Class A capsid assembly modulator apoptotic elimination of hepatocytes with high HBV core antigen level in vivo is dependent on de novo core protein translation.

Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.

Role of core protein mutations in the development of occult HBV infection.

BACKGROUND & AIMS: Occult HBV infection (OBI) is associated with transfusion-transmitted HBV infection and hepatocellular carcinoma. Studies on OBI genesis have concentrated on mutations in the S region and the regulatory elements. Herein, we aimed to determine the role of mutations in the core region on OBIs. METHODS: An OBI strain (SZA) carrying 9 amino acid (aa) substitutions in the core protein/capsid (Cp) was selected by sequence alignment and Western blot analysis from 26 genotype B OBI samples to extensively explore the impact of Cp mutations on viral antigen production in vitro and in vivo. RESULTS: A large panel of 30 Cp replicons were generated by a replication-competent pHBV1.3 carrying SZA or wild-type (WT) Cp in a 1.3-fold over-length of HBV genome, in which the various Cp mutants were individually introduced by repairing site mutations of SZA-Cp or creating site mutations of WT-Cp by site-directed mutagenesis. The expression of HBcAg, HBeAg, and HBsAg and viral RNA was quantified from individual SZA and WT Cp mutant replicons in transfected Huh7 cells or infected mice, respectively. An analysis of the effect of Cp mutants on intracellular or extracellular viral protein production indicated that the W62R mutation in Cp had a critical impact on the reduction of HBcAg and HBeAg production during HBV replication, whereas P50H and/or S74G mutations played a limited role in influencing viral protein production invivo. CONCLUSIONS: W62R and its combination mutations in HBV Cp might massively affect HBcAg and HBeAg production during viral replication, which, in turn, might contribute to the occurrence of OBI. LAY SUMMARY: Occult hepatitis B virus infections (OBIs) have been found to be associated with amino acid mutations in the S region of the HBV, but the role of mutations in the core protein (Cp) remains unclear. In this study, an OBI strain (SZA) carrying 9 amino acid substitutions in Cp has been examined comprehensively in vitro and in vivo. The W62R mutation in Cp majorly reduces HBcAg and HBeAg production during HBV replication, potentially contributing to the occurrence of OBI.

In vitro preparation of uniform and nucleic acid free hepatitis B core particles through an optimized disassembly-purification-reassembly process.

Structure heterogeneity and host nucleic acids contamination are two major problems for virus-like particles (VLPs) produced by various host cells. In this study, an in vitro optimized disassembly-purification-reassembly process was developed to obtain uniform and nucleic acid free hepatitis B core (HBc) based VLPs from E. coli fermentation. The process started with ammonium sulfate precipitation of all heterogeneous HBc structures after cell disintegration. Then, dissolution and disassembly of pellets into basic subunits were carried out under the optimized disassembly condition. All contaminants, including host nucleic acids and proteins, were efficiently removed with affinity chromatography. The purified subunits reassembled into VLPs by final removal of the chaotropic agent. Two uniform and nucleic acid free HBc-based VLPs, truncated HBc149 and chimeric HBc183-MAGE3 I, were successfully prepared. It was found that disassembly degree of HBc-based VLPs had a great influence on the protein yield, nucleic acid removal and reassembly efficiency. 4 M urea was optimal because lower concentration would not disassemble the particles completely while higher concentration would further denature the subunits into disordered aggregate and could not be purified and reassembled efficiently. For removal of strong binding nucleic acids such as in the case of HBc183-MAGE3 I, benzonase nuclease was added to the disassembly buffer before affinity purification. Through the optimized downstream process, uniform and nucleic acid free HBc149 VLPs and HBc183-MAGE3 I VLPs were obtained with purities above 90% and yields of 55.2 and 43.0 mg/L, respectively. This study would be a reference for efficient preparation of other VLPs.

Negative HBcAg in immunohistochemistry assay of liver biopsy is a predictive factor for the treatment of patients with nucleos(t)ide analogue therapy.

The hepatitis B core antigen (HBcAg) is an important target for antiviral response in chronic hepatitis B (CHB) patients. However, the correlation between HBcAg in the hepatocyte nucleus and nucleos(t)ide analogue (NA) therapeutic response is unclear. We sought to evaluate the role of HBcAg by analysing liver biopsies for viral response in NA-naïve hepatitis B e antigen (HBeAg) positive (+) CHB patients via immunohistochemistry (IHC). A total of 48 HBcAg-negative (-) patients and 48 HBcAg (+) patients with matching baseline characteristics were retrospectively analysed for up to 288 weeks. Virological response (VR) rates of patients in the HBcAg (-) group were significantly higher at week 48 and 96 than the HBcAg (+) group (77.1% versus 45.8% at week 48, respectively, P = 0.002 and 95.3% versus 83.3% at week 96, respectively, P = 0.045). The serological negative conversion rate of HBeAg was significantly higher in the HBcAg (-) than in the HBcAg (+) group from week 96 to 288 (35.4 % versus 14.6% at week 96, respectively, P = 0.018; 60.4% versus 14.6%, respectively, P < 0.001 at week 144; 72.9% versus 35.4%, respectively, P < 0.001 at week 288). The cumulative frequencies of VR and lack of HBeAg were higher in the HBcAg (-) group (both P < 0.05). Binary logistic regression analysis showed that HBcAg (-) was the predictor for the lack of HBeAg (OR 4.482, 95% CI: 1.58-12.68). In summary, the absence of HBcAg in the hepatocyte nucleus could be an independent predictor for HBeAg seroconversion rates during NA-naïve treatment in HBeAg (+) CHB patients.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication.

PTD-fused p53 as a potential antiviral agent directly suppresses HBV transcription and expression.

In Hepatitis B virus (HBV) infection, the virus generates numerous viral mRNAs/proteins and viral loads, which plays a major role in driving T cell tolerance, viral persistence, and hepatocellular carcinoma. However, currently available anti-HBV agents have no direct effect on viral mRNA transcription and protein expression. In this study, we designed a recombinant fusion of p53 protein with the cell-penetrating peptide PTD (protein transduction domain of trans-activator of transcription), which mediated p53 internalization into hepatocytes. PTD-p53 effectively suppressed HBV transcription and antigen expression by interaction with viral enhancers. We further provide evidence that PTD-p53 counteracts the viral transcription feedback loop and effectively suppressed HBV production of viral mRNAs, as well as HBsAg, HBeAg, and HBcAg, both in vitro and in vivo. Our results thereby provide a basis for developing a new therapeutic approach against HBV infection.

SplitCore Technology Allows Efficient Production of Virus-Like Particles Presenting a Receptor-Contacting Epitope of Human IgE.

Immunoglobulin E (IgE) plays a central role in type I hypersensitivity including allergy and asthma. Novel treatment strategy envisages development of a therapeutic vaccine designed to elicit autologous blocking antibodies against the IgE. We sought to develop an IgE-epitope antigen that induces antibodies against a receptor-contacting epitope on human IgE molecule. We designed the VLP immunogens which utilize hepatitis B virus core protein (HBcAg) as a carrier, and present arrays of the receptor-contacting epitopes of the human IgE on their surfaces. FG loop from the IgE domain Cε3 was engineered into the HBcAg. Two constructs explore a well-established approach of insertion into a main immunodominant region of the HBcAg. Third construct is different in that the carrier is produced in a form of an assembly of two polypeptide chains which upon expression remain associated in a stable VLP-forming subunit (SplitCore technology). No VLPs were isolated from E.coli expressing the IgE-epitope antigens with contiguous sequences. On the contrary, the SplitCore antigen carrying the FG loop efficiently formed the VLPs. Immunization of mice with the VLPs presenting receptor-contacting epitope of the IgE elicited antibodies recognizing the human IgE in ELISA.

Meeting report VLPNPV: Session 5: Plant based technology.

The VLPNPV 2014 Conference that was convened at the Salk institute was the second conference of its kind to focus on advances in production, purification, and delivery of virus-like particles (VLPs) and nanoparticles. Many exciting developments were reported and discussed in this interdisciplinary arena, but here we report specifically on the contributions of plant-based platforms to VLP vaccine technology as reported in the section of the conference devoted to the topic as well in additional presentations throughout the meeting. The increasing popularity of plant production platforms is due to their lower cost, scalability, and lack of contaminating animal pathogens seen with other systems. Reports include production of complex VLPs consisting of 4 proteins expressed at finely-tuned expression levels, a prime-boost strategy for HIV vaccination using plant-made VLPs and a live viral vector, and the characterization and development of plant viral nanoparticles for use in cancer vaccines, drug delivery, and bioimaging.

Native agarose gel electrophoresis and electroelution: A fast and cost-effective method to separate the small and large hepatitis B capsids.

Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self-assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost-effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE-EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE-EE method are monodisperse with polydispersity values of ∼15% and ∼13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE-EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ∼84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.

[Impacts on hepatitis B virus replication by gene engineering at apical loop region of capsid protein].

Hepatitis B virus (HBV) DNA replication takes place in the viral capsid that consists of 180 or 240 copies of HBV capsid (HBc or core) protein. The monomeric core protein contains an apical loop region that forms the spikes on the surface of viral capsid upon core dimerization and capsid assembly. To investigate the impact on HBV DNA replication through gene engineering at the spike of HBV capsid. plasmids expressing engineered HBc with linker-fused enhanced green fluorescent protein (EGFP) or shortened EGFP insertion at the spike region were constructed by Restriction Digestion and Ligation-independent Cloning (RLIC). The wildtype or mutant HBc construct was cotransfected with HBV1.1c(-), a plasmid containing 1.1 unit-length HBV genome with deficiency in HBc expression, into HEK293 cells, respectively. GFP signal was observed through a fluorescence microscope and HBV DNA replicative intermediates were assayed by Southern blotting to determine the expression and functions of different recombinants. Our results demonstrated that the RLIC method was effective to generate deletion or insertion in the apical loop region of HBc. Both HBc-EGFP recombinants with different linkers produced green fluorescence but with different subcellular distribution pattern. However, HBV DNA replication was not detected with the trans-complementation of these two HBc recombinants. In addition, other recombinants including the one only with the deletion of aa79-80 failed to support HBV replication. Taken together, our results suggest that RLIC is a robust method which can be broadly applied in gene engineering; different peptide linkers may have different influences on the functions of an engineered fusion protein; and HBc aa79-80 play a critical role for HBc to support HBV DNA replication.

Inhibition of hepatitis B virus gene expression by small interfering RNAs targeting cccDNA and X antigen.

To test the possible inhibition of hepatitis B virus (HBV) replication and expression by small interfering RNAs (siRNAs) targeting simultaneously covalenthy closed circular DNA (dnacccDNA) and X antigen, corresponding recombinant plasmids were transfected into HepG2.2.15 cells and the levels of cccDNA, HBXAg, HBcAg, and HBeAg were assayed at various times post transfection. As expected, the single siRNAs showed marked inhibitory effects but their combination was even more efficient. These results provide a new insight into the development of a potential anti-HBV strategy of enhancing the efficacy of individual antivirals and overcoming the high mutation rate of HBV.

The effect of the G1888A mutation of subgenotype A1 of hepatitis B virus on the translation of the core protein.

A distinctive characteristic of subgenotype A1 of hepatitis B virus is G1888A in the precore region. This transition introduces an out-of-frame AUG, creating an overlapping upstream open reading frame (uORF), terminating five nucleotides downstream from the core AUG. This uORF can potentially be translated into a seven amino acid peptide. In addition to stabilizing the encapsidation signal by forming a base pair with T1871, this mutation may affect translation of the core protein. The aim of this study was to use reporter constructs to determine whether G1888A had any modulating effect on core protein translation. The complete core gene with part of the precore of subgenotype A1 was cloned into the amino terminal of a green fluorescent protein (GFP) plasmid. Core/GFP fusion protein expression was measured using flow cytometry following transfection of Huh 7 cells. The introduction of uORF resulted in an 18.75% reduction of core gene expression. When the suboptimal Kozak sequence of the 1888 AUG was replaced with an optimal one, this reduction was enhanced (64.84%). By increasing the distance between the stop of the overlapping uORF and the core AUG, by a minimum of 15 nucleotides, core/GFP expression was almost doubled, indicating that stalling of ribosomes at the stop of the uORF may be interfering with initiation at the core AUG through steric hindrance. Our findings indicate that the G1888A mutation, may interfere with initiation at the downstream 1901 core AUG, decreasing core protein translation. This decrease may account for the relatively low viral loads seen in individuals infected with subgenotype A1.

Characterization of the pleiotropic effects of the genotype G-specific 36-nucleotide insertion in the context of other hepatitis B virus genotypes.

The pregenomic RNA (pgRNA) of hepatitis B virus (HBV) serves as the messenger for both core and P proteins, with the downstream P gene translated by ribosomal leaky scanning. HBV replication begins with packaging of the pgRNA and P protein into core protein particles, followed by conversion of RNA into DNA. Genotype G has a low replication capacity due to a low pgRNA level. It has a 36-nucleotide (nt) insertion in the 5' end of the core gene, adding 12 residues to the core protein. The insertion is needed to maintain efficient core protein expression and genome replication but causes inefficient virion secretion yet high maturity of virion DNA. In the present study, we confirmed that the 36-nt insertion had similar effects on core protein expression and virion secretion when it was introduced into genotype A and D clones but no impact on virion genome maturity. Surprisingly, the insertion impaired genome replication in both genotypes. Transcomplementation assays suggest that increased efficiency of core protein translation diminishes ribosomal scanning toward the downstream P gene. Indeed, mutating the core gene Kozak sequence restored core protein to lower levels but increased replication of the insertion mutant. Similar mutations impaired replication in genotype G. On the other hand, replacement of the core promoter sequence of genotype G with genotype A sequence increased pgRNA transcription and genome replication, implicating this region in the low replication capacity of genotype G. Why the 36-nt insertion is present in genotype G but absent in other genotypes is discussed.

[Immunization with recombinant HBcAg and ß-amyloid peptide fusion protein promotes clearance of intrahippocampally injected ß-amyloid peptide in rats].

OBJECTIVE: To study the effects of immunization with the fusion protein CAC (a product of prokaryotic expression of recombinant HBcAg and ß-amyloid peptide fusion gene) against the toxicity induced by intrahippocampal injection of aggregated ß-amyloid peptide (Aß) in rats. METHODS: SD rats were immunized intraperitoneally with the fusion protein CAC, and the titer of anti-Aß antibody was evaluated by ELISA. When the titers of the anti-Aß antibody reached 1:3 000, aggregated Aß was injected into the CA1 region of the rat hippocampus. Two weeks after Aß injection, the rats underwent morris water maze test before sacrificed to prepare the brain slices with Congo red and haematoxylin staining. RESULTS: The titer of anti-Aß antibody reached 1:3 000 after 5 immunizations with the fusion protein. After Aß injection, the saline-immunized rats showed a reduced cognitive behavior in the Morris water maze test compared to the CAC-immunized rats. In the saline-immunized rats, the neurons around the site of Aß injection exhibited obvious cell damages with Aß deposits and glial infiltration, whereas in CAC-immunized rats, Aß deposits were significantly reduced or even absent. CONCLUSION: Immunization with the fusion protein CAC can inhibit the toxicity induced by intrahippocampal aggregated Aß injection.

Relation of hepatitis B core antigen expression with histological activity, serum HBeAg, and HBV DNA levels.

BACKGROUND: Hepatitis B virus (HBV) is a potentially life-threatening liver infection which may progress to liver failure and cirrhosis. Intrahepatic expression patterns of viral antigens detected by immunohistochemistry may have prognostic implications in disease process. AIM: In this study, we aimed to investigate the relationship between the HBV core antigen (HBcAg) expression and histological activity index (HAI), fibrosis, serum hepatitis B e-antigen (HBeAg) status and HBV DNA levels in patients with chronic HBV infection. MATERIALS AND METHODS: A total of 114 liver biopsies from patients with chronic HBV infection were included in the study. Immunohistochemical expression of HBcAg and its relation with HAI, fibrosis, serum alanine aminotransferase (ALT) levels, HBeAg status and HBV DNA levels were assessed. RESULTS: The presence of nuclear expression of HBcAg did not show any correlations with ALT levels, HAI and fibrosis score. When the groups were categorized according to the HBeAg status, nuclear HBcAg expression was found to be high in HBeAg positive patients. However, HBcAg nuclear expression showed significant correlations with HBV DNA levels and fibrosis scores in HBeAg negative but not HBeAg positive patients. HBV DNA levels were also significantly associated with HAI and fibrosis scores in HBeAg negative patients. CONCLUSIONS: Significant differences found between HBeAg positive and negative patients suggest that HBeAg negative disease is different from HBeAg positive disease, and also point outs that in HBeAg negative disease, patients with nuclear HBcAg expression and increased levels of HBV DNA levels are at a higher risk of developing progressive liver disease.

Development of cell cultures that express hepatitis B virus to high levels and accumulate cccDNA.

Establishment of an infection with hepatitis B virus (HBV) requires synthesis and maintenance of a covalently closed circular DNA (cccDNA) form of the viral genome in the nucleus of host cells. To facilitate the investigation of the synthesis of cccDNA, cell cultures were developed that express HBV to high levels. Cell lines derived from hepatoma cells Huh7 and HepG2 were created that express Epstein-Barr virus (EBV) nuclear antigen-1 and a fusion protein of the Tet repressor and Kox1 transcriptional repression domain stably. Transfection of these cell lines with an expression plasmid for HBV that contains the origin of plasmid replication of EBV (oriP) led to increases in the intracellular levels of HBV core protein ( approximately 8- to 51-fold) and encapsidated HBV DNA ( approximately 3- to 12-fold) in comparison to Huh7 and HepG2 cells. Virion production was also increased ( approximately 3- to 12-fold) in these cell cultures and an increase in the level of cccDNA ( approximately 3-fold) was observed in the Huh7-derived cell lines. In addition, these cell lines maintained the HBV expression plasmid upon selection and expressed HBV conditionally. Thus, these cell cultures exhibit several features that facilitate study of the synthesis of cccDNA and other aspects of replication of HBV.

[Expression of hepatitis B virus core antigen gene region in yeast cell]. / Hepatit B virusu kor antijeni gen bölgesinin mayada ekspresyonu.

In this study, the core antigen (HBcAg) gene region of hepatitis B virus (HBV) was transformed and expressed into an eukaryotic expression vector by recombinant DNA technology in order to obtain the protein used in anti-HBc tests which is being one of the most important marker for the serodiagnosis of HBV infections. For this purpose, HBV-DNA positive patient sera were used as the source of viral nucleic acids, and the primers coding HBcAg gene region have been designed. After the amplification of HBcAg gene region by polymerase chain reaction (PCR), the amplicons purified by Invisorb Spin Rapid PCR Kit" (Invitek, Germany), were cloned to pYES2.1 plasmid via the TOPO TA expression kit (Invitrogen, USA) and this plasmid was transformed to competent bacteria (TOPO 10F' Escherichia coli) by CaCl2 method. After competent bacteria were grown on LB (Lysogeny Broth) agar media supplemented with ampicillin, the plasmid "pYES2.1 + HBcAg" were isolated and transformed to Saccaromyces cerevisiae via the "S.c. EasyComp Transformation Kit" (Invitrogen, USA). Finally, the expression of HBcAg by the yeast was confirmed with the use of in house ELISA method. Since the diagnostic kits used in our country for hepatitis B serology are usually imported products, this creates a great economical burden. Thus, the experience and knowledge that builds up following such studies will help to produce our own diagnostic products using our equity.

Exploring different strategies to express Dengue virus envelope protein in a plant system.

Dengue virus envelope glycoprotein (E-protein) is the main protein associated with immunity induction. To produce a candidate for subunit vaccines and to provide an antigen for diagnostic kits, it was expressed in a novel plant system using deconstructed viral modules. A truncated version of the E-protein was designed to be expressed alone and co-expressed with Dengue virus structural proteins. As well, the critical domain III of E-protein was fused to hepatitis B core antigen (HBcore). The recombinant proteins were produced in Nicotiana benthamiana plants and were reactive with the anti-E antibody. The fusion was reactive with both anti-E and anti-HBcore antibodies.

Immunopathology on hepatocyte expression of HBV surface, core, and x antigens in chronic hepatitis B: clinical and virological correlation.

PURPOSE: Hepatocyte expression of HBV surface, core, and x antigens (HBsAg, HBcAg and HBxAg), semi-quantitated by immunopathology, were correlated with clinical and virological data in 80 patients with chronic hepatitis B. RESULTS: Seventy patients were HBsAg positive in cytoplasm, 61 were HBcAg positive, including 45 in both nucleus and cytoplasm and 16 in cytoplasm only, and 47 were HBxAg positive in cytoplasm. The detection rates for HBcAg increased while those for HBsAg and HBxAg decreased with HBV DNA levels. Positive HBcAg staining usually suggested the presence of HBV DNA levels >10(6) copies/ml. HBcAg, HBsAg, and HBxAg expressions showed no significant differences between patients with genotype B and C. Serum HBeAg and HBV DNA levels correlated positively with nuclear or cytoplasmic HBcAg expression but inversely with HBsAg expression. By multiple regression analysis, HBV DNA levels correlated significantly only with nuclear HBcAg expression. ALT levels and inflammatory grades correlated with cytoplasmic HBcAg expression. There was an inverse quantitative relationship between HBcAg and HBsAg expression. Furthermore, HBxAg expression correlated significantly with HBsAg expression as well as male gender. CONCLUSIONS: With diminishing HBV DNA levels following HBeAg seroconversion, HBcAg expression decreased but HBsAg expression increased with a concomitant increase in HBxAg expression. Whether the finding that a significantly higher expression of HBxAg observed in males than females may account for the gender difference in long-term sequelae of chronic HBV infection needs further investigation.

[Effect of HBc-antigen expression on replication of recombinant Salmonella strains in vivo and in vitro].

The development of therapeutic vaccines against chronic hepatitis B is a major challenge facing modern medicine. HBc antigen-expressing DNA-vaccines designed to be transported into the organism by attenuated Salmonella appear to be good candidates for the purpose. Expression of HBc-antigen was earlier shown to lead to the loss of ability of attenuated Salmonella bearing the DNA-vaccine to multiply in the host lymphoid tissue of mice after per rectum immunization and in human monocyte culture. The aim of the study was to assess the influence of HBc-antigen expression on replication of a virulent strain of wild-type Salmonella serotype Enteritidis in the murine lymphoid tissue after per rectum infection and in human blood monocytes. HBc expression was shown to inhibit propagation of Salmonella in both model systems despite its high virulence.