Hepatitis B virus impairs TLR9 expression and function in plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) play a key role in detecting pathogens by producing large amounts of type I interferon (IFN) by sensing the presence of viral infections through the Toll-Like Receptor (TLR) pathway. TLR9 is a sensor of viral and bacterial DNA motifs and activates the IRF7 transcription factor which leads to type I IFN secretion by pDCs. However, during chronic hepatitis B virus (HBV) infection, pDCs display an impaired ability to secrete IFN-α following ex vivo stimulation with TLR9 ligands. Here we highlight several strategies used by HBV to block IFN-α production through a specific impairment of the TLR9 signaling. Our results show that HBV particle internalisation could inhibit TLR9- but not TLR7-mediated secretion of IFN-α by pDCs. We observed that HBV down-regulated TLR9 transcriptional activity in pDCs and B cells in which TLR9 mRNA and protein levels were reduced. HBV can interfere with TLR9 activity by blocking the MyD88-IRAK4 axis and Sendai virus targeting IRF7 to block IFN-α production. Neutralising CpG motif sequences were identified within HBV DNA genome of genotypes A to H which displayed a suppressive effect on TLR9-immune activation. Moreover, TLR9 mRNA and protein were downregulated in PBMCs from patients with HBV-associated chronic hepatitis and hepatocellular carcinoma. Thus HBV has developed several escape mechanisms to avoid TLR9 activation in both pDCs and B lymphocytes, which may in turn contribute to the establishment and/or persistence of chronic infection.

Hepatitis B virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model.

UNLABELLED: The development of new anti-hepatitis B virus (HBV) therapies, especially immunotherapeutic approaches, has been limited by the lack of a primate model more accessible than chimpanzees. We have previously demonstrated that sylvanus and cynomolgus macaques are susceptible to in vivo HBV infection after intrahepatic HBV DNA inoculation. In this study, we evaluated the susceptibility of primary macaque hepatocytes (PMHs) to HBV infection with a highly efficient HBV genome-mediated transfer system via a recombinant baculovirus (Bac-HBV). Freshly prepared PMHs, isolated from macaque liver tissue by collagenase perfusion, were transduced with Bac-HBV, and intermediates of replication were followed for 9 days post-transduction. Evidence of HBV replication (hepatitis B surface antigen secretion, viral DNA, RNA, and covalently closed circular DNA) was detected from day 1 to day 9 post-transduction. HBV markers were dose-dependent and still detectable at a multiplicity of infection of 10. Importantly, transduced PMHs secreted all typical forms of HBV particles, as evidenced by a cesium chloride gradient as well as transmission electron microscopy. Furthermore, the Toll-like receptor 9 (TLR9) ligand was used to stimulate freshly prepared macaque peripheral blood mononuclear cells to generate TLR9-induced cytokines. We then demonstrated the antiviral effects of both TLR9-induced cytokines and nucleoside analogue (lamivudine) on HBV replication in transduced PMHs. CONCLUSION: Baculovirus-mediated genome transfer initiated a full HBV replication cycle in PMHs; thus highlighted both the baculovirus efficiency in crossing the species barrier and macaque susceptibility to HBV infection. Moreover, our results demonstrate the relevance of thus system for antiviral compound evaluations with either nucleoside analogues or inhibitory cytokines. Cynomolgus macaques are readily available, are immunologically closely related to humans, and may therefore represent a promising model for the development of new immunotherapeutic strategies.

Inhibitory effect of the combination of CpG-induced cytokines with lamivudine against hepatitis B virus replication in vitro.

BACKGROUND: Currently approved antiviral monotherapies against chronic hepatitis B fail to eradicate hepatitis B virus (HBV), to overcome the defects in HBV-specific immune responses and to prevent HBV relapse after cessation of therapy. CpG oligodesoxynucleotides (CpG ODN) are synthetic agonists of Toll-like receptor 9 and potent inducers of innate and acquired immunity. Our aim was to establish the proof of concept of the antiviral benefit of combining a nucleoside analogue with CpG-induced cytokines on HBV replication in vitro. METHODS: Peripheral blood mononuclear cells from HBV-negative individuals were stimulated with CpG ODN to generate CpG-induced cytokine supernatants. Proliferating HepaRG and HepG2 cells were transduced with recombinant HBV baculovirus and differentiated HepaRG cells were inoculated with HBV virions. Antiviral effects of CpG-induced cytokine with or without lamivudine were evaluated by analysing HBV DNA, HBV RNA and antigen secretion (hepatitis B surface antigen [HBsAg] and hepatitis B e antigen [HBeAg]). RESULTS: Following transduction or HBV inoculation, CpG-induced cytokines strongly inhibited HBV viral intermediates of replication, as well as HBsAg and HBeAg secretion from infected cells. Strikingly, in transduced HepaRG cells, the combination of CpG-induced cytokines with lamivudine reduced the 50% effective concentration of lamivudine by 100-fold. Importantly, the treatment of CpG-induced cytokines prior to HBV inoculation conferred a partial protection against infection to hepatocytes. CONCLUSIONS: CpG-induced cytokines associated with polymerase inhibitors represent a promising combination to suppress HBV replication. Such an immunotherapeutic strategy should be evaluated in vivo to assess restoration and duration of anti-HBV-specific immune responses.

Essential role of antigen-presenting cell-derived BAFF for antibody responses.

Antigen-presenting cells (APC) are directly involved in survival, growth and differentiation of naive B cells and in immunoglobulin class switch recombination. Less is known about the contribution of APC to memory B cell responses. We employed an in vitro model to investigate the secondary humoral response against foot-and-mouth disease virus, with cells from a natural host of the virus - the pig. This response is T cell-dependent. Under conditions of limited T cell help, defined as a low T-to-B cell ratio or by the replacement of T cells with interleukin-2 only, the antibody response was dependent on APC. These included monocytes and monocyte-derived DC, but not plasmacytoid DC. APC mediated their help through soluble factors, particularly soluble B cell-activating factor belonging to the TNF family (BAFF). Our results suggest that the 'ménage à trois' concept, saying that both APC and T cells have a direct effect in B cell activation, is also valid for secondary B cell responses, and imply an important role for BAFF under conditions that might be physiologically relevant in secondary lymphoid organs.

Silencing of natural interferon producing cell activation by porcine circovirus type 2 DNA.

Porcine circovirus type 2 (PCV2) infection of natural interferon producing cells (NIPCs) impairs the induction of interferon (IFN)-alpha and tumour necrosis factor (TNF)-alpha by cytosine-phosphorothioate-guanine (CpG) oligodeoxynucleotides (ODNs), thereby preventing both their autocrine maturation and the paracrine maturation of myeloid dendritic cells (DCs). The present study shows that the PCV2-mediated inhibition of NIPCs was mediated by viral DNA, although it was independent of virus replication. The inhibitory effect of PCV2 DNA was more diversified than if it had simply targeted CpG-ODN-induced cytokines (IFN-alpha, TNF-alpha, interleukin-6, IL-12). A broad spectrum inhibition was noted, affecting responses induced by toll-like receptor (TLR)-7 and TLR9 agonists, as well as viruses including pseudorabies virus, transmissible gastroenteritis virus and classical swine fever virus. From these results, it would appear that PCV2 DNA can induce a dominant negative signal influencing independent pattern recognition receptor-induced activation cascades. Despite a concomitant internalization of PCV2 DNA and CpG-ODNs, no colocalization was observed, indicating that PCV2 DNA and CPG-ODNs may not target the same receptor. This study describes a novel modulation of the innate immune response, which would render the host more susceptible to secondary or concomitant microbial infections.

Subset-dependent modulation of dendritic cell activity by circovirus type 2.

Viral interactions with dendritic cells (DCs) have important consequences for immune defence function. Certain single-stranded DNA viruses that associate with a number of species, including humans and pigs, exhibit interesting characteristics in this context. Porcine circovirus type 2 (PCV2) can persist within myeloid DCs in the absence of virus replication. Internalization was observed with both conventional blood DCs and plasmacytoid DCs [natural interferon-producing cells (NIPCs)], as well as DC precursors. This PCV2-DC interaction neither induced nor inhibited DC differentiation. The maturation of myeloid DCs induced by a cocktail of interferon-alpha/tumour necrosis factor-alpha (IFN-alpha/TNF-alpha), and the ability to process and present antigen to T lymphocytes, remained intact in the presence of PCV2. The virus was clearly internalized by the DCs, a process noted with both mature and immature cells. This suggested a non-macropinocytic uptake, confirmed by an insensitivity to wortmannin but sensitivity to cytochalasin D, chlorpromazine and bafilomycin. Nevertheless, PCV2 was immunomodulatory, being effected through the reaction of NIPC to danger signals. When NIPCs responded to the CpG-oligonucleotide (CpG-ODN), their costimulatory function which induces myeloid DC maturation was clearly impaired by the presence of PCV2. This was caused by a PCV2-induced inhibition of the IFN-alpha and TNF-alpha normally produced following interaction with CpG-ODN. Thus, the immunomodulatory activity of PCV2 is mediated through the disruption of NIPC function. This would impair the maturation of associated myeloid DC and have major implications for the efficient recognition of viral and bacterial danger signals, favouring the establishment of infections additional to that of PCV2.