RESUMO
The discovery of a novel series of highly potent quinazoline TLR 7/8 agonists is described. The synthesis and structure-activity relationship is presented. Structural requirements and optimization of this series toward TLR 7 selectivity afforded the potent agonist 48. Pharmacokinetic and pharmacodynamic studies highlighted 48 as an orally available endogenous interferon (IFN-α) inducer in mice.
Assuntos
Glicoproteínas de Membrana/agonistas , Quinazolinas/farmacologia , Receptor 7 Toll-Like/agonistas , Animais , Inibidores das Enzimas do Citocromo P-450/síntese química , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Inibidores das Enzimas do Citocromo P-450/farmacologia , Células HEK293 , Meia-Vida , Humanos , Interferon-alfa/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Quinazolinas/síntese química , Quinazolinas/química , Quinazolinas/farmacocinética , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Receptor 8 Toll-Like/agonistasRESUMO
Chronic infection with hepatitis B virus (HBV) develops in millions of patients per year, despite the availability of effective prophylactic vaccines. Patients who resolve acute HBV infection develop HBV-specific polyfunctional T cells accompanied by neutralizing antibodies, while in patients with chronic hepatitis B (CHB), immune cells are dysfunctional and impaired. We describe a lipid nanoparticle (LNP)-formulated mRNA vaccine, optimized for the expression of HBV core, polymerase, and surface (preS2-S) antigens with the aim of inducing an effective immune response in patients with CHB. Prime and prime/boost vaccination with LNP-formulated mRNA encoding for core, pol, and/or preS2-S dosing strategies were compared in naive C57BL/6 and BALB/c mice. Immune responses were assessed by IFN-γ ELISpot, intracellular cytokine staining (ICS), and ELISA for antibody production, whereas anti-viral efficacy was evaluated in the AAV-HBV mouse model. The mRNA vaccine induced strong antigen-specific polyfunctional T cell responses in these mouse models, accompanied by the emergence of anti-HBs and anti-HBe antibodies. After three immunizations, the antigen-specific immune stimulation resulted in up to 1.7 log10 IU/mL reduction in systemic HBV surface antigen (HBsAg), accompanied by a transient drop in systemic HBeAg, and this was observed in 50% of the AAV-HBV-transduced mice in the absence of additional modalities such as adjuvants, HBsAg reducing agents, or checkpoint inhibitors. However, no treatment-related effect on viremia was observed in the liver. These results warrant further optimization and evaluation of this mRNA vaccine as a candidate in a multimodal therapeutic regimen for the treatment of chronic HBV infection.
RESUMO
Research on liver-related conditions requires a robust and efficient method to purify viable hepatocytes, lymphocytes and all other liver resident cells, such as Kupffer or liver sinusoidal endothelial cells. Here we describe a novel purification method using liver enzymatic digestion, followed by a downstream optimized purification. Using this enzymatic digestion protocol, the resident liver cells as well as viable hepatocytes could be captured, compared to the classical mechanical liver disruption method. Moreover, single-cell RNA-sequencing demonstrated higher quality lymphocyte data in downstream analyses after the liver enzymatic digestion, allowing for studying of immunological responses or changes. In order to also understand the peripheral immune landscape, a protocol for lymphocyte purification from mouse systemic whole blood was optimized, allowing for efficient removal of red blood cells. The combination of microbeads and mRNA blockers allowed for a clean blood sample, enabling robust single-cell RNA-sequencing data. These two protocols for blood and liver provide important new methodologies for liver-related studies such as NASH, hepatitis virus infections or cancer research but also for immunology where high-quality cells are indispensable for further downstream assays.
Assuntos
Leucócitos , Fígado , Animais , Camundongos , Fígado/imunologia , Leucócitos/imunologia , Hepatócitos/imunologia , Separação Celular/métodos , Análise de Célula Única/métodos , Camundongos Endogâmicos C57BL , Masculino , Linfócitos/imunologia , Linfócitos/citologiaRESUMO
BACKGROUND: Suppression of HBV DNA, inhibition of HBV surface (HBsAg) production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve a functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBV when HBsAg levels are low. Using a single-cell approach, we investigated the liver immune environment with different levels of HBsAg and sustained HBsAg loss through treatment with a GalNAc-HBV-siRNA followed by therapeutic vaccination. METHODS: AAV-HBV-transduced C57BL/6 mice were treated with GalNAc-HBV-siRNA to lower HBsAg levels and then vaccinated using a DNA vaccine. We used single-cell RNA and V(D)J sequencing to understand liver immune microenvironment changes. RESULTS: GalNAc-HBV-siRNA, followed by therapeutic vaccination, achieved sustained HBsAg loss in all mice. This was accompanied by CD4 follicular helper T-cell induction, polyclonal activation of CD8 T cells and clonal expansion of plasma cells that were responsible for antibody production. CONCLUSIONS: This study provides novel insights into liver immune changes at the single-cell level, highlighting the correlation between induced reduction of HBsAg levels and clonal expansion of CD4, CD8 T cells and plasma cells in the liver upon HBV siRNA and subsequent therapeutic vaccination.
RESUMO
Despite the availability of an effective prophylactic vaccine for more than 30 years, nearly 300 million people worldwide are chronically infected with the hepatitis B virus (HBV), leading to 1 death every 30 s mainly from viral hepatitis-related cirrhosis and liver cancer. Chronic HBV patients exhibit weak, transient, or dysfunctional CD8+ T-cell responses to HBV, which contrasts with high CD8+ T-cell responses seen for resolvers of acute HBV infection. Therefore, a therapeutic DNA vaccine was designed, expressing both HBV core and polymerase proteins, and was sequence optimized to ensure high protein expression and secretion. Although the vaccine, administered intramuscularly via electroporation, had no effect on plasma viral parameters in a mouse model of persistent HBV infection, it did induce robust HBV-specific immune responses in healthy and adeno-associated hepatitis B virus (AAV-HBV) infected mice as well as in healthy non-human primates.
RESUMO
A novel series of 2,4-diaminoquinazolines was identified as potent dual Toll-like receptor (TLR) 7 and 8 agonists with reduced off-target activity. The stereochemistry of the amino alcohol was found to influence the TLR7/8 selectivity with the ( R) isomer resulting in selective TLR8 agonism. Lead optimization toward a dual agonist afforded ( S)-3-((2-amino-8-fluoroquinazolin-4-yl)amino)hexanol 31 as a potent analog, being structurally different from previously described dual agonists ( McGowan J. Med. Chem. 2016 , 59 , 7936 ). Pharmacokinetic and pharmacodynamic (PK/PD) studies revealed the desired high first pass profile aimed at limiting systemic cytokine activation. In vivo pharmacodynamic studies with lead compound 31 demonstrated production of cytokines consistent with TLR7/8 activation in mice and cynomolgus monkeys and ex vivo inhibition of hepatitis B virus (HBV).
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Quinazolinas/farmacologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Animais , Antivirais/química , Antivirais/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Quinazolinas/química , Quinazolinas/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Receptor 7 Toll-Like/química , Receptor 8 Toll-Like/químicaRESUMO
Pyrrolo[3,2-d]pyrimidines were identified as a new series of potent and selective TLR7 agonists. Compounds were optimized for their activity and selectivity over TLR8. This presents an advantage over recently described scaffolds that have residual TLR8 activity, which may be detrimental to the tolerability of the candidate drug. Oral administration of the lead compound 54 effectively induced a transient interferon stimulated gene (ISG) response in mice and cynomolgus monkeys. We aimed for a high first pass effect, limiting cytokine induction systemically, and demonstrated the potential for the immunotherapy of viral hepatitis.
Assuntos
Antivirais/síntese química , Hepatite B/tratamento farmacológico , Pirimidinas/síntese química , Pirróis/síntese química , Receptor 7 Toll-Like/agonistas , Administração Oral , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Cães , Feminino , Genes Reporter , Células HEK293 , Hepatite B/imunologia , Humanos , Imunoterapia , Interferons/biossíntese , Macaca fascicularis , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Pirróis/farmacocinética , Pirróis/farmacologia , Ratos , Relação Estrutura-Atividade , Receptor 7 Toll-Like/genética , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/genéticaRESUMO
Toll-like receptor (TLR) 7 and 8 agonists can potentially be used in the treatment of viral infections and are particularly promising for chronic hepatitis B virus (HBV) infection. An internal screening effort identified a pyrimidine Toll-like receptor 7 and 8 dual agonist. This provided a novel alternative over the previously reported adenine and pteridone type of agonists. Structure-activity relationship, lead optimization, in silico docking, pharmacokinetics, and demonstration of ex vivo and in vivo cytokine production of the lead compound are presented.