Mucosal immunogenicity of the hepatitis B core antigen

The hepatitis B virus (HBV) core antigen (HBcAg) is a potent immunogen in animal models and humans and has been used as a carrier for several antigens, however, the mucosal immunogenicity of HBcAg or chimeric HBcAg proteins has been poorly studied and only using the truncated variant of the HBcAg. In this study we explored the mucosal immunogenicity in mice of the recombinant complete nucleocapside of HBcAg. The antigen was administered by different mucosal and parenteral routes. The antibody response in sera was evaluated after each immunization and mucosal lavages were tested with the final extraction. To characterize the immune response, the serum IgG antibody response was tested during six months and also the ratio IgG2a to IgG1 was determined. The results obtained evidenced that the mucosal immunogenicity of HBcAg depended on the administration route, being the intranasal (i.n.) route the one that generated the higher IgG responses in sera, similar in intensity and duration to parenteral administrations. The IgA response in mucosal washes was superior for nasally immunized mice compared to the rest of mucosal and parenteral groups. The nasal route also induced the higher IgG2a to IgG1 ratio, evidencing a Th1-like Ab subclass pattern. In addition to the high Ab responses, preliminary results of the cellular response induced by nasal administration evidenced the induction of strong lymphoproliferative responses in spleen cells.     

In vitro production of antibody to hepatitis B core and E antigens by peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection

To evaluate the relative immunogenicity of and the mechanism for production of antibody to hepatitis B core (HBc) and hepatitis B e (HBe) Ag, we investigated the in vitro anti-HBc and anti-HBe production by PBMC from 25 patients with chronic active hepatitis (CAH) (15 with HBeAg and 10 with anti-HBe) and 12 ASC (5 with HBeAg and 7 with anti-HBe) in the presence of PWM, rHBcAg, or purified HBeAg. PWM-stimulated culture produced higher titer anti-HBc (mean % inhibition +/- SD = 73 +/- 23%, p less than 0.001) than anti-HBe (34 +/- 17%). HBcAg stimulation elicited greater anti-HBc response (43 +/- 26%, p less than 0.001) than did HBeAg for anti-HBe (26 +/- 12%). Both HBcAg and HBeAg induced equivalent anti-HBe response. Anti-HBc production in response to HBcAg was higher in CAH patients (51 to 55%) than in asymptomatic carriers of hepatitis B surface Ag (22 to 28%) irrespective of their HBeAg/anti-HBe status, but reflecting serum anti-HBc value. Similar findings were noted in HBeAg-stimulated anti-HBe production for the two patient groups. In HBeAg- and anti-HBe-positive CAH, HBcAG-stimulated anti-HBc production was similar in T (1.4 x 10(6)) and B (0.6 x 10(6)) cells coculture, and B cells (2 x 10(6)) alone culture. However, in the HBeAg-stimulated culture, T plus B cells produced significantly higher titer anti-HBe than B cells alone did. These results indicate that HBcAg has a relatively higher immunogenicity in terms of antibody production as compared to HBeAg. Furthermore, HBcAg was shown to function as a T cell-dependent and -independent Ag, whereas HBeAg is T cell-dependent during chronic hepatitis virus B infection in man.     

Multivalent DNA-based immunization against hepatitis B virus with plasmids encoding surface and core antigens

The immune response against hepatitis B surface and core antigens was evaluated by either coinoculation or independent intramuscular administration of pAEC compact DNA immunization vectors carrying their genes. The pAEC vectors bear just the essential elements for mammalian expression and bacterial amplification. Balb/c mice were immunized with 100 microg of each construct, either alone or in combination. In spite of lacking known immunostimulatory sequences (e.g., AACGTT), significant cellular (proliferative) and humoral immune responses were raised against both antigens. Coadministration of both plasmids maintained the immune response against the two antigens, without interference between them. Modulation of the antigen expression and further immune response, by using the Kozak's translation initiation sequence, was also analyzed. No differences due to its presence or absence were observed.     

The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and l-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed inE. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCI density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBcAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B144C191. Using those fusion proteins, ELISA for screening of antibodies against both HBV and HCV in human sera was also established.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d     

Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences.

Amphotropic retroviral expression systems were used to synthesize hepatitis B virus surface antigen (HBsAg) and core antigen. The vectors permitted establishment of cell lines which expressed antigen from either the retroviral long terminal repeat or the mouse metallothionein-I promoter. HBsAgs were synthesized containing no pre-S sequences, pre-S(2) sequences alone, or pre-S(1) plus pre-S(2) sequences. Inclusion of pre-S(2) sequences did not affect the secretion or density of HBsAg particles but did reduce their mass by approximately 30%. Addition of pre-S(1) sequences almost completely abolished secretion of HBsAg and resulted in its localization in an aqueous-nonextractable pre- or early-Golgi cellular compartment. HBsAg was localized to the cytoplasm of the cell. This localization was unaffected by the presence of pre-S sequences in the antigen. Cell lines synthesizing hepatitis B antigens from core DNA fragments, containing or not containing precore sequences, secreted hepatitis B e antigen. However, the absence of precore DNA sequences resulted in additional synthesis of hepatitis core antigen, which was predominantly nuclear in localization.     

Inhibition of hepatitis B virus assembly with synthetic peptides derived from the viral surface and core antigens

The long surface antigen (L-HBsAg) of hepatitis B virus (HBV) plays a central role in the production of infectious virions. During HBV morphogenesis, both the PreS and S domains of L-HBsAg form docking sites for the viral nucleocapsids. Thus, a compound that disrupts the interaction between the L-HBsAg and nucleocapsids could serve as a therapeutic agent against the virus based upon inhibition of morphogenesis. Synthetic peptides correspond to the binding sites in L-HBsAg inhibited the association of L-HBsAg with core antigen (HBcAg). A synthetic peptide carrying the epitope for a monoclonal antibody to the PreS1 domain competed weakly with L-HBsAg for HBcAg, but peptides corresponding to a linear sequence at the tip of the nucleocapsid spike did not, showing that the competing peptide does not resemble the tip of the spike.     

Variability and conservation in hepatitis B virus core protein

Background  

Hepatitis B core protein (HBVc) has been extensively studied from both a structural and immunological point of view, but the evolutionary forces driving sequence variation within core are incompletely understood.     

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.     

Phosphorylation of hepatitis B virus precore and core proteins.

Hepatitis B virus precore and core proteins are related. The precore protein contains the entire sequence of the core protein plus an amino-terminal extension of 29 amino acids. The amino-terminal extension of the precore protein contains a signal sequence for the secretion of the precore protein. This signal sequence is removed after the translocation of the precore protein across the endoplasmic reticulum membrane to produce the precore protein derivative named P22. We demonstrate that both P22 and the core protein can be phosphorylated in cells. Microsomal fractionation and trypsin digestion experiments demonstrate that a fraction of phosphorylated P22 is located in the endoplasmic reticulum lumen. Phosphorylation of P22 likely occurs in the carboxy terminus, since the P22 derivative P16, which lacks the carboxy terminus of P22, is not phosphorylated. Linking the carboxy terminus of the precore-core protein to heterologous secretory and cytosolic proteins led to the phosphorylation of the resulting chimeric proteins. These results indicate that phosphorylation of P22 and the core protein is likely mediated by cellular kinases.     

Novel chitosan derivative nanoparticles enhance the immunogenicity of a DNA vaccine encoding hepatitis B virus core antigen in mice

BACKGROUND: Chitosan has been shown to possess useful properties such as non-toxicity, high biocompatibility and non-antigenicity that offer advantages for vaccine delivery systems. In this study, we prepared novel chitosan derivative nanoparticles as DNA vaccine carriers and the potential and mechanism of the DNA-nanoparticle complexes in inducing augmented immune responses were explored. METHODS: The pVAX(HBc)DNA-nanoparticle complexes as vaccine delivery systems were studied in several aspects: the protection against DNase I degradation was measured by an in vitro inhibition assay; the sustained expression of the plasmid in vivo was determined by RT-PCR; the elevated uptake efficiency by phagocytes was observed with confocal microscopy; the biocompatibility was evaluated by cytotoxicity and histology assay; the complexes were administrated to C57BL/6 mice and the humoral and cellular immune responses were evaluated by ELISA, IFN-gamma production and cytolytic T lymphocyte (CTL)-specific lysis assay. RESULTS: The remaining relative activity of DNase I after inhibition varied from 32.3% to 77.6%. The complexes were observed with higher uptake efficiency by phagocytes than naked DNA. Three types of nanoparticles did not induce significant cytotoxicity at concentrations相似文献   

Comparative antigenicity and immunogenicity of hepadnavirus core proteins

The hepatitis B virus core protein (HBcAg) is a uniquely immunogenic particulate antigen and as such has been used as a vaccine carrier platform. The use of other hepadnavirus core proteins as vaccine carriers has not been explored. To determine whether the rodent hepadnavirus core proteins derived from the woodchuck (WHcAg), ground squirrel (GScAg), and arctic squirrel (AScAg) viruses possess immunogen characteristics similar to those of HBcAg, comparative antigenicity and immunogenicity studies were performed. The results indicate that (i) the rodent core proteins are equal in immunogenicity to or more immunogenic than HBcAg at the B-cell and T-cell levels; (ii) major histocompatibility complex (MHC) genes influence the immune response to the rodent core proteins (however, nonresponder haplotypes were not identified); (iii) WHcAg can behave as a T-cell-independent antigen in athymic mice; (iv) the rodent core proteins are not significantly cross-reactive with the HBcAg at the antibody level (however, the nonparticulate "eAgs" do appear to be cross-reactive); (v) the rodent core proteins are only partially cross-reactive with HBcAg at the CD4+ T-cell level, depending on MHC haplotype; and (vi) the rodent core proteins are competent to function as vaccine carrier platforms for heterologous, B-cell epitopes. These results have implications for the selection of an optimal hepadnavirus core protein for vaccine design, especially in view of the "preexisting" immunity problem that is inherent in the use of HBcAg for human vaccine development.     

Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection

The proliferative response of PBMC to hepatitis B virus (HBV) envelope, core, and e Ag was analyzed prospectively in 21 patients with acute self-limited HBV infection and compared with the response of patients with chronic HBV infection and different levels of HBV replication (i.e., hepatitis e Ag (HBeAg)- or anti-HBe-positive) and liver damage (i.e., chronic active hepatitis or chronic asymptomatic carriers). Our results indicate that: 1) HBV-infected subjects who develop a self-limited acute hepatitis show a vigorous PBMC response to hepatitis B core Ag and HBeAg, as expression of T cell activation; 2) appearance of a detectable lymphocyte response to HBV nucleocapsid Ag is temporally associated with the clearance of HBV envelope Ag; 3) in patients with chronic HBV infection the level of T cell responsiveness to hepatitis B core Ag and to HBeAg is significantly lower than that observed during acute infection; 4) T cell sensitization to HBV envelope Ag in acute and chronic HBV infection is usually undetectable and when measurable is expressed transiently and at low levels. These results may reflect immune events of pathogenetic relevance with respect to evolution of disease and viral clearance.     

Structural relationships between the surface antigens of ground squirrel hepatitis virus and human hepatitis B virus.

Several physical, chemical, and serological properties of surface antigen particles from ground squirrel hepatitis virus (GSHsAg) and human hepatitis B virus (HBsAg) were compared. GSHsAg and HBsAg particles were purified from positive sera by gel chromatography and isopycnic centrifugation. Both antigens consisted mainly of spherical particles with an average diameter of approximately 20 nm and a buoyant density in CsCl of approximately 1.19 g/ml. Their UV absorption spectra indicated the presence of more tryptophane than tyrosine and the absence of detectable nucleic acid. GSHsAg was found to contain two major polypeptides of approximately 23,000 and 27,000 daltons, with electrophoretic migration rates distinctly faster than those of the two major polypeptides of HBsAg particles. After radiolabeling of purified antigen preparations with Bolton-Hunter reagent, the two major polypeptides of GSHsAg showed almost identical tryptic peptide maps. The tryptic peptide map of the major polypeptide from GSHsAg contained 13 of 37 spots also present in the map of the major HBsAg polypeptide, and 13 of 27 spots in the map of the major HBsAg polypeptide were also present in the map of the major GSHsAg polypeptide. This suggests considerable sequence homology between the major surface antigen polypeptides of the two viruses. However, there was only a weak serological cross-reactivity between antigens of the two viruses. Using an anti-HBs-containing serum with a relatively strong cross-reactivity, GSHsAg was found to consist of at least two antigenically different subspecies. The more strongly cross-reacting from had a slightly higher buoyant density than the other antigenic form.     

Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein

We have demonstrated previously that the core protein of hepatitis C virus (HCV) exhibits suppression activity on gene expression and replication of hepatitis B virus (HBV). Here we further elucidated the suppression mechanism of HCV core protein. We demonstrated that HCV core protein retained the inhibitory effect on HBV gene expression and replication when expressed as part of the full length of HCV polyprotein. Based on the substitution mutational analysis, our results suggested that mutation introduced into the bipartite nuclear localization signal of the HCV core protein resulted in the cytoplasmic localization of core protein but did not affect its suppression ability on HBV gene expression. Mutational studies also indicated that almost all dibasic residue mutations within the N-terminal 101-amino acid segment of the HCV core protein (except Arg(39)-Arg(40)) impaired the suppression activity on HBV replication but not HBV gene expression. The integrity of Arg residues at positions 101, 113, 114, and 115 was found to be essential for both suppressive effects, whereas the Arg residue at position 104 was important only in the suppression of HBV gene expression. Moreover, our results indicated that the suppression on HBV gene expression was mediated through the direct interaction of HCV core protein with the trans-activator HBx protein, whereas the suppression of HBV replication involved the complex formation between HBV polymerase (pol) and the HCV core protein, resulting in the structural incompetence for the HBV pol to bind the package signal and consequently abolished the formation of the HBV virion. Altogether, this study suggests that these two suppression effects on HBV elicited by the HCV core protein likely depend on different structural context but not on nuclear localization of the core protein, and the two effects can be decoupled as revealed by its differential targets (HBx or HBV pol) on these two processes of the HBV life cycle.     

Antigenic and structural relationships of the surface antigens of hepatitis B virus, ground squirrel hepatitis virus, and woodchuck hepatitis virus.

The surface antigens of human hepatitis B (HBsAg), ground squirrel hepatitis (GSHsAg), and woodchuck hepatitis (WHsAg) viruses were compared serologically, and their major polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping. Results showed that both GSHsAg and WHsAg are antigenically cross-reactive, that their major pairs of polypeptides have identical mobilities on sodium dodecyl sulfate gels, and that the major polypeptides of GSHsAg and WHsAg migrate faster in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than do the corresponding bands of HBsAg. The peptide maps of the major (P-22) surface antigen polypeptides of GSHsAg and WHsAg showed that they shared over half of their spots. Peptide mapping of HBsAg subtypes indicated a close relationship between the major polypeptides (P-24) of adw and adr and a more distal relationship to ayw. Only about 25% of the spots shared by the combined HBsAg subtypes were also found in the peptide maps of GSHsAg and WHsAg, indicating at least some structural homology among the major polypeptides of the human and animal virus surface antigen particles. This is also reflected in the serological cross-reactivity among HBsAg, GSHsAg, and WHsAg. Further, the detection of ground squirrel and woodchuck antigens by Ausria II radioimmunoassay, combined with peptide mapping data indicating the common origin of these viruses, suggests that the common a determinant is shared by each and is restricted to approximately 25% of the sequences in their major polypeptides.     

Immune tolerance split between hepatitis B virus precore and core proteins

The function of the hepatitis B virus (HBV) precore or HBeAg is largely unknown because it is not required for viral assembly, infection, or replication. However, the HBeAg does appear to play a role in viral persistence. It has been suggested that the HBeAg may promote HBV chronicity by functioning as an immunoregulatory protein. As a model of chronic HBeAg exposure and to examine the tolerogenic potential of the HBV precore and core (HBcAg) proteins, HBc/HBeAg-transgenic (Tg) mice crossed with T cell receptor (TCR)-Tg mice expressing receptors for the HBc/HBeAgs (i.e., TCR-antigen double-Tg pairs) were produced. This study revealed three phenotypes of HBe/HBcAg-specific T-cell tolerance: (i) profound T-cell tolerance most likely mediated by clonal deletion, (ii) T-cell clonal ignorance, and (iii) nondeletional T-cell tolerance mediated by clonal anergy and dependent on the structure, location, and concentration of the tolerogen. The secreted HBeAg is significantly more efficient than the intracellular HBcAg at eliciting T-cell tolerance. The split T-cell tolerance between the HBeAg and the HBcAg and the clonal heterogeneity of HBc/HBeAg-specific T-cell tolerance may have significant implications for natural HBV infection and especially for precore-negative chronic hepatitis.