Virus pre-S2 Research Articles

Vaccine Based on Recombinant Fusion Protein Combining Hepatitis B Virus PreS with SARS-CoV-2 Wild-Type- and Omicron-Derived Receptor Binding Domain Strongly Induces Omicron-Neutralizing Antibodies in a Murine Model.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants have dominated in the spread of the disease. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently, we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro using protein chemical methods, mass spectrometry, and circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum-hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study the specific antibody and cytokine responses, safety and Omicron neutralization. Defined and pure immunogens could be produced in significant quantities as secreted and folded proteins in mammalian cells. The antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, the RBD-specific IgG levels induced with the different vaccines were comparable, but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were seven- and twofold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively, and they were six-fold higher than those of the bivalent vaccine. Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

Increased plasma levels of monocyte chemoattractant protein-1 in patients with hepatitis B virus pre-S2 gene deletion mutation predict a higher risk of hepatocellular carcinoma recurrence after curative surgical resection.

Despite resection surgery as a curative therapy for hepatocellular carcinoma (HCC), the high rate of postoperative HCC recurrence remains a big challenge for patient survival. Chronic hepatitis B virus (HBV) infection is the most important risk factor for HCC. Deletion mutation in the HBV pre-S2 gene leads to expression of an essential viral oncoprotein called pre-S2 mutant and represents an independent prognostic biomarker for HCC recurrence after curative surgical resection. Additionally, cytokines are multifunctional secreted proteins and implicated in all stages of HBV-related HCC tumorigenesis. This study aimed to identify the cytokines whose plasma levels were associated with pre-S2 gene deletion mutation and HCC recurrence and evaluate their potential to be combined with pre-S2 gene deletion mutation in predicting HCC recurrence. Among a panel of 27 cytokines examined, plasma levels of monocyte chemoattractant protein-1 (MCP-1) were significantly upregulated in patients with pre-S2 gene deletion mutation or HCC recurrence. MCP-1 was validated as an independent prognostic biomarker for HCC recurrence. Moreover, patients with both the presence of pre-S2 gene deletion mutation and high levels of MCP-1 displayed a higher risk of HCC recurrence than patients with either one or none of these two biomarkers. The combination of pre-S2 gene deletion mutation and MCP-1 levels exhibited a better prognostic performance for HCC recurrence than each biomarker alone. This study discovered that MCP-1 levels had a significance to be as a combination biomarker with pre-S2 gene deletion mutation providing an improved performance in predicting HCC recurrence after curative surgical resection.

Combination of Hepatitis B Virus Pre-S2 Gene Deletion Mutation and Tumor-Node-Metastasis Stage Predicts Higher Hepatocellular Carcinoma Recurrence after Curative Surgical Resection

Hepatocellular carcinoma (HCC) is one of the most frequent and life-threatening human cancers worldwide. Despite curative resection surgery, the high recurrence rate of HCC leads to poor patient survival. Chronic hepatitis B virus (HBV) infection is a major etiological factor for HCC. HBV pre-S2 gene deletion mutation leads to the expression of an important oncoprotein called a pre-S2 mutant. It represents an independent prognostic biomarker for HCC recurrence. This study aimed to identify other independent prognostic biomarkers from clinicopathological characteristics of 75 HBV-related HCC patients receiving resection surgery and to validate their potential to be combined with pre-S2 gene deletion mutation as a combination biomarker for HCC recurrence. Patients with both the presence of pre-S2 gene deletion mutation and tumor-node-metastasis (TNM) stage IIIA-IIIC had a higher HCC recurrence risk than patients with either one or none of these two factors. Moreover, the combination of pre-S2 gene deletion mutation and TNM stage exhibited better performance than either of these two factors alone in discriminating patients from patients without HCC recurrence. Collectively, this study proposed that the TNM stage held significance as a combination biomarker with pre-S2 gene deletion mutation with a greater performance in predicting HCC recurrence after curative surgical resection.

Analysis of Hepatitis B Virus Pre-S Gene in Hepatocellular Carcinoma and Paracancerous Tissues from Southern China

Analysis of Hepatitis B Virus Pre-S Gene in Hepatocellular Carcinoma and Paracancerous Tissues from Southern China

Occult infection with hepatitis B virus PreS variants synergistically promotes hepatocellular carcinoma development in a high-fat diet context by generating abnormal ceramides

BackgroundSome occult hepatitis B virus (HBV) infections are resulted from PreS mutations that reduce secretion of envelope protein (HBsAg). We investigated the ceramide amounts and species in hepatocytes infected with PreS variants that were isolated from HBsAg-seronegative patients with hepatocellular carcinoma (HCC) and the ceramide effects on autochthonous HCC development in murine models.MethodsHBV PreS/S regions from 35 HBsAg-seronegative HCC patients were sequenced. Hepatocyte cell lines and male C57BL/6J mouse livers were transfected with two PreS variant representatives. The ceramides with variated lengths of fatty acyl chains were quantified. Tumour development was examined in the HBV-transfected mice fed different diet types.ResultsIn HBsAg-seronegative HCC patients, nonneoplastic liver tissues harboured HBsAg and replication-competent HBV. The most frequently detected PreS/S variants carried mutations of altered amino acid properties in HBsAg compared with an isolate from one HBsAg-seronegative HCC patient. Hepatocyte infection with PreS variants caused HBsAg retention within the endoplasmic reticulum and generated more amounts of ceramides with C16:0 ceramide elevated the highest. Saturated fatty acids aggravated the PreS variant-infected hepatocytes to generate abnormal amounts and species of ceramides, which with HBV proteins synergistically activated NLRP3 inflammasome in liver inflammatory macrophages. Liver tumours were only detected in HBV-transfected mice fed high-fat diet, with higher tumour loads in the PreS variant-transfected, associated with abnormal ceramide generation.ConclusionsHBV PreS mutations which altered amino acid properties of envelope proteins inhibited HBsAg secretion. Hepatocyte infection with PreS variants generated abnormal ceramides which with HBV proteins coactivated NLRP3 inflammasome in liver macrophages to promote autochthonous HCC development.

HBV preS Mutations Promote Hepatocarcinogenesis by Inducing Endoplasmic Reticulum Stress and Upregulating Inflammatory Signaling.

This study aimed to elucidate the effects and underlying mechanisms of hepatitis B virus (HBV) preS mutations on hepatocarcinogenesis. The effect of the preS mutations on hepatocellular carcinoma (HCC) occurrence was evaluated using a prospective cohort study with 2114 HBV-infected patients, of whom 612 received antiviral treatments. The oncogenic functions of HBV preS mutations were investigated using cancer cell lines and Sleeping Beauty (SB) mouse models. RNA-sequencing and microarray were applied to identify key molecules involved in the mutant-induced carcinogenesis. Combo mutations G2950A/G2951A/A2962G/C2964A and C3116T/T31C significantly increased HCC risk in patients without antiviral treatment, whereas the preS2 deletion significantly increased HCC risk in patients with antiviral treatment. In SB mice, the preS1/preS2/S mutants induced a higher rate of tumor and higher serum levels of inflammatory cytokines than did wild-type counterpart. The preS1/preS2/S mutants induced altered gene expression profiles in the inflammation- and metabolism-related pathways, activated pathways of endoplasmic reticulum (ER) stress, affected the response to hypoxia, and upregulated the protein level of STAT3. Inhibiting the STAT3 pathway attenuated the effects of the preS1/preS2/S mutants on cell proliferation. G2950A/G2951A/A2962G/C2964A, C3116T/T31C, and preS2 deletion promote hepatocarcinogenesis via inducing ER stress, metabolism alteration, and STAT3 pathways, which might be translated into HCC prophylaxis.

Association of Increased Programmed Death Ligand 1 Expression and Regulatory T Cells Infiltration with Higher Hepatocellular Carcinoma Recurrence in Patients with Hepatitis B Virus Pre-S2 Mutant after Curative Surgical Resection.

Although surgical resection is available as a potentially curative therapy for hepatocellular carcinoma (HCC), high recurrence of HCC after surgery remains a serious obstacle for long-term patient survival. Therefore, the discovery of valuable prognostic biomarkers for HCC recurrence is urgently needed. Pre-S2 mutant is a mutant form of hepatitis B virus (HBV) large surface protein which is expressed from the HBV surface gene harboring deletion mutations spanning the pre-S2 gene segment. Pre-S2 mutant-positive HCC patients have been regarded as a high-risk population of HCC recurrence after resection surgery and display increased immune checkpoint programmed death ligand 1 (PD-L1) expression and pro-tumor regulatory T cells (Tregs) infiltration in tumor tissues. In this study, the association of higher levels of PD-L1 expression and Tregs infiltration in tumor tissues with post-operative HCC recurrence in pre-S2 mutant-positive HCC patients was evaluated. We found that patients with pre-S2 mutant in combination with higher levels of PD-L1 expression and Tregs infiltration in tumor tissues were independently associated with a higher risk of HCC recurrence (hazard ratio, 4.109; p value = 0.0011) and poorer recurrence-free survival (median, 8.2 versus 18.0 months; p value = 0.0004) than those of patients with either one or two of these three biomarkers. Furthermore, a combination of pre-S2 mutant, intra-tumoral PD-L1 expression, and tumor-infiltrating Tregs exhibited superior performance in identifying patients at a higher risk of HCC recurrence (area under the receiver operating characteristic curve, 0.8400). Collectively, this study suggests that higher levels of PD-L1 expression and Tregs infiltration in tumor tissues predicted a higher risk of HCC recurrence in pre-S2 mutant-positive HCC patients after curative surgical resection.

Approaches for Detection of Hepatitis B Virus Pre-S Gene Deletions and Pre-S Deleted Proteins and Their Application in Prediction of Higher Risk of Hepatocellular Carcinoma Development and Recurrence.

Hepatocellular carcinoma (HCC) is among the most common and lethal human cancers worldwide and is closely associated with chronic hepatitis B virus (HBV) infection. Pre-S deleted proteins are naturally occurring mutant forms of HBV large surface proteins that are expressed by HBV surface genes harboring deletion mutations over the pre-S gene segments. It has been well demonstrated that HBV pre-S deleted proteins function as important oncoproteins, which promote malignant phenotypes of hepatocytes through the activation of multiple oncogenic signaling pathways and result in HCC formation. The oncogenic signaling pathways activated by pre-S deleted proteins have been verified as potential therapeutic targets for the prevention of HCC development. Moreover, the presence of pre-S gene deletions and the expression of pre-S deleted proteins in the blood and liver tissues of HBV-infected patients have been evaluated as valuable biomarkers for predicting a higher risk of HCC development and recurrence after curative surgical resection. Therefore, the precise detection of pre-S gene deletions and pre-S deleted proteins holds great promise as regards identifying the patients at higher risk of HCC development and recurrence, thus aiding in more timely and better treatments to improve their survival. This review summarizes the major approaches used for the detection of pre-S gene deletions and pre-S deleted proteins, including the approaches based on Sanger DNA sequencing, pre-S gene chips, next-generation sequencing and immunohistochemistry staining, and it highlights their important applications in the prediction of higher risks of HCC development and recurrence.

Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: The myristoylated pre-S1 peptide (Myr47) synthesized to mimic pre-S1 domain (2-48) in large (L) surface protein of hepatitis B virus (HBV) prevents HBV infection to hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP). We previously demonstrated that yeast-derived nanoparticles containing L protein (bio-nanocapsules: BNCs) bind scavenger receptor class B type 1 (SR-B1). In this study, we examined the binding of Mry47 to SR-B1. (2) Methods: The binding and endocytosis of fluorescence-labeled Myr47 to SR-B1 (and its mutants)-green fluorescence protein (GFP) fusion proteins expressed in HEK293T cells were analyzed using flow cytometry and laser scanning microscopy (LSM). Various ligand-binding properties were compared between SR-B1-GFP and NTCP-GFP. Furthermore, the binding of biotinylated Myr47 to SR-B1-GFP expressed on HEK293T cells was analyzed via pull-down assays using a crosslinker and streptavidin-conjugated beads. (3) Conclusions: SR-B1 bound not only Myr47 but also its myristoylated analog and BNCs, but failed to bind a peptide without myristoylation. However, NTCP only bound Myr47 among the ligands tested. Studies using SR-B1 mutants suggested that both BNCs and Myr47 bind to similar sites of SR-B1. Crosslinking studies indicated that Myr47 binds preferentially SR-B1 multimer than monomer in both HEK293T and HepG2 cells.

Construction of an anti-hepatitis B virus preS1 antibody and usefulness of preS1 measurement for chronic hepatitis B patients: Anti-HBV PreS1 antibody

The preS1 region plays an essential role in hepatitis B virus (HBV) infection. We construct an antibody that binds to preS1 and a measurement system for serum preS1 in chronic HBV-infected patients. Hybridoma clones that produce anti-preS1 antibodies were obtained by the iliac lymph node method. Epitope mapping was conducted, and an enzyme-linked immunosorbent assay (ELISA)-based method was developed. Using this ELISA system, serum preS1 levels were measured in 200 chronic HBV-infected patients. Eight types of hybridomas were obtained, of which antibody 3-55 using amino acids 38-47 as the epitope showed high binding affinity to preS1. Serum preS1 levels measured by ELISA using 3-55 antibody were correlated with HBsAg, HBcrAg and HBV DNA levels. Among HBeAg-negative patients without antiviral therapeutic objective (HBV DNA <3.3logIU/mL or alanine aminotransferase ≤30U/L), preS1 was significantly higher in subjects who had progressed to the point of requiring antiviral therapy compared to subjects who had maintained their status for the preceding three years (p<0.01). We constructed an antibody against preS1 and an ELISA system capable of measuring serum preS1 levels. PreS1 may serve as a novel tool to predict the need for antiviral therapy in HBeAg-negative HBV-infected patients.

Nucleotide variants in hepatitis B virus preS region predict the recurrence of hepatocellular carcinoma.

Background: Hepatitis B virus (HBV) variants in the preS region have been associated with hepatocellular carcinoma (HCC). However, the effect of the preS variants on HCC prognosis remains largely unknown. We aimed to identify the preS variants that reliably predict postoperative prognosis in HCC.Methods: RNA-seq data of 203 HCC patients retrieved from public database were screened for the preS variants related to HCC prognosis. The variants in the sera and tumors were then validated in our prospective cohort enrolling 103 HBV-associated HCC patients.Results: By analyzing prognosis-related gene sets in the RNA-seq data, 12 HBV preS variants were associated with HCC recurrence. Of those, G40C and C147T in the sera predicted an unfavorable recurrence-free survival in our cohort (hazard ratio [HR]=2.18, 95% confidence interval [CI]=1.37-3.47, p=0.001 for G40C; HR=1.84, 95% CI=1.15-2.92, p=0.012 for C147T). G40C and C147T were significantly associated with microscopic vascular invasion, larger tumor size, and abnormal liver function. Multivariate Cox regression analysis showed that G40C significantly increased the risk of HCC recurrence in patients with postoperative antiviral treatment. The HCC prognosis-prediction model consisting of α-fetoprotein and G40C in the sera achieved the best performance (sensitivity=0.80, specificity=0.70, and area under the curve=0.79). Functional analysis indicated that these two variants were associated with cell proliferation, chromosome instability, carcinogenesis, metastasis, and anticancer drug resistance.Conclusions: G40C and C147T are serological biomarkers for HCC prognosis and the prognostic model consisting of serological α-fetoprotein and G40C achieved the best performance in predicting postoperative prognosis.

Association of Low Serum Albumin Level with Higher Hepatocellular Carcinoma Recurrence in Patients with Hepatitis B Virus Pre-S2 Mutant after Curative Surgical Resection.

Hepatocellular carcinoma (HCC) is, globally, one of the most prevalent and deadly human cancers; despite curative surgical resection, its high recurrence rate after surgery remains a large threat, resulting in poor patient survival. The hepatitis B virus (HBV) pre-S2 mutant that harbors deletions spanning the pre-S2 gene segment has emerged as an important oncoprotein for HCC development and a valuable prognostic biomarker for HCC recurrence; however, its relationship with clinicopathological factors is largely unexplored. In this study, the correlation of the deletion spanning the pre-S2 gene segment with clinicopathological factors and the association of such correlation with HCC recurrence after curative surgical resection were examined in HBV-related HCC patients. Inverse correlation between serum albumin level and the deletion spanning the pre-S2 gene segment was identified. HCC patients with the presence of the deletion spanning the pre-S2 gene segment and a low serum albumin level were associated with higher HCC recurrence than patients with either factor alone or neither factor were. Moreover, a combination of the serum albumin level and the deletion spanning the pre-S2 gene segment exhibited better performance than that of either factor alone in predicting HCC recurrence. Collectively, this study shows an association of low serum albumin level with pre-S2 mutant-positive HCC patients, and validates the prognostic value of this association in identifying patients with higher HCC recurrence after curative surgical resection.

HBV Pre-S1-Derived Myristoylated Peptide (Myr47): Identification of the Inhibitory Activity on the Cellular Uptake of Lipid Nanoparticles.

The Myr47 lipopeptide, consisting of hepatitis B virus (HBV) pre-S1 domain (myristoylated 2–48 peptide), is an effective commercialized anti-HBV drug that prevents the interaction of HBV with sodium taurocholate cotransporting polypeptide (NTCP) on human hepatocytes, an activity which requires both N-myristoylation residue and specific amino acid sequences. We recently reported that Myr47 reduces the cellular uptake of HBV surface antigen (HBsAg, subviral particle of HBV) in the absence of NTCP expression. In this study, we analyzed how Myr47 reduces the cellular uptake of lipid nanoparticles (including liposomes (LPs) and HBsAg) without NTCP expression. By using Myr47 mutants lacking the HBV infection inhibitory activity, they could reduce the cellular uptake of LPs in an N-myristoylation-dependent manner and an amino acid sequence-independent manner, not only in human liver-derived cells but also in human non-liver-derived cells. Moreover, Myr47 and its mutants could reduce the interaction of LPs with apolipoprotein E3 (ApoE3) in an N-myristoylation-dependent manner regardless of their amino acid sequences. From these results, lipopeptides are generally anchored by inserting their myristoyl residue into the lipid bilayer and can inhibit the interaction of LPs/HBsAg with apolipoprotein, thereby reducing the cellular uptake of LPs/HBsAg. Similarly, Myr47 would interact with HBV, inhibiting the uptake of HBV into human hepatic cells, while the inhibitory effect of Myr47 may be secondary to its ability to protect against HBV infection.

Hepatitis B Virus Pre-S Gene Deletions and Pre-S Deleted Proteins: Clinical and Molecular Implications in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most frequent and fatal human cancers worldwide and its development and prognosis are intimately associated with chronic infection with hepatitis B virus (HBV). The identification of genetic mutations and molecular mechanisms that mediate HBV-induced tumorigenesis therefore holds promise for the development of potential biomarkers and targets for HCC prevention and therapy. The presence of HBV pre-S gene deletions in the blood and the expression of pre-S deleted proteins in the liver tissues of patients with chronic hepatitis B and HBV-related HCC have emerged as valuable biomarkers for higher incidence rates of HCC development and a higher risk of HCC recurrence after curative surgical resection, respectively. Moreover, pre-S deleted proteins are regarded as important oncoproteins that activate multiple signaling pathways to induce DNA damage and promote growth and proliferation in hepatocytes, leading to HCC development. The signaling molecules dysregulated by pre-S deleted proteins have also been validated as potential targets for the prevention of HCC development. In this review, we summarize the clinical and molecular implications of HBV pre-S gene deletions and pre-S deleted proteins in HCC development and recurrence and highlight their potential applications in HCC prevention and therapy.

A Novel DNA Vaccine Against SARS-CoV-2 Encoding a Chimeric Protein of Its Receptor-Binding Domain (RBD) Fused to the Amino-Terminal Region of Hepatitis B Virus preS1 With a W4P Mutation.

A coronavirus SARS-CoV-2, which has caused the pandemic viral pneumonia disease COVID-19, significantly threatens global public health, highlighting the need to develop effective and safe vaccines against its infection. In this study, we developed a novel DNA vaccine candidate against SARS-CoV-2 by expressing a chimeric protein of its receptor-binding domain (RBD) fused to a 33-bp sequence (11 aa) from the hepatitis B virus (HBV) preS1 region with a W4P mutation (W4P-RBD) at the N-terminal region and evaluated its immunogenicity. In vitro transfection experiments in multiple cell lines demonstrated that W4P-RBD vs. wild-type RBD protein (W-RBD) led to enhanced production of IL-6 and TNFα at the transcription and translation levels, suggesting the adjuvant potential of N-terminal HBV preS1 sequences for DNA vaccines against SARS-CoV-2. W4P-RBD also led to enhanced production of IgG and IgA, which can neutralize and block SARS-CoV-2 infection in both blood sera and bronchoalveolar lavage (BAL) fluid from the lung in vaccinated mice. Additionally, W4P-RBD led to an enhanced T-cell-mediated cellular immune response under S1 protein stimulation. In summary, W4P-RBD led to robust humoral and cell-mediated immune responses against SARS-CoV-2 in vaccinated mice, highlighting its feasibility as a novel DNA vaccine to protect against SARS-CoV-2 infection.

Hepatitis B Virus preS2 Domain Promotes Angiogenesis in Hepatocellular Carcinoma via Transcriptional Activation of VEGFA Promoter

: Angiogenesis is closely related to the development and progression of hepatocellular carcinoma (HCC). Angiogenic factors have been confirmed to be overexpressed in HCC. The hepatitis B virus preS2 domain is a transactivator that plays an important role in hepatitis B virus (HBV)-related HCC. Here, we aimed to investigate the potential of the preS2 domain in inducing angiogenesis in HCC. A total of 25 cases of pathologically confirmed HCC were screened. The levels of preS2, CD34, and vascular endothelial growth factor A (VEGFA) in HCC samples were evaluated by immunohistochemistry (IHC). The proliferation of vascular endothelial cells was detected by CCK-8. Besides, VEGFA was analyzed by Western blot in HCC cells. The effect of preS2 on the VEGFA promoter was measured by dual-luciferase reporter assays. We found that preS2 domain-positive HCCs had significantly higher microvessel density (MVD) and VEGFA expression than preS2 domain-negative HCCs. Overexpression of preS2 upregulated VEGFA expression in HepG2 and activated vascular endothelial cell proliferation. However, blocking preS2 expression reduced VEGFA expression in HepG2.2.15 and inhibited the proliferation of vascular endothelial cells. In addition, a dual-luciferase assay indicated that the preS2 domain could activate VEGFA promoter activity. In conclusion, we showed that the expression of the preS2 domain promotes angiogenesis by transactivating the VEGFA promoter in HCC.

The Strategy of Conditionally Replicating Adenovirus-Mediated PreS2 Mini-Antibody Expression Has Dual Effects of Inhibiting HBV Infection and Preventing Hepatocellular Carcinoma.

AimTo investigate the inhibitory effect of hepatitis B virus (HBV) preS2 mini-antibody (mPreS2) against HBV infection, HBV-associated liver injury and HBV-associated hepatic carcinogenesis.MethodsA recombinant adenovirus vector with the human survivin promoter and mPreS2 gene, Ad5SVP-mPreS2, was constructed. Fluorescence microscopy examination and TCID 50 analysis were utilized to determine the specific proliferation of recombinant adenovirus in liver cancer cells. Western blot analysis was used to determine the mPreS2 expression levels. Enzyme-linked immunosorbent assay (ELISA) was used to examine HBsAg levels to evaluate the inhibitory effect of mPreS2 against HBV infection. The protective effects on hepatic function and preventive effects against hepatic carcinogenesis of Ad5SVP-mPreS2 were studied in diethylnitrosamine (DEN)-treated HBV transgenic Imprinting Control Region mice.ResultsThe recombinant adenovirus regulated by the human survivin promoter proliferated exclusively in liver cancer cells rather than normal liver cells. The expression levels of mPreS2 were increased in liver cancer cells compared with normal liver cells, and mPreS2 could be used to recognize liver cells from HBV transgenic mice. ELISA showed that HBsAg levels were decreased in the group treated with Ad5SVP-mPreS2. Ad5SVP-mPreS2 had a protective effect on hepatic function in a DEN-induced liver injury model because of lower serum levels of alanine transaminase and aspartate transaminase. Additionally, HBV transgenic mice treated with Ad5SVP-mPreS2 had fewer and smaller cancerous nodes after induction with DEN than untreated mice.ConclusionConditionally replicating adenovirus-mediated mPreS2 expression inhibited HBV infection and had an inhibitory effect on liver injury and hepatocellular carcinogenesis in HBV transgenic mice.

Increased infiltration of regulatory T cells in hepatocellular carcinoma of patients with hepatitis B virus pre-S2 mutant

Hepatocellular carcinoma (HCC) is a frequent and deadly human cancer worldwide that is intimately associated with chronic hepatitis B virus (HBV) infection. Pre-S2 mutant is a HBV oncoprotein that plays important roles in HCC development and is linked to poor prognosis in HCC patients. However, the profiles of tumor-infiltrating lymphocytes in HCC tissues of pre-S2 mutant-positive patients remain unknown. In this study, we performed fluorescent immunohistochemistry staining to detect the infiltration of ‘anti-tumor’ cytotoxic T lymphocytes (CTLs) and ‘pro-tumor’ regulatory T cells (Tregs) in pre-S2 mutant-positive and -negative HCC patients. We showed that pre-S2 mutant-positive patients had a significantly higher infiltration of CD4+CD25+ cells and forkhead box P3 (Foxp3)-expressing cells but similar CTLs and lower granzyme B-expressing cells in HCC tissues compared with pre-S2 mutant-negative patients. Moreover, the percentage of pre-S2 plus pre-S1 + pre-S2 deletion (pre-S2 mutant) was positively correlated with the density of CD4+CD25+ cells and Foxp3-expressing cells but negatively with granzyme B-expressing cells in HCC tissues. Considering that increased intratumoral Tregs have been shown to promote tumor immune evasion, our data may provide new insights into the pathogenesis of HBV pre-S2 mutant-induced HCC and suggest that therapeutics targeting Tregs may be a promising strategy for treating pre-S2 mutant-positive high-risk patient population.

Increased Expression of Programmed Death Ligand 1 in Hepatocellular Carcinoma of Patients with Hepatitis B Virus Pre-S2 Mutant.

PurposeChronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), a leading cause of cancer-related death worldwide. The HCC patients who harbor HBV pre-S2 mutant, an oncoprotein that plays key roles in HCC development, have been closely associated with a worse prognosis after curative surgical resection, suggesting an urgent need for alternative therapeutic options to improve their survival. In this study, we aimed to evaluate the expression profiles of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1), two of the most well-studied immune checkpoint molecules that promote tumor immune evasion, in tumor of the pre-S2 mutant-positive/high HCC patients.MethodsWe classified 40 HBV-related HCC patients into the pre-S2-positive/high and -negative/low groups by a next-generation sequencing-based approach. The fluorescent immunohistochemistry staining was performed to detect the expression of PD-1 and PD-L1 in HCC tissues of patients.ResultsWe showed that patients with either deletion spanning pre-S2 gene segment or high percentage of pre-S2 plus pre-S1+pre-S2 deletion (the pre-S2 mutant-positive/high group) exhibited a significantly higher density of PD-L1-positive cells in HCC tissues than those without. Moreover, the percentage of pre-S2 plus pre-S1+pre-S2 deletion displayed a high positive correlation with the density of PD-L1-positive cells in HCC tissues.ConclusionThe increased expression of PD-L1 in tumor tissues of the pre-S2 mutant-positive HCC patients suggest that pre-S2 mutant may play a potential role in dysregulation of tumor immune microenvironment in the progression of HBV-related HCC, implicating for the development of future therapeutic strategies.

TIMM29 interacts with hepatitis B virus preS1 to modulate the HBV life cycle.

Hepatitis B virus (HBV), a major global health problem, can cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinomas in chronically infected patients. However, before HBV infection can be adequately controlled, many mysteries about the HBV life cycle must be solved. In this study, TIMM29, an inner mitochondrial membrane protein, was identified as an interaction partner of the preS1 region of the HBV large S protein. The interaction was verified by both an immunoprecipitation with preS1 peptides and a GST-pulldown assay. Immunofluorescence studies also showed colocalization of preS1 and TIMM29. Moreover, it was determined that the preS1 bound with amino acids 92-189 of the TIMM29 protein. Infection of HBV in TIMM29-overexpressing NTCP/G2 cells resulted in a significant decrease of HBeAg and both extracellular particle-associated and core particle-associated HBV DNA without affecting cccDNA formation. Comparable results were obtained with TIMM29-overexpressing HB611 cells, which constitutively produce HBV. In contrast, knockout of TIMM29 in NTCP/G2 cells led to a higher production of HBV including HBeAg expression, as did knockout of TIMM29 in HB611. Collectively, these results suggested that TIMM29 interacts with the preS1 region of the HBV large S protein and modulates HBV amplification.