pre-S Protein Research Articles

Therapeutic vaccination with lentiviral vector in HBV-persistent mice and two inactive HBsAg carriers

Immunotherapy for chronic hepatitis B virus (HBV) infection has not yet demonstrated sufficient efficacy. We developed a non-integrative lentiviral-vectored therapeutic vaccine for chronic hepatitis B and tested its antiviral effects in HBV-persistent mice and two inactive HBsAg carriers. Lentiviral vectors (LVs) encoding the core, preS1, or large HBsAg (LHBs) proteins of HBV were evaluated for immunogenicity in HBV-naïve mice and therapeutic efficacy in a murine model of chronic HBV infection. In addition, two inactive HBsAg carriers each received two doses of 5×107 transduction units (TU) or 1×108 TU of lentiviral-vectored LHBs (LV-LHBs), respectively. The endpoints were safety, LHBs-specific T-cell responses, and serum HBsAg levels during a 24-week follow-up. In the mouse models, LV-LHBs was the most promising in eliciting robust antigen-specific T cells and in reducing the levels of serum HBsAg and viral load. By the end of the 34-week observation period, six out of ten (60%) HBV-persistent mice vaccinated with LV-LHBs achieved serum HBsAg loss and significant depletion of HBV-positive hepatocytes in the liver. In the two inactive HBsAg carriers, vaccination with LV-LHBs induced a considerable increase in the number of peripheral LHBs-specific T cells in one patient, and a weak but detectable response in the other, accompanied by a sustained reduction of HBsAg (-0.31 log10 IU/ml and -0.46 log10 IU/ml, respectively) from baseline to nadir. A lentiviral-vectored therapeutic vaccine for chronic HBV infection demonstrated the potential to improve HBV-specific T-cell responses and deplete HBV-positive hepatocytes, leading to a sustained loss or reduction of serum HBsAg. Chronic HBV infection is characterized by an extremely low number and profound hypo-responsiveness of HBV-specific T cells. Therapeutic vaccines are designed to improve HBV-specific T-cell responses. We show that immunization with a lentiviral-vectored therapeutic HBV vaccine was able to expand HBV-specific T cells invivo, leading to reductions of HBV-positive hepatocytes and serum HBsAg.

Genetic Heterogeneity and Mutated PreS Analysis of Duck Hepatitis B Virus Recently Isolated from Ducks and Geese in China.

In this study, we detected 12 duck and 11 goose flocks that were positive for duck hepatitis B virus (DHBV) using polymerase chain reaction and isolated 23 strains between 2020 and 2022 in China. The complete genomes of goose strains E200801 and E210501 shared the highest identity (99.9%), whereas those of strains Y220217 and E210526 shared the lowest identity (91.39%). The phylogenetic tree constructed based on the genome sequences of these strains and reference strains was classified into three major clusters: the Chinese branch DHBV-I, the Chinese branch DHBV-II, and the Western branch DHBV-III. Furthermore, the duck-origin strain Y200122 was clustered into a separate branch and was predicted to be a recombinant strain derived from DHBV-M32990 (belonging to the Chinese branch DHBV-I) and Y220201 (belonging to the Chinese branch DHBV-II). Additionally, preS protein analysis of the 23 DHBV strains revealed extensive mutation sites, almost half of which were of duck origin. All goose-origin DHBV contained the mutation site G133E, which is related to increased viral pathogenicity. These data are expected to promote further research on the epidemiology and evolution of DHBV. Continuing DHBV surveillance in poultry will enhance the understanding of the evolution of HBV.

Recombinant measles virus expressing prefusion spike protein stabilized by six rather than two prolines is more efficacious against SARS-CoV-2 infection.

Measles virus (MeV) has been an excellent vector platform for delivering vaccines against many pathogens because of its high safety and efficacy, and induction of long-lived immunity. Early in the COVID-19 pandemic, a recombinant MeV (rMeV) expressing the prefusion full-length spike protein stabilized by two prolines (TMV-083) was developed and tested in phase 1 and 1/2 clinical trials but was discontinued because of insufficient immunogenicity and a low seroconversion rate in adults. Here, we compared the immunogenicity of rMeV expressing a soluble prefusion spike (preS) protein stabilized by two prolines (rMeV-preS-2P) with a rMeV expressing a soluble preS protein stabilized by six prolines (rMeV-preS-6P). We found that rMeV-preS-6P expressed approximately five times more preS than rMeV-preS-2P in cell culture. Importantly, rMeV-preS-6P induced 30-60 and six times more serum immunoglobulin G and neutralizing antibody than rMeV-preS-2P, respectively, in IFNAR-/- mice. IFNAR-/- mice immunized with rMeV-preS-6P were completely protected from challenge with a mouse-adapted SARS-CoV-2, whereas those immunized with rMeV-preS-2P were partially protected. In addition, hamsters immunized with rMeV-preS-6P were completely protected from the challenge with a Delta variant of SARS-CoV-2. Our results demonstrate that rMeV-preS-6P is significantly more efficacious than rMeV-preS-2P, highlighting the value of using preS-6P as the antigen for developing vaccines against SARS-CoV-2.

Hepatitis B virus PreS2 mutants in liver cancer patients

Liver cancer, or Hepatocellular Carcinoma (HCC), is one of the most common cancers in the world. Chronic infection with the hepatitis B virus (HBV) is one of the most critical factors that cause it. During chronic infection with HBV, variants of the virus are created. They may have deletion mutations in the PreS2 region. These variants may play a role in the occurrence of HCC. This study aims to determine the presence of these mutants in patients with liver cancer in China. For this purpose, virus DNA was extracted from the serum of 10 patients with HCC. After amplifying the PreS region and determining the sequence of this region from the genome, the presence of PreS2 mutants in these patients was investigated compared to the database. The results showed that a point mutation was observed at the level of the start codon of PreS2 in two samples. In three of the isolates, several amino acids were deleted at the end of the PreS2 region. In PreS2 deletion mutants, the T-cell and B-cell epitopes on the PreS2 region product are generally deleted. As a result, conditions are created where the virus can escape from the immune system. These mutant PreS2 proteins accumulate in the endoplasmic reticulum (ER) network and cause ER stress. In this way, in addition to creating unstable conditions in the cell genome, the proliferation of hepatocytes is stimulated indirectly. As a result, there is a possibility that the cells will progress toward becoming cancerous.

SARS-CoV-2 prefusion spike protein stabilized by six rather than two prolines is more potent for inducing antibodies that neutralize viral variants of concern

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (104 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 103 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.

Designing Next-Generation Vaccines Against Common Pan-Allergens Using In Silico Approaches.

Next-generation allergy vaccines refer to allergen-derived attenuated molecules that can boost allergen-blocking IgG response. These IgG antibodies are specifically directed toward the IgE epitope of allergens and interfere in allergen-IgE interaction. Our study is a computational approach to design such vaccines against four widespread pan-allergens families. Pan-allergens display extensive immunological cross-reactivity due to the presence of conserved IgE epitope and T cell epitope. In this study, the vaccine design is based on hapten-carrier concept in which the carrier protein is an immunogenic component providing T cell help. Either PreS protein of hepatitis B or cholera enterotoxin B (CTB) fused with three tetanus toxoid fragments (TTFrC) was used here as the carrier. The hapten components are nonanaphylactic peptides (NAPs) derived from experimentally determined antigenic regions of the allergens. The charged residues of NAPs are selectively modified to obliterate IgE, as well as T cell reaction, and hence, are safe to apply in allergy patients. Various combinations of vaccine constructs (PreS/CTB+TTFrC and NAPs) were designed with intermediate linker motifs. Screening of constructs was performed through a three-step method such as physicochemical parameters, secondary structures, and tertiary structures using various bioinformatic tools. The final construct with best quality and stability was selected for each allergen family. Suitability of these constructs for being expressed in recombinant form was checked at DNA, RNA, and protein level. Presence of putative epitopes inducing tolerogenic interleukin-10 was also predicted for these constructs. The present work led to the design of putative vaccines with immunotherapeutic potential and broad applicability for allergic diseases caused by a wide array of cross-reactive allergens.

Elucidating Structural, Functional and Phylogenetic Relationship of Large Envelope Protein of Hepatitis B V irus

Objectives: To predict the structure of L-HBsAg envelope protein of Hepatitis B Virus and its evolutionary relationship. Methods: Bioinformatics Computational methods were used for the structure prediction and evolutionary conservation analysis of large envelope protein of Hepatitis B virus (L-HBsAg). Structure prediction L-HBsAg envelope protein was done using Multi-template homology modeling method using Schrodinger software. Phylogenetic tree was constructed by MEGA tool to identify protein similarity of L-HBsAg protein with other genotypes of HBV. Findings: Modelled structure of L-HBsAg protein shows that this protein has single alpha-helix and loop structure. Multiple sequence alignment result shows that L-HBsAg belongs to family of vMSA (pfam00695, major surface antigen from hepadnavirus) conserved domain. Phylogenetic tree reveals that L-HBsAg shows similarity with other proteins of HBV protein like preS protein and envelope protein. Novelty: Structural analysis identifies the binding properties of L-HBsAg protein. This study concluded that large envelope protein of HBV can be potent drug target for designing a novel drug. Keywords: LHBsAg; HBV; evolutionary analysis; structure prediction; dane particles

Serodiagnosis of Hepatitis B Virus Using PreS Antigen from Pakistani Isolate SBS001

Background: An estimated 325 million people worldwide live with hepatitis B and/or C and approximately, 5 million people are affected with hepatitis B. in Pakistan.This study aimed at developing PreS protein from Hepatitis B Virus Pakistani isolate (SBS001) with enhanced sensitivity to detect antibodies in serum as a diagnostic method.
 Methods: Gene encoding PreS region from hepatitis B Virus was cloned and expressed in Escherchia coli. The recombinant protein preS-His was purified by Ni-IDA affinity chromatography. Antibodies were raised in rabbit. This protein was screened for detection of antibodies in HBV patients’ sera through ELISA. This ELISA procedure was compared with commercially available Kit used for diagnosis of HBV infection.
 Results: Single band purified recombinant PreS protein was obtained with high titer of antibodies raised in rabbits. This recombinant protein was used in ELISA as antigen coated on the plate. That efficiently detected antibodies present in HBV patients. It was concluded that preS-His antigen/ protein A HRP-conjugate ELISA method was more sensitive than the commercial kit for detecting the antibodies present in HBV patient sera.
 Conclusion: It was concluded that SBS001 PreS recombinant protein can be used in ELISA kits for detection of HBV in Pakistani population.

Sequence Analysis of PreS2 Region of Hepatitis B Virus Genotype D Isolates

Hepatitis B virus (HBV) is a well known agent of liver diseases. HBV disease burden varies across theglobe with regions from low to high endemicity. Pakistan lies in the intermediate endemic zone, withhigh rate of mortality due to liver disease, cirrhosis and hepatocellular carcinoma. There is a wide rangeof heterogeneity in relation to HBV genotypes and sub-genotypes and in their patterns of pathogenesis,virulence and response to antiviral therapy. A large number of HBV genomic variations are associatedwith clinical outcomes such as hepatocellular carcinoma and liver cirrhosis. Thus, the present study aimsto analyze PreS2 gene sequences from HBV isolates and their phylogeny. To investigate this, a study wasconducted on twenty one HBV chronically infected individuals, serum samples were subjected to PCRwith specific primers for PreS2 region of HBV genotype D and then sequenced. Point mutations: A39V,P41H and L42I were found in cell permeability domain of PreS2 protein. However, MHC class I and IIepitopes were conserved in all sequences. Phylogenetic analysis was carried out by comparing thenucleotide sequence with 22 reference sequences of HBV sub-genotype D retrieved from the GeneBank.Phylogenetic analysis showed that two of our isolates, ASAB1 (2266) and ASAB3 (PIMS 7) sharedcluster 1 with China D1, Pakistan D1, Iran D1 and Turkey D1. Meanwhile, ASAB2 (HF2) was grouped incluster 2 with Lebanese D2 and Brazil D2.

Expression of Hepatitis B viral surface antigen (Pre S1) in E. coli

Hepatitis B is the most common liver diseases, which caused by hepatitis B virus (HBV) infection. There are around 257 million people around the world suffer from severe chronic hepatitis B infection. Therefore, it is necessary to develop a vaccine to prevent viral infection. PreS1 is one of the HBV envelope proteins that have been proved to be an effective vaccine. Accordingly, Viral DNA was purified from patients’ serums and amplified by PCR using specific primers. Amplicons of 324 bp bands of PreS1 was observed on gel electrophoresis. The PreS1 was cloned into pTXB21 plasmid to form the recombinant plasmid pTXB1_PreS1 and transformed into DH5α E. coli. Screening of transformants was done using Colony PCR and Sequencing. Alignment of 26 polypeptide sequences showed conservation of this region. The pTXB1_PreS1 was retransformed into T7 Express Competent E. coli and screened using colony PCR. The PreS1 was expressed as a recombinant protein fused to an intein tag with a molecular weight of ~ 39.5 kD. The PreS1 protein was purified by a single affinity chromatography step and after cleaved from intein tag by Dithiothreitol the obtained protein had a molecular weight of ~ 11.5 kD. Only one protein band was observed on the SDS-page gel. The PreS1 protein was successfully cloned and expressed in E. coli, which can be used as a vaccine against HBV.

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.

Development of antiviral drugs against hepatitis B virus

Hepatitis B virus (HBV) infection is prevalent throughout the world, with approximately 300 million people chronically infected. HBV infection can cause acute and chronic liver hepatitis. Long-term chronic infection can increase the incidence of liver cirrhosis and hepatocellular carcinoma (HCC). At present, there are two types of approved drugs for chronic HBV infection: nucleos(t)ide analogs (NAs) and interferon α (IFNα), currently pegylated IFNα (PegIFNα). Although these drugs can inhibit viral replication and reduce the risk of liver cirrhosis and HCC, the limitations are obvious: HBV surface antigen (HBsAg) and covalently closed circular DNA (cccDNA) pool cannot be efficiently eliminated. To overcome these limitations, new drugs are required urgently to be designed and developed. In recent years, with the deepening of research on HBV, drugs targeting different targets of viral complex replication mechanisms and interactions with the host immune system have been developed. Drugs targeting viral complex replication mechanisms include HBV entry inhibitors, cccDNA formation and transcription inhibitors, gene editing targeting HBV DNA, HBV specific siRNAs, capsid inhibitors, polymerase inhibitors and HBV secretion inhibitors. Entry inhibitors have been used successfully in treating viral infections. Myrcludex B, a synthetic N-myristoylated lipopeptide derived from HBV preS1 protein, competes with the virus for binding to the solute carrier family 10 member 1 (SLC10A1 or NTCP). This agent prevents HBV and Hepatitis D virus (HDV) infection and in cells and animal models. cccDNA is the transcriptional template of HBV RNAs and is an ideal target for clearing HBV. DNA ligase inhibitors, which can inhibit cccDNA formation and are being developed as anticancer agents, may be tested for treatment of chronic HBV infection, if the side effects are tolerable. The genome editing tool, clustered regularly interspaced short palindromic repeats associated nuclease 9 (CRISPR/Cas9), has many innovative applications in different fields because of its potential for gene therapy. These have provided a clear proof of concept that this approach has the potential to treat or even cure patients with chronic HBV infection. RNA interference technology can be used to manipulate virus gene expression and may be developed for HBV treatment. HBV nucleocapsid formation and pgRNA packaging are critical steps of viral life cycle that may be targeted by antiviral agents. Inside the viral capsid, pgRNA is reverse transcribed into minus-strand DNA by the viral polymerase, with its terminal protein domain acting as a primer for initiation of reverse transcription. HBV polymerase is also a good drug target. Drugs targeting the host immune system include that stimulating the innate immune system, like IFNα, Toll-like receptor (TLR) agonists and stimulating cytoplasmic nucleic acid sensors, and that activating an adaptive immune response, like checkpoint inhibitors, T-cell redirection and therapeutic vaccines. IFN-based therapies are considered as immunomodulatory strategies. TLRs are expressed either at the cell surface or on endosome membranes, where they recognize molecular patterns associated with invading pathogens. Stimulation of TLR1, TLR2, TLR3, and TLR4 on hepatocytes has direct antiviral effects against HBV. The RNA helicases DExD/H-box helicase 58 (DDX58, also called RIG-I) and IFN induced with helicase C domain 1 (IFIH1, also called MDA5) are RNA sensors in the cytoplasm that induce production of cytokines by activating IRF3 and NF-kB. Activation of RIG-I induces an antiviral response against HBV. PD-1 is the inhibitory receptor expressed by most HBV specific T cells, so agents are in development to disrupt the interaction between PD-1 and its ligand, PD-L1. A therapeutic vaccine against HBV needs to overcome mechanisms of HBV-specific immune tolerance. Because natural resolution of HBV infection is accompanied by lifelong T-cell- and antibody-mediated immunity, these vaccines should induce HBV-specific B- and T-cell immune responses. Here, we review recent advances in development of directing-acting antiviral (DAA), host-targeting agents and immunomodulatory therapies. Some of which have entered clinical trials. We also discuss strategies for unbiased high-throughput screens to identify compounds that inhibit HBV and for repurposing existing drugs.

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.

Correlation between HBV protein preS2 and tumor markers of hepatocellular carcinoma

BackgroundAlpha-fetoprotein (AFP) and Glypican 3 (GPC3) are both oncogenes and reactivated in hepatocellular carcinoma (HCC). PreS2 has been proved to be an important transactivator in HCC. In this study, we aim to provide evidence that HBV protein preS2 is responsible for AFP and GPC3′s reactivation in HCC. MethodsTotally Sixty-three cases of HCC, aged 34–79, who were surgically treated and pathologically confirmed were enrolled. The levels of AFP in peripheral serum were detected with electrochemical luminescence method before surgery. Levels of GPC3 in HCC samples were evaluated by immunohistochemistry. Luciferase reporter assays were used to measure the effect of preS2 on AFP and GPC3 promoters. ResultsAFP level and GPC3 but not albumin were significantly higher in preS2-positive HCC samples than preS2-negative HCC samples. And the preS2 protein expression was positively related with serum AFP level and GPC3 expression. Furtherly, dual luciferase assay showed that preS2 activated AFP and GPC3 promoter activity. ConclusionThe expression of preS2 protein relates closely to HCC markers AFP and GPC3.

Molecular analysis of Hepatitis B virus sub-genotypes and incidence of preS1/preS2 region mutations in HBV-infected Egyptian patients from Mansoura.

Hepatitis B virus (HBV) is one of the major causes of viral hepatitis worldwide. Despite the prevalence of HBV infection in Egypt, few studies have focused on sub-genotyping of the virus. Moreover, no studies are available regarding the mutational analysis of the preS1/preS2 region of the viral genome, or its impact on hepatocellular carcinoma (HCC) development in Egypt. In this study, we have analyzed the sub-genotypes and incidence of mutations in the preS1/preS2 region of HBV present in HBV-infected patients, from Mansoura city (located in the center of Nile Delta region of Egypt), via partial sequencing of this specific region. Moreover, we have investigated the impact of these mutations on HCC development by measuring serum alpha fetoprotein (AFP) level and abdominal ultrasound examination of the HBV-infected patients. According to our results, all samples were genotype D in which sub-genotype D1 was predominant. In addition, the results revealed mutations in the preS1/preS2 region, which could result in either immature preS1 protein or completely inhibit the translation of the preS2 protein. However, there was no incidence of HCC development in patients infected with mutated HBV in the preS1/preS2 region. In summary, for the first time our work has proved the predominance of sub-genotype D1 among HBV-infected Egyptian patients in Mansoura city, Nile Delta region, Egypt, and incidence of mutations in the preS1/preS2 region of HBV genome. This current study opens up research opportunities to discuss the impact of HBV mutations on the development of HCC in Egypt.

Hepatitis B virus PreS1 facilitates hepatocellular carcinoma development by promoting appearance and self-renewal of liver cancer stem cells

Hepatitis B virus (HBV) is a major etiologic agent of hepatocellular carcinoma (HCC). However, the molecular mechanism by which HBV infection contributes to HCC development is not fully understood. Here, we initially showed that HBV stimulates the production of cancer stem cells (CSCs)-related markers (CD133, CD117 and CD90) and CSCs-related genes (Klf4, Sox2, Nanog, c-Myc and Oct4) and facilitates the self-renewal of CSCs in human hepatoma cells. Cellular and clinical studies revealed that HBV facilitates hepatoma cell growth and migration, enhances white blood cell (WBC) production in the sera of patients, stimulates CD133 and CD117 expression in HCC tissues, and promotes the CSCs generation of human hepatoma cells and clinical cancer tissues. Detailed studies revealed that PreS1 protein of HBV is required for HBV-mediated CSCs generation. PreS1 activates CD133, CD117 and CD90 expression in normal hepatocyte derived cell line (L02) and human hepatoma cell line (HepG2 and Huh-7); facilitates L02 cells migration, growth and sphere formation; and finally enhances the abilities of L02 cells and HepG2 cells to induce tumorigeneses in nude mice. Thus, PreS1 acts as a new oncoprotein to play a key role in the appearance and self-renewal of CSCs during HCC development.

Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7.

The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection.

Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals.

Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21–47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21–47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants.

Correlation between hepatitis B virus DNA levels and diagnostic tests for HBsAg, HBeAg, and PreS1-Ag in chronic hepatitis B.

The aim of this study was to investigate the diagnostic value of the serum markers HBsAg and HBeAg and PreS1 protein (PreS1-Ag) in quantifying the levels of hepatitis B virus (HBV) DNA in patients with chronic hepatitis B (CHB). One thousand CHB patients were recruited from Beijing You'an Hospital between June and December 2012. Serum HBsAg and HBeAg levels were detected by electrochemiluminescence immunoassay. Enzyme-linked immunosorbent assay and fluorescence quantitative PCR were used to determine the level of PreS1-Ag and HBV DNA, respectively. We observed a low correlation between HBsAg and HBV DNA (r = 0.172, P < 0.001) expression; however, the correlation coefficient increased gradually with the increase in HBV DNA levels, and was more significant when HBV DNA log10 > 7 (r = 0.597, P < 0.001). Additionally, HBsAg and HBV DNA showed a significant positive correlation in the HBeAg+ group (r = 0.321, P < 0.001), whereas no correlation was observed in the HBeAg- group (r = -0.016, P = 0.825). HBV DNA expression was correlated with HBeAg (χ2 = 83.07, P < 0.001) and PreS1-Ag (χ2 = 36.01, P < 0.001). HBV DNA-positive rate was higher in HBeAg/PreS1-Ag++ patients (72.26%) than that in the single-positive groups (P < 0.001). Therefore, serum HBsAg is not a good marker for the prediction of HBV replication, and co-detection of HBeAg and PreS1-Ag, which can better predict HBV DNA replication, can be used as a reliable method for the clinical diagnosis and treatment of CHB.

Identification and genotyping of hepatitis B virus by PCR assay using genotype specific primers

Eight genotypes of hepatitis B virus are recognized which show deference in geographical distribution. Differences in genotypes affect the severity, complication, course and treatment of disease. This study was established in Sulaimani city, Kurdistan region, Iraq to detect HBV genotypes by rapid and specific method using PCR, which based on type specific primers. A total of 19 suspected hepatitis B patients including 14 males (73.68%) and 5 females (26.32%) were examined by PCR assay with universal outer primer used for detection of all HBV genotypes. The positive samples then examined by PCR assay using specific primers for each genotype. The result showed that 15 cases were negative and 4 cases were positives for HBV. The 4 positive HBV samples were subjected to genotyping. 4 genotypes (A, B, C, D) out of 6 genotypes, which can be determined by this method, were identified. Unique results were detected. Firstly, all of the samples showed mix infection, in which 1 case was mix genotype B+C+D and 3 cases were mix genotypes of A+B+C+ D. Secondly, sequencing of D genotype showed 120 nucleotide deletion mutations which encode overlapping gene of polymerase and pre-S protein. These results were not or rarely reported