Yongjun Tian

The Amino Acid Residues at Positions 120 to 123 Are Crucial for the Antigenicity of Hepatitis B Surface Antigen

ABSTRACT The major hydrophilic region (MHR) of hepatitis B surface antigen (HBsAg) harbors conformational B-cell epitopes and is the major target of neutralizing antibodies to HBsAg (anti-HBs). Mutant HBsAg (mtHBsAg) with amino acid substitutions such as G145R is known to affect the binding of specific anti-HB antibodies and their detection by conventional diagnostic assays. In the present study, we focused on the role of the amino acid positions 120 to 123, which are around MHR 2 according to the spectrum of recently identified, naturally occurring mtHBsAg. Strikingly, the amino acid substitution K122I abolished the reactivity of HBsAg in all immunoassays tested so far. Also, mtHBsAg G145R could be clearly detected with four different enzyme-linked immunosorbent assays that were based on monoclonal anti-HB antibodies (MAbs) with high affinity. Positive immunofluorescence staining of mtHBsAg K122I was achieved only by polyclonal anti-HBs, while all MAbs tested failed. mtHBsAg T123N showed a low reactivity in immunoassays and appeared to be secretion defective. The amino acid substitution P120T reduced the binding of anti-HBs but did not completely prevent the detection of mtHBsAg by anti-HB MAbs. The testing of naturally occurring mtHBsAg confirmed that the presence of amino acid substitutions within the region of 120 to 123 is strongly associated with impaired detection in immunoassays. In conclusion, MHR 2 is essential for HBsAg antigenicity, a fact that has not been recognized before.

Biological significance of amino acid substitutions in hepatitis B surface antigen (HBsAg) for glycosylation, secretion, antigenicity and immunogenicity of HBsAg and hepatitis B virus replication.

Amino acid substitutions of hepatitis B surface antigen (HBsAg) may affect the antigenicity and immunogenicity of HBsAg, leading to immune escape and diagnostic failure. The amino acid positions 122 and 160 are known as determinants for HBsAg subtypes d/y and w/r, respectively. The substitution K122I has been shown to strongly affect HBsAg antigenicity. In this study, we investigated the significance of naturally occurring amino acid substitutions K122I, T123N, A159G and K160N. Both T123N and K160N substitutions resulted in additional N-glycosylated forms of HBsAg, while the other mutations produced more glycosylated HBsAg compared with the wild type (wt). Detection of HBsAg by ELISA and immunofluorescence staining indicated that variant HBsAg (vtHBsAg) with K122I was not recognized by HBsAg immunoassays, while vtHBsAg with T123N, A159G, K160N and A159G/K160N had reduced antigenicity. DNA immunization in BALB/c mice revealed that wtHBsAg and vtHBsAg with T123N and K160N are able to induce antibodies to HBsAg (anti-HBs), whereas K122I and A159G greatly impair the ability of HBsAg to trigger anti-HBs responses. The cellular immune response to the HBsAg aa 29-38 epitope was enhanced by the K160N substitution. Using replication competent clones of hepatitis B virus (HBV), T123N and A159G substitutions were shown to strongly reduce virion assembly. The amino acid substitution K160N appeared to compensate for the negative effect of A159G on virion production. These results reveal complex effects of amino acid substitutions on biochemical properties of HBsAg, on antigenicity and immunogenicity, and on the replication of HBV.

Inhibition of hepatitis B virus gene expression and replication by endoribonuclease-prepared siRNA.

Abstract Endoribonuclease-prepared siRNA (esiRNA) is an alternative tool to chemical synthetic siRNA for gene silencing. Since esiRNAs are directed against long target sequences, the genetic variations in the target sequences will have little influence on their effectiveness. The ability of esiRNAs to inhibit hepatitis B virus (HBV) gene expression and replication was tested. EsiRNAs targeting the coding region of HBV surface antigen (HBsAg) and the nucleocapsid (HBcAg) inhibited specifically the expression of HBsAg and HBcAg when cotransfected with the respective expression plasmids. Both esiRNAs reduced the HBV transcripts and replication intermediates in transient transfected cells and cells with HBV genomes integrated stably. Compared with synthetic siRNA, esiRNA targeting HBsAg was less effective than the selected synthetic siRNA in terms of the inhibition of HBV gene expression and replication. However, while the ability of synthetic siRNAs for specific gene silencing was impaired strongly by the nucleotide substitutions within the target sequences. The efficiency of gene silencing by esiRNAs was not influenced by sequence variation. The transfection of esiRNA did not induce interferon-stimulated genes (ISGs) like STAT1 and ISG15, indicating the absence of off-target effects. In general, esiRNAs strongly inhibited HBV gene expression and replication and may have an advantage against HBV strains which are variable genetically.

The expression of PD-1 ligands and their involvement in regulation of T cell functions in acute and chronic woodchuck hepatitis virus infection.

Background The programmed cell death 1 (PD-1)/programmed death-1 ligand 1 (PD-L1) system may play a role in the negative regulation of T cell functions in hepatitis B virus (HBV) infection. Thus, it is important to study its role in the widely used animal model for HBV infection of woodchucks with woodchuck hepatitis virus (WHV). Methods Woodchuck PD-L1 (wPD-L1) and -L2 (wPD-L2) were cloned and characterized. The levels of wPD-L1 expression in primary woodchuck hepatocytes (PWH), peripheral blood mononuclear cells (PBMCs), and liver tissue of naive and WHV-infected woodchucks were examined by real time reverse transcription (RT)-PCR and flow cytometry. Using antibodies against wPD-L1 and -L2, the effect of blocking PD-1/PD-L1/PD-L2 interaction on the proliferation and degranulation of woodchuck PBMCs was examined. Principal Findings Both wPD-L1 and -L2 showed a high homology to their counterparts of other mammalian species and humans. WPD-L1 expression in PWH and PBMCs of naive animals was low but could be stimulated by Toll-like receptor (TLR) ligands and interferons (IFN). WPD-L1 expression in liver tissue was significantly higher than that measured in PWHs and was slightly elevated during acute and chronic WHV infection. However, wPD-1 and wPD-L1 expression on PBMCs was strongly up-regulated during acute and chronic infection. In vitro blockade with antibodies against wPD-L1 and -L2 partially enhanced proliferation and degranulation of PBMCs from WHV-infected woodchucks. Conclusions Our results demonstrated that wPD-1/wPD-L1 expression in hepatocytes and PBMCs can be induced by different inflammatory stimuli and is up-regulated mainly on PBMCs during WHV infection. A blockade of the woodchuck PD-1/PD-L pathway could partially enhance T cell functions in WHV infection.

Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance

Summary.  Seroconversion from hepatitis B surface antigen (HBsAg) to antibodies against HBsAg (anti‐HBs) usually indicates resolution of hepatitis B virus (HBV) infection. Here, two HBV‐infected patients with seroconversion to anti‐HBs were found to be persistently positive for HBeAg and HBV DNA. Immunohistology of liver biopsies confirmed the expression of HBV proteins in the liver of one patient. The neutralizing ability of anti‐HBs in patient sera was demonstrated by blocking HBV infection of primary tupaia hepatocytes. Analysis of the HBsAg‐encoding region of HBV isolates from patients indicated the coexistence of heterogeneous HBV genomes in patients. The majority of recombinant variant HBsAg was reactive in HBsAg assays and was able to bind to anti‐HBs. Circulating immune complexes (CIC) of HBsAg in patient sera could be detected by polyethylene glycol precipitation and trypsin digestion. Thus, neutralizing anti‐HBs may appear in chronic HBV carriers for long periods but does not necessarily lead to complete viral clearance.

Establishing a new animal model for hepadnaviral infection: susceptibility of Chinese Marmota-species to woodchuck hepatitis virus infection

Hepatitis B virus infection (HBV) is a major medical problem in China. The lack of a suitable infection model in China is recognized as an obstacle for research on HBV in China. Chinese Marmota-species is phylogenetically closely related to Marmota monax, thus, it might be suitable to serve as an animal model for HBV infection. Therefore, we attempted to prove the claim about the existence of woodchuck hepatitis virus (WHV)-like viruses in Chinese Marmota-species and to determine the susceptibility of these species to experimental WHV infection. In the present study, 653 sera from three Chinese Marmota-species, Marmota himalayana, Marmota baibacina and Marmota bobak, were screened for WHV-like viruses by serological and molecular assays. The susceptibility to WHV of three species was investigated by experimental infection and monitored by testing of anti-WHc and WHsAg by ELISA, detection of WHV DNA by PCR, and detection of WHV replication intermediates and antigens in liver samples. No evidence for the existence of a genetically closely related virus to WHV in three Chinese Marmota-species was found by serological assays and PCR. M. himalayana was susceptible to WHV infection as inoculated animals became positive for anti-WHc, WHsAg and WHV DNA. Further, WHV replication intermediates and proteins were detected in liver samples. In contrast, M. baibacina remained negative for tested virological parameters. M. bobak species showed a limited susceptibility to WHV. Our data do not support early reports about WHV-like viruses in China. M. himalayana is suitable for the establishment of a model for hepadnaviral infection.

Reduced Antigenicity of Naturally Occurring Hepatitis B Surface Antigen Variants with Substitutions at the Amino Acid Residue 126

Background: Substitutions at amino acid residue 126 of hepatitis B surface antigen (HBsAg) occur frequently in hepatitis B virus (HBV) isolates from patients with chronic HBV infection. These substitutions occur naturally, but their significance for viral persistence is unclear and requires further investigation. Methods: We amplified coding regions of HBsAg by PCR using sera from 1 patient chronically infected with HBV. Three representative clones of HBsAg with amino acid residues 126Ile (I), 126Thr (T) and 126Ser (S) were selected from sequenced clones. HBsAg 126Ala (A) mutants of subtype C/adr and D/adw were generated by site-directed mutagenesis. The HBsAg expression vectors were constructed and transiently transfected into HepG2 cells. The binding reactivity of HBsAg to anti-HBs antibodies was tested by chemiluminescent microparticle immunoassay and by immunofluorescence staining with polyclonal and monoclonal anti-HBs antibodies. Results: Diverse HBsAg variants with substitutions at amino acid residue 126 co-existed in a chronically infected HBV patient. HBsAg with the substitution 126S showed a significantly low antigenicity, while the binding reactivity to anti-HBs of other HBsAg with 126I, 126T and 126A were comparable. Conclusion: HBsAg with the 126S substitution has a reduced antigenicity, which may contribute to immune escape in chronic HBV infection.

HBsAg/HBsAb Double Positive Hepatitis B Virus Infection Model in vitro and in vivo *

The pathogenesis of HBsAg (+)/HBsAb (+) double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.SummaryThe pathogenesis of HBsAg (+)/HBsAb (+) double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.