-J), and to genotype C additively. Considering that genotypes A-D are common, whereas genotypes E and F are occasionally represented in small patient population, the combination of these two antibodies might block the entry of most virus genotypes and thus broadly neutralize HBV infection.

Malaria sporozoite protein expression enhances baculovirus-mediated gene transfer to hepatocytes.

Baculovirus vector (BV) is able to transduce foreign genes into mammalian cells efficiently and safely by incorporating a mammalian promoter. In the present study, we tailored the surface proteins expressed by malaria sporozoites to enhance hepatocyte transduction. Sporozoites infect hepatocytes within minutes of initial entry into the blood circulation. Infectivity and hepatocyte-specific selectivity are mediated by the interplay between hepatocytes and sporozoite surface proteins. The circumsporozoite protein (CSP) and the thrombospondin-related anonymous protein (TRAP) bind to the heparan sulfate proteoglycan on the hepatocyte surface and contribute to sporozoite infection and hepatocyte selectivity. BVs displaying an ectodomain consisting of three different CSP variants (full-length, N-terminal and C-terminal) or TRAP on the virus envelope were constructed, and the resulting in vitro hepatocyte transduction efficiency was evaluated. We demonstrated improved hepatocyte transduction efficiency in BVs expressing CSP or TRAP ectodomains compared to BVs without malaria surface proteins. In addition, gene transduction efficiencies for BVs displaying CSP or TRAP are higher than those expressing the preS1 antigen of the hepatitis B virus. BVs expressing CSP or TRAP in the ectodomain could represent a promising hepatocyte-specific gene delivery methodology. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a more efficient hepatitis B virus vaccine by targeting hepatitis B virus preS to dendritic cells

BackgroundConventional hepatitis B virus (HBV) vaccines fail to induce protective antibody titers in 5–10% of immune-competent vaccinees. Therefore, there remains an urgent need to develop a safe and effective HBV vaccine. MethodsIn this study, we developed an effective and economical method for producing the HBV vaccine by using the high binding capacity of biotin–streptavidin. The preS antigen of HBV was fused with the core streptavidin (cSA) moiety (preS-cSA) and highly expressed in Escherichia coli. We investigated whether the preS-cSA protein could target dendritic cells (DCs) by binding a biotinylated antibody against the DC receptor CD205 (biotin-αCD205). Moreover, we evaluated the preS-cSA/biotin-αCD205 complex as a candidate vaccine by detecting the humoral and cellular immune responses elicited by this vaccine. ResultsOur data show that the preS-cSA/biotin-αCD205 complex targeted DCs and induced high anti-HBV antibody titers of IgG2a, IgG1, and IgG in vivo. Furthermore, vaccination with the preS-cSA/biotin-αCD205 complex prevented HBV infection in female mice. More interestingly, this novel vaccine exerted a therapeutic role in mice with HBV infection. ConclusionsTaken together, our results reveal that the preS-cSA/biotin-αCD205 vaccine induces effective immunological protection against HBV, and is a promising candidate for preventing HBV infections.

Mandibular metastasis as an initial manifestation of hepatocellular carcinoma: A report of two cases.

The present study reports two cases of mandibular metastasis from hepatocellular carcinoma (HCC), including the clinical presentation, and computed tomography (CT), histopathology and immunohistochemistry results. Space-occupying lesions occurred unilaterally as an initial mandibular manifestation. HCC metastasis was confirmed by post-surgical examination, and the primary tumor was found using CT scans. Hepatitis B virus infection history and positive results of hepatitis B surface antigen, hepatitis B e-antibody, hepatitis B core antibody and hepatitis B virus pre-S1 antigen further supported the pathogenesis of HCC. Based on the clinical findings, the characteristics of the CT scans and the histopathology and immunohistochemistry results, the mechanisms of HCC metastasis and its management are also discussed.