Is the function of the HBeAg really unknown?

The immune response to the hepatitis B virus (HBV) vaccine in newborns of hepatitis B e antigen (HBeAg)-positive or HBeAg-negative mothers is the subject of Huang et al. The authors report no correlation between the HBeAg status of the mothers/cord blood and the newborns immune response to the vaccine, but, unfortunately, draw unfounded conclusions regarding the tolerogenic potential of in utero exposure to HBeAg. In this reply, I address the possible influence of in utero exposure to the HBeAg, and briefly review other characteristics of the HBeAg, that may promote HBV chronicity. I argue that the function of HBeAg should no longer be considered "unknown" and that immunotolerance/immunomodulation represent the dominant functions of the HBeAg in viral-host interactions.

P. falciparum and P. vivax Epitope-Focused VLPs Elicit Sterile Immunity to Blood Stage Infections.

In order to design P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS) T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg) VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf) sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs). Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer >1x10(6)) and provided 80-100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78), which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine.

Palivizumab epitope-displaying virus-like particles protect rodents from RSV challenge.

Respiratory syncytial virus (RSV) is the most common cause of serious viral bronchiolitis in infants, young children, and the elderly. Currently, there is not an FDA-approved vaccine available for RSV, though the mAb palivizumab is licensed to reduce the incidence of RSV disease in premature or at-risk infants. The palivizumab epitope is a well-characterized, approximately 24-aa helix-loop-helix structure on the RSV fusion (F) protein (F254-277). Here, we genetically inserted this epitope and multiple site variants of this epitope within a versatile woodchuck hepadnavirus core-based virus-like particle (WHcAg-VLP) to generate hybrid VLPs that each bears 240 copies of the RSV epitope in a highly immunogenic arrayed format. A challenge of such an epitope-focused approach is that to be effective, the conformational F254-277 epitope must elicit antibodies that recognize the intact virus. A number of hybrid VLPs containing RSV F254-277 were recognized by palivizumab in vitro and elicited high-titer and protective neutralizing antibody in rodents. Together, the results from this proof-of-principle study suggest that the WHcAg-VLP technology may be an applicable approach to eliciting a response to other structural epitopes.

Functional aspects of intrahepatic hepatitis B virus-specific T cells induced by therapeutic DNA vaccination.

Current therapies for the hepatitis B virus (HBV), a major cause of severe liver disease, suppress viral replication but replication rebounds if therapy is withdrawn. It is widely accepted that immune activation is needed to control replication off-therapy. To specifically activate T cells crossreactive between the hepatitis B core and e antigens (HBcAg/HBeAg) in chronically infected patients, we developed a therapeutic vaccine candidate. The vaccine encompass codon-optimized HBcAg and IL-12 expressing plasmids delivered using targeted high-pressure injection combined with in vivo electroporation. One dose of the vaccine primed a B-cell-independent polyfunctional T-cell response, in wild-type, and in HBeAg-transgenic mice with an impaired ability to respond to HBc/eAg. The response peaked at 2 weeks and contracted at week 6 after vaccination. Coadministration of IL-12 improved antibody levels, and T-cell expansion and functionality. The vaccine primed T cells that, 2 weeks after a single dose, cleared hepatocytes transiently expressing HBcAg in vaccinated wild-type and HBeAg-transgenic mice. However, 4 weeks later, these functional responses were lost. Booster doses after 8-12 weeks effectively restored function and expansion of the rapidly contracting T cells. Thus, this vaccine strategy primes functional HBcAg-specific T cells in a host with dysfunctional response to HBV.

Identification of a unique double-negative regulatory T-cell population.

Regulatory T (Treg) cells represent one of the main mechanisms of regulating self-reactive immune cells. Treg cells are thought to play a role in down-regulating immune responses to self or allogeneic antigens in the periphery. Although the function of Treg cells has been demonstrated in many experimental settings, the precise mechanisms and antigen specificity often remain unclear. In a hepatitis B e antigen-T-cell receptor (HBeAg-TCR) double transgenic mouse model, we observed a phenotypically unique (TCR+) CD4- /CD8- CD25(+/-) GITR(high) PD-1(high) FoxP3-) HBeAg-specific population that demonstrates immune regulatory function. This HBeAg-specific double-negative regulatory cell population proliferates vigorously in vitro, in contrast to any other known regulatory population, in an interleukin-2-independent manner.

Heterologous T cells can help restore function in dysfunctional hepatitis C virus nonstructural 3/4A-specific T cells during therapeutic vaccination.

The hepatitis C virus (HCV)-specific T cell response in patients with chronic HCV is dysfunctional. In this study, we aimed at restoring immunological function through therapeutic vaccination in a transgenic mouse model with impaired HCV-specific T cell responses due to a persistent presence of hepatic HCV nonstructural (NS)3/4A Ags. The HCV-specific T cells have an actively maintained dysfunction reflected in reduced frequency, impaired cytokine production, and impaired effector function in vivo, which can be partially restored by blocking regulatory T cells or programmed cell death ligand 1. We hypothesized that the impairment could be corrected by including sequences that created a normal priming environment by recruiting "healthy" heterologous T cells and by activating innate signaling. Endogenously expressed hepatitis B core Ag (HBcAg) can recruit heterologous T cells and activate TLR (TLR7) signaling. Hence, by combining HCV NS3/4A with different forms of HBcAg we found that heterologous sequences somewhat improved activation and expansion of NS3/4A-specific T cells in a wild-type host. Importantly, the signals provided by HBcAg effectively restored the activation of HCV-specific T cells in a tolerant NS3/4A-transgenic mouse model. The adjuvant effect could also be transferred to the priming of dysfunctional HLA-A2-restricted NS3-specific T cells in vivo. Thus, recruiting healthy heterologous T cells to the site of priming may also help restore HCV-specific responses present in a chronically infected host.

Improving on the ability of endogenous hepatitis B core antigen to prime cytotoxic T lymphocytes.

Hepatitis B virus core antigen (HBcAg) is thought to be a major target for specific cytotoxic T cells (CTLs) in hepatitis B virus infections. A single dose of hepatitis C virus nonstructural 3/4A DNA (<5 microg) effectively primes functional specific CTLs, independently of CD4(+) T helper cells and by different routes of immunization. In contrast, HBcAg-specific CTL priming was T helper cell dependent and highly sensitive to the dose and route of delivery. Although CTL priming was improved 10-fold by codon optimization and in vivo electroporation, low levels of DNA still failed to prime CTLs effectively. Only high doses (5 microg) of codon-optimized HBcAg delivered by in vivo electroporation primed in vivo lytic and polyfunctional CTLs. The ability of endogenous HBcAg to prime CTLs is surprisingly inefficient and differs from that of nonstructural 3/4A. This has important implications for the design of HBcAg-based therapeutic vaccines in humans.

Use of hepadnavirus core proteins as vaccine platforms.

The first virus-like particle to be tested for use as a vaccine carrier was based on the hepatitis B virus nucleocapsid protein. This viral subunit, while not infectious on its own, is a 36-nm particle that is highly immunogenic during a natural infection. The self-assembly and high degree of immunogenicity is maintained when expressed as a recombinant protein and, moreover, can confer a high degree of immunogenicity on foreign antigens linked to the particle, either chemically or genetically. This review describes the current state of the hepadnaviral core protein as a vaccine carrier.

Interaction of the hepatitis B core antigen and the innate immune system.

Previous studies demonstrated that the primary APCs for the hepatitis B core Ag (HBcAg) were B cells and not dendritic cells (DC). We now report that splenic B1a and B1b cells more efficiently present soluble HBcAg to naive CD4(+) T cells than splenic B2 cells. This was demonstrated by direct HBcAg-biotin-binding studies and by HBcAg-specific T cell activation in vitro in cultures of naive HBcAg-specific T cells and resting B cell subpopulations. The inability of DC to function as APCs for exogenous HBcAg relates to lack of uptake of HBcAg, not to processing or presentation, because HBcAg/anti-HBc immune complexes can be efficiently presented by DC. Furthermore, HBcAg-specific CD4(+) and CD8(+) T cell priming with DNA encoding HBcAg does not require B cell APCs. TLR activation, another innate immune response, was also examined. Full-length (HBcAg(183)), truncated (HBcAg(149)), and the nonparticulate HBeAg were screened for TLR stimulation via NF-kappaB activation in HEK293 cells expressing human TLRs. None of the HBc/HBeAgs activated human TLRs. Therefore, the HBc/HBeAg proteins are not ligands for human TLRs. However, the ssRNA contained within HBcAg(183) does function as a TLR-7 ligand, as demonstrated at the T and B cell levels in TLR-7 knockout mice. Bacterial, yeast, and mammalian ssRNA encapsidated within HBcAg(183) all function as TLR-7 ligands. These studies indicate that innate immune mechanisms bridge to and enhance the adaptive immune response to HBcAg and have important implications for the use of hepadnavirus core proteins as vaccine carrier platforms.

A mechanism to explain the selection of the hepatitis e antigen-negative mutant during chronic hepatitis B virus infection.

Hepatitis B virus (HBV) expresses two structural forms of the nucleoprotein, the intracellular nucleocapsid (hepatitis core antigen [HBcAg]) and the secreted nonparticulate form (hepatitis e antigen [HBeAg]). The aim of this study was to evaluate the ability of HBcAg- and HBeAg-specific genetic immunogens to induce HBc/HBeAg-specific CD4(+)/CD8(+) T-cell immune responses and the potential to induce liver injury in HBV-transgenic (Tg) mice. Both the HBcAg- and HBeAg-specific plasmids primed comparable immune responses. Both CD4(+) and CD8(+) T cells were important for priming/effector functions of HBc/HBeAg-specific cytotoxic T-lymphocyte (CTL) responses. However, a unique two-step immunization protocol was necessary to elicit maximal CTL priming. Genetic vaccination did not prime CTLs in HBe- or HBc/HBeAg-dbl-Tg mice but elicited a weak CTL response in HBcAg-Tg mice. When HBc/HBeAg-specific CTLs were adoptively transferred into HBc-, HBe-, and HBc/HBeAg-dbl-Tg mice, the durations of the liver injury and inflammation were significantly greater in HBeAg-Tg recipient mice than in HBcAg-Tg mice. Importantly, liver injury in HBc/HBeAg-dbl-Tg mice was similar to the injury observed in HBeAg-Tg mice. Loss of HBeAg synthesis commonly occurs during chronic HBV infection; however, the mechanism of selection of HBeAg-negative variants is unknown. The finding that hepatocytes expressing wild-type HBV (containing both HBcAg and HBeAg) are more susceptible to CTL-mediated clearance than hepatocytes expressing only HBcAg suggest that the HBeAg-negative variant may have a selective advantage over wild-type HBV within the livers of patients with chronic infection during an immune response and may represent a CTL escape mutant.

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.

A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen.

A unique characteristic of the hepatitis B virus is the production of a secreted form (precore or HBeAg) of the structural nucleocapsid (core or HBcAg). By using T cell receptor (TCR) transgenic (Tg) and TCR x HBc/HBeAg double- and triple-Tg pairs, we demonstrate that HBeAg elicits T cell tolerance, whereas HBcAg is nontolerogenic in this system. In fact, TCR x HBc double-Tg mice spontaneously seroconvert to IgG anti-HBc positivity at an early age. However, the presence of HBeAg in the serum of TCR x HBc x HBe triple-Tg mice prevents anti-HBc seroconversion. HBeAg mediates its immunoregulatory effect by eliciting tolerance in HBc/HBeAg-specific T cells. The results suggest that hepadnaviruses have retained a secretory form of the nucleoprotein because it functions as a T cell tolerogen and regulates the immune response to the intracellular nucleocapsid. This HBeAg-mediated immune regulation may predispose to chronicity during perinatal infections and prevent severe liver injury during adult infections.

Immunogenetics of the response to HBsAg vaccination.

The major envelope protein of the hepatitis B virus (HBV), the HBsAg, constitutes the current preventative vaccine, which represents the first subunit viral vaccine developed. The genetics of the immune response to HBsAg has been extensively studied both in humans and mice. Murine studies begun over 20 years ago indicated that at least two MHC class II and one MHC class III genes regulate anti-HBs immune responses. Additional MHC-linked genes influence the immune responses to the higher molecular weight (pre-S) components of the HBV envelope. The murine studies predicted even more complex MHC gene regulation of human immune responses to the HBsAg and that complexity certainly has been demonstrated during the ensuing years. This brief review is an attempt to summarize our current understanding of the MHC genes that influence the immune response to the HBsAg and possible mechanisms of action.

Priming of cytotoxic T cell responses to exogenous hepatitis B virus core antigen is B cell dependent.

The hepatitis B virus (HBV) core antigen (HBcAg) has a unique ability to bind a high frequency of naive human and murine B cells. The role of HBcAg-binding naive B cells in the immunogenicity of HBcAg is not clear. The HBcAg-binding properties of naive B cells were characterized using HBcAg particles with mutated spike region (residues 76-85) sequences. Deletion of residues 76-85 (HBcDelta76-85) destroyed naive B cell binding, whereas deletion of residues 79-85 did not. HBcAg particles with an Ile instead of the natural Ala at position 80 did not bind naive B cells, whereas reversion of Ile80-->Ala restored B cell binding. Destroying the B cell-binding ability of HBcAg had a marginal effect on the overall B cell immunogenicity of the different particles, suggesting that they were equally efficient in priming T helper cells. Therefore, the importance of HBcAg-binding B cells is studied with relation to the priming of HBcAg-specific cytotoxic T cells (CTLs). The role of HBcAg-binding B cells in the priming of HBcAg-specific CTLs was evaluated by immunization with endogenous HBcAg (DNA immunization) and exogenous recombinant HBcAg particles. Endogenous HBcAg primed HBcAg-specific CTLs in wild-type and B cell-deficient mice, whereas exogenous HBcAg primed HBcAg-specific CTLs only in wild-type mice. Importantly, HBcDelta76-85 did not prime CTLs despite the presence of B cells. Thus, the ability of exogenous HBcAg particles to prime specific CTLs is B cell dependent, suggesting a possible role for HBcAg-binding B cells in HBV infections.

A malaria vaccine candidate based on a hepatitis B virus core platform.

OBJECTIVE: The recent success of a Plasmodium falciparum malaria vaccine consisting of circumsporozoite (CS) protein (CSP) T and B cell epitopes has rekindled interest in the development of a pre-erythrocytic vaccine. Our goal was to design an efficient delivery system for known neutralizing epitopes. METHODS: Well-characterized CSP-specific neutralizing B cell epitopes and a 'universal' T cell epitope were combined with a particulate carrier platform, the hepatitis B core antigen (HBcAg), to produce a novel pre-erythrocytic vaccine candidate. RESULTS: The vaccine candidate V12.PF3.1 is a potent immunogen in mice, eliciting unprecedented levels (greater than 106 titers) of sporozoite-binding antibodies after only two doses. The antisporozoite antibodies are long-lasting and 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. CONCLUSION: These characteristics suggest that V12.PF3.1 represents an efficient and economical P. falciparum vaccine candidate for use separately or in combination with other formulations.

Humoral and CD4(+) T helper (Th) cell responses to the hepatitis C virus non-structural 3 (NS3) protein: NS3 primes Th1-like responses more effectively as a DNA-based immunogen than as a recombinant protein.

The non-structural 3 (NS3) protein is one of the most conserved proteins of hepatitis C virus, and T helper 1 (Th1)-like responses to NS3 in humans correlate with clearance of infection. Several studies have proposed that DNA-based immunizations are highly immunogenic and prime Th1-like responses, although few head-to-head comparisons with exogenous protein immunizations have been described. A full-length NS3/NS4A gene was cloned in eukaryotic vectors with expression directed to different subcellular compartments. Inbred mice were immunized twice in regenerating tibialis anterior (TA) muscles with either plasmid DNA or recombinant NS3 (rNS3). After two 100 micrograms DNA immunizations, specific antibody titres of up to 12960 were detected at week 5, dominated by IgG2a and IgG2b. NS3-specific CD4(+) T cell responses in DNA-immunized mice peaked at day 13, as measured by proliferation and IL-2 and IFN-gamma production. Mice immunized with 1-10 micrograms rNS3 without adjuvant developed antibody titres comparable to those of the DNA-immunized mice, but dominated instead by IgG1. CD4(+) T cell responses in these mice showed peaks of IL-2 response at day 3 and IL-6 and IFN-gamma responses at day 6. With adjuvant, rNS3 was around 10-fold more immunogenic with respect to speed and magnitude of the immune responses. Thus, immunization with rNS3 in adjuvant is superior to DNA immunization with respect to kinetics and quantity in priming specific antibodies and CD4(+) T cells. However, as a DNA immunogen, NS3 elicits stronger Th1-like immune responses, whereas rNS3 primes a mixed Th1/Th2-like response regardless of the route, dose or adjuvant.