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6.1. TLR7 agonists
GS-9620, a potent and orally available TLR7 agonist, is the front runner of PRR agonists under development for treatment of chronic hepatitis B. Its great therapeutic potential has been demonstrated in preclinical studies in woodchucks infected with woodchuck hepatitis virus (WHV) and HBV-infected chimpanzees. Specifically, treatment of woodchucks chronically infected by WHV with varying dose frequencies of GS-9620 for 4–8 weeks resulted in a greater than 6 log reduction of viral load. Intriguingly, while 15 out of 19 animals had dramatic viral load reductions during treatment, the suppressive effect on viral load was sustained in 12 of these 15 animals, and 13 of the 15 sustained WHsAg loss after cessation of treatment. Moreover, for those with sustained WHsAg loss, 8 of 13 developed an antibody against the surface antigen (Menne et al., 2015). This result is in marked contrast to treatment with nucleoside analogs and IFN-α, which rarely resulted in WHsAg seroconversion (Fletcher et al., 2012 and Menne and Cote, 2007). Mechanism of action studies suggested that consistent with activation of TLR-7 signaling, the antiviral response induced by GS-9620 is likely mediated by both the cytolytic activity of CD8+ T cells and/or NK cells, and type I/II IFN-mediated non-cytolytic activity, as well as activation of B cells, in the liver microenvironment.
In a chimpanzee study, GS-9620 treatment of three HBV chronically infected animals for 8 weeks also reduced viral load by more than 2 logs and resulted in greater than a 50% reduction in HBsAg and HBeAg serum levels. While reduction of viral load by 1 log persisted, no HBsAg seroconversion occurred in any of the treated animals (Lanford et al., 2013). Although IFN-α was transiently induced, the suppression of HBV/HBsAg coincided with NK/T cell activation. Hence, it is most likely that the TLR-7 therapeutic effect relies on not only induction of IFNs, but also activation of other branches of intrahepatic innate immune responses.
Pharmacokinetic and pharmacodynamic studies in healthy volunteers suggested that at low oral doses, GS-9620 induces a type I interferon-dependent antiviral innate immune response without the induction of systemic IFN-α. This presystemic response is likely due to its high intestinal absorption and activation of TLR7 locally via oral administration ( Fosdick et al., 2014). In two phase 1b studies reported recently, one or two low doses of GS-9620 administered once a week were safe and well tolerated ( Gane et al., 2015). However, phase 1 studies did not show evidence of clinical efficacy of GS-9620 in terms of HBV DNA decline, HBsAg reduction ( Gane et al., 2015) or decrease in HCV RNA ( Lawitz et al., 2014). Further clinical investigations are certainly warranted to optimize the dosage and treatment schedules.
6.2. TLR8 agonists
A recent report indicates that TLR8 is a more important PRR than TLR7 in human livers (Jo et al., 2014). Specifically, TLR8 agonist ssRNA40 selectively activated liver-resident, and to a lesser extent, the blood-derived, NKT mucosal-associated invariant T and CD56 (bright) NK cells to produce IFN-γ in both healthy livers and HBV- or HCV-infected livers. This was mediated by the production of IL-12 and IL-18 by intrahepatic monocytes. This work thus suggests that TLR8 agonists might be ideal candidates to activate intrahepatic immunity in patients with chronic hepatitis B (Jo et al., 2014).
6.3. TLR3 agonists
A recent report showed that intrahepatic delivery of poly(I:C), a TLR3 agonist, by hydrodynamic injection, efficiently enhanced intrahepatic innate and adaptive immune responses, and accelerated the clearance of HBV in a mouse model established by the hydrodynamic injection of pAAV–HBV1.2 (Wu et al., 2014). The clearance of HBV was dependent on both type I and type II IFNs, indicating a coordinated action of innate and adaptive immune responses. Moreover, T cell recruitment appeared to be critical for the success of TLR3-mediated antiviral action. These findings suggest that intrahepatic delivery of TLR3 agonists might have a good potential for treating chronic hepatitis B.
6.4. TLR9 agonists
The liver is an immunologically unique organ, with many layers of inhibitory mechanisms that prevent the local population expansion and execution of effector functions of CTLs. This may protect the infected liver from over whelming immunopathology, but may also functionally compromise pathogen-specific CTLs and favor the development of chronic infection (Knolle and Thimme, 2014 and Pallett et al., 2015). An elegant study reported by Knolle’s laboratory recently showed that treatment of mice with the agonist of TLR9, but not TLR3 or TLR7, induced the formation of intrahepatic CD11b+MHCII+ myeloid cell aggregates, designated by the authors as “intrahepatic myeloid-cell aggregates for T cell population expansion” (iMATEs), that support massive expansion of the CTL population locally in the liver (Huang et al., 2013). The iMATEs rapidly formed within 2 days after TLR9 agonist injection and provided an anatomic structure for local proliferation of CTLs dependent on the T-cell costimulatory receptor OX40.
Intriguingly, acute but not chronic lymphocytic choriomeningitis virus (LCMV) infection induced intrahepatic iMATEs formation. However, TLR9 agonist treatment of mice chronically infected with LCMV resulted in iMATEs formation and expansion of virus-specific CLTs and subsequent control of infection. Furthermore, using a model of chronic HBV infection of immunocompetent mice established by Ad-HBV infection (Huang et al., 2012), the authors showed that injection of TLR9 agonist at day 12 after vaccination with a plasmid expressing HBcAg resulted in expansion of the H-2 Kb-restricted CTL population specific for HBcAg amino acids 93–100 (HBc93) in the livers of the mice. The antiviral immune response reduced HBV antigenemia and eventually eliminated HBV-replicating hepatocytes (Huang et al., 2013). This work implies that a combination of DNA vaccination and TLR9 agonist therapy induces intrahepatic iMATEs-facilitated expansion of the vaccination-induced HBV-specific CTL population, which subsequently resolves chronic HBV infection.
6.5. STING agonists
Stimulator of interferon genes (STING) is the adaptor protein of multiple cytoplasmic DNA receptors and a PRR recognizing the bacterial second messengers, cyclic di-adenosine monophosphate (c-di-AMP) and cyclic di-guanosine monophosphate (c-di-GMP) (Burdette and Vance, 2013). It was discovered recently that cytoplasmic DNA activates cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) to produce cGAMP, which subsequently binds to STING and induces IFNs and other cytokines (Gao et al., 2013 and Ishikawa and Barber, 2008). The fact that STING can be activated by cyclic di-nucleotides implies that like TLR7 and TLR8, STING might be activated by other small molecules and thus be a potential target for pharmacological activation of innate immune responses, as well as priming of an adaptive immune response.
Indeed, we recently showed that 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a mouse STING agonist, induced a type I-IFN-dominant cytokine response in macrophages, which potently suppressed HBV replication in mouse hepatocytes by reducing the amount of cytoplasmic viral nucleocapsids. Moreover, intraperitoneal administration of DMXAA significantly induced the expression of IFN-stimulated genes and reduced HBV DNA replication intermediates in the livers of HBV-hydrodynamically injected mice. Our study thus provides proof of concept that activation of the STING pathway induces a potent innate antiviral response, and that the development of small-molecule human STING agonists as immunotherapeutic agents for the treatment of chronic hepatitis B is warranted (Guo et al., 2015).
Many PRR agonists have thus far been tested in animal models or in clinical studies for their effects on chronic viral infections of the liver, including hepatitis B. Although promising results have been obtained, the search for the proper agonists that fine-tune host immune responses and cure chronic HBV infection is still under way. In addition, there are many studies reporting HBV evasion and antagonization of innate immune pathways under certain experimental conditions (reviewed in (Chang et al., 2012)). Further investigation is required to re-examine such phenomena in the context of viral replication and in human hepatocytes during natural infection. If confirmed, pharmacological interruption of HBV antagonism of host innate immune responses should be an ideal therapeutic strategy to restore innate, and possibly also adaptive immunity to HBV infection. |
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发表于 2015-8-13 11:53
