Designing a therapeutic hepatitis B vaccine to circumvent immune tolerance.

An effective prophylactic hepatitis B virus (HBV) vaccine has long been available but is ineffective for chronic infection. The primary cause of chronic hepatitis B (CHB) and greatest impediment for a therapeutic vaccine is the direct and indirect effects of immune tolerance to HBV antigens. The resulting defective CD4+/CD8+ T cell response, poor cytokine production, insufficient neutralizing antibody (nAb) and poor response to HBsAg vaccination characterize CHB infection. The objective of this study was to develop virus-like-particles (VLPs) that elicit nAb to prevent viral spread and prime CD4+/CD8+ T cells to eradicate intracellular HBV. Eight neutralizing B cell epitopes from the envelope PreS1 region were consolidated onto a species-variant of the HBV core protein, the woodchuck hepatitis core antigen (WHcAg). PreS1-specific B cell epitopes were chosen because of preferential expression on HBV virions. Because WHcAg and HBcAg are not crossreactive at the B cell level and only partially cross-reactive at the CD4+/CD8+ T cell level, CD4+ T cells specific for WHcAg-unique T cell sites can provide cognate T-B cell help for anti-PreS1 Ab production that is not curtailed by immune tolerance. Immunization of immune tolerant HBV transgenic (Tg) mice with PreS1-WHc VLPs elicited levels of high titer anti-PreS1 nAbs equivalent to wildtype mice. Passive transfer of PreS1 nAbs into human-liver chimeric mice prevented acute infection and cleared serum HBV from mice previously infected with HBV in a model of CHB. At the T cell level, PreS1-WHc VLPs and hybrid WHcAg/HBcAg DNA immunogens elicited HBcAg-specific CD4+ Th and CD8+ CTL responses.

The hepatitis C virus and immune evasion: non-structural 3/4A transgenic mice are resistant to lethal tumour necrosis factor alpha mediated liver disease.

BACKGROUND: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. AIM: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. METHODS: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl(4)), lipopolysaccaride (LPS), tumour necrosis factor alpha (TNFalpha), and anti-Fas antibody. RESULTS: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl(4), LPS, TNFalpha, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFalpha, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). CONCLUSIONS: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFalpha induced liver damage in vivo. The TNFalpha resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.

Relation between viral fitness and immune escape within the hepatitis C virus protease.

BACKGROUND: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. AIMS: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073-1081 of the NS3 protease was limited by viral fitness. PATIENTS: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection. METHODS: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. RESULTS: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073-1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073-1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. CONCLUSIONS: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.

Conversion of poorly immunogenic malaria repeat sequences into a highly immunogenic vaccine candidate.

The recent success of a Plasmodium falciparum malaria vaccine consisting of circumsporozoite protein (CSP) T and B cell epitopes has rekindled interest in the development of a pre-erythrocytic vaccine. In order to optimize immunogenicity, well-characterized CSP-specific neutralizing B cell epitopes and a universal T cell epitope were combined with an efficient and flexible particulate carrier platform, the hepatitis B core antigen (HBcAg), to produce a novel pre-erythrocytic vaccine candidate. The vaccine candidate, V12.PF3.1, is a potent immunogen in mice eliciting unprecedented levels (greater than 10(6) titers) of sporozoite-binding antibodies after only two doses. The anti-sporozoite antibodies are long lasting, represent all IgG isotypes, and antibody production is not genetically restricted. CSP-specific CD4+ T cells are also primed by V12.PF3.1 immunization in a majority of murine strains. Furthermore, the hybrid HBcAg-CS particles can be produced inexpensively in bacterial expression systems. These and other characteristics suggest that V12.PF3.1 represents an efficient and economical P. falciparum vaccine candidate for use separately or in combination with other formulations.

Flow cytometric determination of cytokine production and proliferation in hepatitis B core antigen specific murine CD4 cells: lack of correlation between number of cytokine producing cells and cytokine levels in supernatant.

Hepatitis B virus (HBV) core antigen (HBcAg) has extraordinary immunostimulatory properties. The majority of studies done so far on HBcAg induced responses have used ELISA or bioassay for cytokine determination and the 3[H]thymidine incorporation assay to measure proliferation. Here multiparameter flow cytometry was used to measure HBcAg induced cytokine production and proliferation of murine T cells. The advantage with this technique was that we could analyse the cytokine phenotype of proliferating cells of a particular cell type. We found that IL-10 expression was strongly induced in CD4+ T cells after HBcAg immunization. Importantly, we found that IL-4 producing HBcAg-specific CD4+ T cells are common after immunization although detection of IL-4 in culture supernatants indicates only low levels of IL-4. In contrast, IFN-gamma producing HBcAg-specific CD4+ T cells were found at lower numbers despite the detection of high levels of IFN-gamma in culture supernatants. Thus, the frequency of these cells is not accurately reflected by the detectability of the respective cytokine in culture supernatants. A low number of specific CD4+ T cells may effectively produce high levels of cytokine. We therefore suggest that different types of cytokine assays are used in order to obtain the most accurate picture of the intrinsic cytokine phenotype of the CD4+ T cells primed by HBcAg.

Modeling the T-helper cell response in acute and chronic hepatitis B virus infection using T-cell receptor transgenic mice.

Chronicity following hepatitis B virus (HBV) infection may be maintained by high levels of viral proteins circulating in the serum. To examine the characteristics of T cells capable of co-existing with the secreted hepatitis B e-antigen (HBeAg), T-cell receptor (TCR) transgenic (Tg) mice were produced. To insure that HBeAg-specific T cells would not be deleted in the presence of serum HBeAg, the TCR alpha and beta-chain genes used to produce the TCR-Tg mice were derived from T-cell hybridomas from HBeAg-Tg mice. A TCR-Tg lineage (11/4-12) was produced that possessed a high frequency (approximately 67%) of CD4(+) T cells that expressed a TCR-Tg specific for the HBeAg. As predicted, when 11/4-12 TCR-Tg mice were bred with HBeAg-Tg mice no deletion of the HBeAg-specific CD4(+) T cells occurred in the thymus or the spleen. Functional analysis of the TCR-Tg T cells revealed that the HBeAg-specific CD4(+) T cells escaped deletion in the thymus and periphery by virtue of low avidity. Regardless of their low avidity, HBeAg-specific TCR-Tg T cells could be activated by exogenous HBeAg as measured by cytokine production in vitro and T-helper cell function for anti-HBe antibody production in vitro and in vivo. Furthermore, activated TCR-Tg HBeAg-specific T cells polarized to the Th(1) subset were able to elicit liver injury when transferred into HBeAg or HBcAg-Tg recipients. Therefore, HBeAg-specific CD4(+) T cells that can survive deletion or anergy in the presence of circulating HBeAg nonetheless are capable of being activated and of mediating liver injury in vivo.

Hepatitis B virus core antigen binds and activates naive human B cells in vivo: studies with a human PBL-NOD/SCID mouse model.

The hepatitis B virus (HBV) core (HBc) antigen (HBcAg) is a highly immunogenic subviral particle. Studies with mice have shown that HBcAg can bind and activate B cells in a T-cell-independent fashion. By using a human peripheral blood leukocyte (hu-PBL)-Nod/LtSz-Prkdc(scid)/Prkdc(scid) (NOD/SCID) mouse model, we show here that HBcAg also activates human B cells in vivo in a T-cell-independent way. HBcAg was capable of inducing the secretion of HBcAg-binding human immunoglobulin M (IgM) in naive human B cells derived from adult human and neonatal (cord blood) donors when these hu-PBL were transferred directly into the spleens of optimally conditioned NOD/SCID mice. No such responses were found in chimeric mice that were given hu-PBL plus HBV e antigen or hu-PBL plus phosphate-buffered saline. In addition, HBcAg activated purified human B cells to produce anti-HBc IgM in the chimeric mice, thus providing evidence that HBcAg behaves as a T-cell-independent antigen in humans. However, HBcAg-activated hu-PBL from naive donors were unable to switch from IgM to IgG production, even after a booster dose of HBcAg. Production of HBcAg-specific IgG could only be induced when hu-PBL from subjects who had recovered from or had an ongoing chronic HBV infection were transferred into NOD/SCID mice. Our data suggest that humans also have a population of naive B cells that can bind HBcAg and is subsequently activated to produce HBcAg-binding IgM.

Molecular basis for the interaction of the hepatitis B virus core antigen with the surface immunoglobulin receptor on naive B cells.

The nucleocapsid of the hepatitis B virus (HBV) is composed of 180 to 240 copies of the HBV core (HBc) protein. HBc antigen (HBcAg) capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors. The molecular basis for the interaction between HBcAg and naive B cells is not known. The functionality of this activation was evidenced in that low concentrations of HBcAg, but not the nonparticulate homologue HBV envelope antigen (HBeAg), could prime naive B cells to produce anti-HBc in vitro with splenocytes from HBcAg- and HBeAg-specific T-cell receptor transgenic mice. The frequency of these HBcAg-binding B cells was estimated by both hybridoma techniques and flow cytometry (B7-2 induction and direct HBcAg binding) to be approximately 4 to 8% of the B cells in a naive spleen. Cloning and sequence analysis of the immunoglobulin heavy- and light-chain variable (VH and VL) domains of seven primary HBcAg-binding hybridomas revealed that six (86%) were related to the murine and human VH1 germ line gene families and one was related to the murine VH3 family. By using synthetic peptides spanning three VH1 sequences, one VH3 sequence, and one VLkappaV sequence, a linear motif in the framework region 1 (FR1)complementarity-determining region 1 (CDR1) junction of the VH1 sequence was identified that bound HBcAg. Interestingly, the HBcAg-binding motif was present in the VL domain of the HBcAg-binding VH3-encoded antibody. Finally, two monoclonal antibodies containing linear HBcAg-binding motifs blocked HBcAg presentation by purified naive B cells to purified HBcAg-primed CD4(+) T cells. Thus, the ability of HBcAg to bind and activate a high frequency of naive B cells seems to be mediated through a linear motif present in the FR1-CDR1 junction of the heavy or light chain of the B-cell surface receptor.

Transgenic technology and the study of hepatitis viruses: a review of what we have learned.

Because of the absence of inbred animal models susceptible to infection by the hepatitis B (HBV), C (HCV) and delta (HDV) viruses, and the inability to culture these viruses, a number of investigators have produced transgenic (Tg) mice that express one or all the viral genes. This review attempts to catalogue and characterize the Tg mice produced to date. The topics addressed are HBV, HCV and HDV gene expression and regulation; HBV replication models and factors that inhibit replication; HBV pathogenesis models; HBV tolerance and persistence models; modulation of the immune response to HBV proteins in Tg mice; T cell receptor Tg mice; and models of hepatocellular carcinoma.

Emergence of the precore mutant late in chronic hepatitis B infection correlates with the severity of liver injury and mutations in the core region.

OBJECTIVE: The reason that precore negative mutants (e-minus DNA) gradually become predominant in some patients during chronic hepatitis B virus infection is not clear. Theoretically, as long as both e-plus and e-minus DNA share the same epitopes in the core region, HBcAg-specific cytotoxic T lymphocytes (CTLs) cannot distinguish between the target peptides expressed by e-plus and e-minus DNA. Therefore, e-minus DNA may be accompanied by additional mutations in the core region, which may affect cytotoxic T lymphocyte recognition. To examine this possibility, the sequences of the precore and the entire core region of the hepatitis B virus genome were analyzed from paired serum samples in CH-B patients who experienced HBeAg to anti-HBe seroconversion (SC). METHODS: Patients were divided into two groups. Group A patients (n = 17) genome-converted to e-minus DNA in the precore region, which abolished HBeAg secretion within 3-4 yr after SC. Group B patients (n = 16) retained precore wild-type DNA for more than 3-4 yr after SC. To investigate the impact of the emergence of precore mutant type DNA on liver injury, alanine aminotransferase (ALT) levels were also examined. RESULTS: ALT flares were more severe among patients in group A than in group B. The average mean ALT level during the HBeAg negative phase of chronic infection was 54 +/- 38 in group A and 28 +/- 24 in group B. The average maximal ALT level during the HBeAg negative phase was 235 +/- 249 in group A and 83 +/- 106 in group B. Furthermore, all 17 patients in group A developed new core mutants during genome conversion. The average number of mutations in the core gene was 0.9 +/- 1.2 before genome conversion (e-plus DNA dominant phase) and increased to 2.8 +/- 1.3 for the 3-4 yr during genome conversion (e-minus DNA dominant phase). In contrast, only 56% (nine of 16) of patients in group B developed new core mutations after the loss of HBeAg. The average number of mutations in the core gene was 1.8 +/- 1.3 before SC (HBeAg-positive and e-plus DNA dominant phase), and decreased to 1.1 +/- 1.1 for 3-4 yr after seroconversion (anti-HBe-positive and e-plus DNA dominant phase). CONCLUSIONS: These data indicate that the emergence of a predominant precore negative genotype late in chronic hepatitis B virus infection is associated with the selection of additional mutations in the core gene, as well as with liver injury.

Nondeletional T-cell receptor transgenic mice: model for the CD4(+) T-cell repertoire in chronic hepatitis B virus infection.

Chronicity after infection with the hepatitis B virus (HBV) can occur for a variety of reasons. However, once established, chronicity may be maintained by high levels of viral proteins circulating in the serum. To examine the characteristics of T cells capable of coexisting with the secreted hepatitis B e antigen (HBeAg), T-cell receptor (TCR) transgenic (Tg) mice were produced. To ensure that HBeAg-specific T cells would not be deleted in the presence of serum HBeAg, the TCR alpha- and beta-chain genes used to produce the TCR-Tg mice were derived from T-cell hybridomas produced from immunizing HBeAg-Tg mice. A TCR-Tg lineage (11/4-12) was produced that possessed a high frequency ( approximately 67%) of CD4(+) T cells that expressed a Tg TCR specific for the HBeAg. As predicted, when 11/4-12 TCR-Tg mice were bred with HBeAg-Tg mice no deletion of the HBeAg-specific CD4(+) T cells occurred in the thymus or the spleen. Functional analysis of the TCR-Tg T cells revealed that the HBeAg-specific CD4(+) T cells escaped deletion in the thymus and periphery by virtue of low avidity. Regardless of their low avidity, HBeAg-specific TCR-Tg T cells could be activated by exogenous HBeAg, as measured by cytokine production in vitro and T-helper-cell function for anti-HBe antibody production in vitro and in vivo. Furthermore, activated TCR-Tg HBeAg-specific T cells polarized to the Th1 subset were able to elicit liver injury when transferred into HBeAg or HBcAg-Tg recipients. Therefore, HBeAg-specific CD4(+) T cells that can survive deletion or anergy in the presence of circulating HBeAg nonetheless are capable of being activated and of mediating liver injury in vivo. The 11/4-12 TCR-Tg lineage may serve as a monoclonal model for the HBe/HBcAg-specific CD4(+) T-cell repertoire present in chronically infected HBV patients.

Limited humoral immunity in hepatitis C virus infection.

BACKGROUND & AIMS: The extremely high rate of chronicity to hepatitis C virus (HVC) infection suggests an inefficient immune response. The humoral immune response to HCV was evaluated in 60 patients with chronic HCV infection and in 12 patients acutely infected with HCV. METHODS: A number of recombinant HCV antigens including the core, envelope 2 (E2), nonstructural (NS) 3, NS4, and NS5 proteins, and NS4a and E2-HVR-1 peptides were used in enzyme-linked immunoassays. RESULTS: Immunoglobulin (Ig) G antibody responses to these viral antigens, except for the HCV core, were highly restricted to the IgG1 isotype. The prevalence of antibodies of the IgG1 isotype specific for the HCV core, E2, E2-HVR1, NS3 (helicase domain), NS4, and NS5 antigens was 97%, 98%, 28%, 88%, 33%, and 68%, respectively. Antibodies of the IgG3 isotype specific for E2, E2-HVR-1, NS3, NS4, and NS5 were detected in a minority of serum samples. The IgG2 and IgG4 isotypes were rarely if ever detected. Furthermore, antibody responses to HCV viral antigens were of relatively low titer and, with the exception of anti-HCV core, were delayed in appearance until the chronic phase of infection. CONCLUSIONS: The IgG1 restriction, low titer, and delayed appearance of antibody responses elicited during HCV infection suggest that the immunogenicity of HCV proteins is limited in the context of natural infection. Inasmuch as recombinant HCV viral antigens perform as relatively normal immunogens in small animals, we suggest that the defective humoral immune responses during HCV infection may be attributable to an "immune avoidance" strategy.

The antiviral compound ribavirin modulates the T helper (Th) 1/Th2 subset balance in hepatitis B and C virus-specific immune responses.

Ribavirin is effective in combination therapies against chronic hepatitis C virus (HCV) infection, although its direct antiviral properties are unclear. We therefore studied the immune-modulatory effects of ribavirin on hepatitis B virus (HBV)- and HCV-specific immune responses. During a 24 week placebo-controlled ribavirin trial in ten patients with chronic HCV infection, HCV antibodies and alanine aminotransferase (ALT) levels decreased transiently whereas the serum levels of HCV RNA remained stable. Effects of ribavirin on human and murine phytohaemagglutinin (PHA)-activated T cells included inhibition of in vitro proliferation and modulation of IL-2, IL-4, IFN-gamma and TNF-alpha levels. HBcAg- and HBeAg-specific IL-2 and IFN-gamma levels were > or = 25-fold higher in mice immunized with HBV core- or e-antigens (HBcAg, HBeAg) while receiving ribavirin compared to untreated mice, but IL-4 and IL-6 remained constant. Concordantly, a slight shift was observed in the IgG subclass distribution of the humoral responses of ribavirin-treated mice to HBeAg and HCV NS3 protein. Ribavirin treatment of HBeAg-transgenic (HBeAg-Tg) mice induced a dose-dependent down-regulation of T helper (Th)2-mediated antibody production to HBeAg. In ribavirin-treated HBeAg-Tg mice anti-HBe IgG1 (positively regulated by Th2 cytokines) decreased simultaneously as both anti-HBe IgG2a (positively regulated by Th1 cytokines) levels and in vitro T-cell IFN-gamma production increased, indicating a change in the Th1/Th2 balance. Thus, the present data suggest that ribavirin is not strictly an antiviral compound, but rather it alters the T-cell balance in the immune system.

Human and murine antibody recognition is focused on the ATPase/helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein.

The hepatitis C virus (HCV) nonstructural (NS) 3 protein has been shown to possess at least two enzymatic domains. The amino terminal third contains a serine-protease domain, whereas the carboxy terminal two thirds is comprised of an adenosine triphosphatase (ATPase)/helicase domain. These domains are essential for the maturation of the carboxy-terminal portion of the HCV polyprotein and catalyze the cap synthesis of the RNA genome. In this report, human and murine antibody responses induced by NS3 were characterized using a recombinant full-length NS3 (NS3-FL) protein, or the isolated protease or ATPase/ helicase domains, expressed and purified from Escherichia coli. Sera from 40 patients with chronic HCV infection were assayed in enzyme-linked immunoassays (EIAs) for antibody binding to the panel of NS3 proteins. Virtually all patient sera contained antibodies specific for NS3-FL and the ATPase/helicase domain, whereas only 10% of sera reacted with the protease domain of NS3. Human antibodies reactive with NS3-FL were highly restricted to the immunoglobulin G1 (IgG1) isotype and were inhibited by soluble ATPase/helicase, but not by the protease domain. The anti-NS3 (ATPase/helicase) reactivity decreased on denaturation by sodium dodecyl sulfate (SDS) and beta-mercaptoethanol (2ME), suggesting the recognition of nonlinear or conformational B-cell determinants. Similar to infected humans, mice immunized with NS3-FL developed high-titered primary antibody responses to the NS3 ATPase/ helicase domain, whereas an anti-NS3 protease response was not observed after primary or secondary immunizations. Thus, the human and murine humoral immune responses to the HCV NS3 protein are focused on the ATPase/helicase domain, are restricted to the IgG1 isotype in humans, and are conformationally dependent. Unexpectedly, in both species, the NS3 protease domain, present in the context of the full-length NS3, appears to possess low intrinsic immunogenicity in terms of antibody production.

The secreted hepatitis B precore antigen can modulate the immune response to the nucleocapsid: a mechanism for persistence.

The hepatitis B precore Ag (HBeAg) is a secreted nonparticulate version of the viral nucleocapsid hepatitis B core Ag (HBcAg), and its function is unknown. A proportion of HBeAg-specific Th cells evade deletion/anergy in HBeAg-transgenic (Tg) mice and mediate anti-HBe "autoantibody" (autoAb) production after in vivo activation with the appropriate Th cell peptide. This model system was used to determine how secretory HBeAg may effect deletion of Th cells in the periphery. For this purpose, HBeAg-Tg mice were bred with Fas and Fas ligand (FasL)-defective lpr/lpr and gld/gld mutant mice. Fas-FasL interactions mediate activation-induced apoptosis in the periphery. In HBeAg-Tg/+ mice, high-titrated anti-HBe autoAb was produced that was exclusively composed of the IgG1 isotype (i.e., Th2-like profile). In contrast, HBeAg-Tg/lpr and HBeAg-Tg/gld mice produced significantly less anti-HBe autoAb, and the IgG isotype patterns were broadened to include IgG2a, IgG2b and IgG3 as well as IgG1 (i.e., mixed Th1/Th2-like profile). These results suggest that HBeAg-specific Th1 cells are preferentially depleted by Fas-FasL-mediated interactions. The effect of circulating HBeAg on HBcAg-specific Th1 cells was also examined by transferring HBe/HBcAg-specific Th cells into dual HBeAg- and HBcAg-expressing Tg recipient mice. The presence of serum HBeAg ablated the expected Th1-mediated anti-HBc Ab response and shifted it toward a Th2 phenotype. These results suggest that in the context of a hepatitis B viral infection, circulating HBeAg has the potential to preferentially deplete inflammatory HBeAg- and HBcAg-specific Th1 cells that are necessary for viral clearance, thereby promoting hepatitis B virus persistence.

Precore wild-type DNA and immune complexes persist in chronic hepatitis B after seroconversion: no association between genome conversion and seroconversion.

Precore hepatitis B virus (HBV) mutants may gradually prevail during or after seroconversion (SC) from hepatitis B e antigen (HBeAg) to hepatitis B e antigen antibody (anti-HBe) status in many chronic hepatitis B (CH-B) patients. However, patients with CH-B still produce anti-HBe more than several years after SC, and the relationship between SC and genome conversion in the precore region has not been clarified. Therefore, in patients with CH-B who had a sustained loss of HBeAg and complete remission of hepatitis after SC, the precore region was sequenced in paired serum samples from 1 year before SC to 3 years after SC. Mutant precore defective HBV DNA was found in only 6 (19%) of 31 CH-B patients who had a complete remission of hepatitis after SC. Mixed-type HBV DNA (precore wild-type and mutant-type) was found in 4 (13%) patients. Wild-type HBV DNA was found in 21 (68%) CH-B patients after SC. Longer-term follow-up of 11 CH-B patients indicated that 3 of 11 patients experienced precore genome conversion 2 to 3 years after SC. E-plus DNA or e-minus DNA was semiquantitated by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays before and after SC. E-plus DNA levels decreased from 10(5.56+/-1.58) to 10(2.45+/-1.61). Similarly, e-minus DNA levels declined from 10(4.25+/-1.56) to 10(1.86+/-1.37). By dot-blot assay, serum HBV DNA became negative soon after SC, as did serum HBeAg. In contrast, HBeAg-containing immune complexes were still detected after SC. Anti-HBe antibody was produced throughout SC and thereafter, as determined by a sensitive experimental assay. Therefore, we conclude that genome-conversion in the precore region is a separate event from HBeAg/anti-HBe seroconversion.

Immune responses to the hepatitis C virus NS4A protein are profoundly influenced by the combination of the viral genotype and the host major histocompatibility complex.

The interaction between the host major histocompatibility complex (MHC) and the genotype of the hepatitis C virus (HCV) was analysed using synthetic full-length non-structural (NS) 4A proteins, residues 1658-1712, of genotypes 1b, 2b, 3a, 4a and 5a. Human and murine antibodies specific for the five NS4A genotypes analysed focused on residues 1688-1707. In immunized B10 H-2 congenic mice, the H-2d, H-2f and H-2s haplotypes were good responders to NS4A, irrespective of the viral genotype. In contrast, the H-2k haplotype was a low or non-responder to all NS4A genotypes, except for genotype 2b. Also, H-2f- and H-2s-restricted NS4A genotype 1b-specific T-cells focused on residues 1670-1679 and 1683-1692, respectively, whereas H-2k-restricted NS4A genotype 2b-specific T-cells focused on the carboxy terminus. Interestingly, H-2f-restricted genotype 1b-specific T-cells did not cross-react with T-cell site analogues of seven other genotypes, whereas the H-2s-restricted, genotype 1b-specific T-cells cross-reacted with genotypes 1a, 4a and 5a. Thus the combination of viral genotype and host MHC profoundly influences the ability to mount an HCV NS4A-specific immune response.

Antibodies to the hepatitis B e antigen (HBeAg) can be induced in HBeAg-transgenic mice by adoptive transfer of a specific T-helper 2 cell clone.

Production of antibody to hepatitis B e antigen (HBeAg); i.e., anti-HBe antibody,) in HBeAg-transgenic mice is believed to be mediated by T-helper 2 (Th2) cells. Injection of an HBeAg-specific Th2 clone into HBeAg-transgenic H-2k mice induced anti-HBe antibody production, confirming the function of Th2 cells in this model system.