Development and Characterization of a Chronic Hepatitis B Murine Model With a Mutation in the START Codon of an HBV Polymerase

Chronic hepatitis B (CHB) is caused by the Hepatitis B virus (HBV) and affects millions of people worldwide. Developing an effective CHB therapy requires using in vivo screening methods, such as mouse models reflecting CHB based on hydrodynamic delivery of plasmid vectors containing a replication-competent HBV genome. However, long-term expression of HBV proteins is accompanied by production of progeny virions, thereby requiring a Biosafety Level (BSL) 3 animal facility. In the present study, we introduced a point mutation in the START codon of the HBV polymerase to develop a mouse model reflecting chronic hepatitis B infection without formation of viral progeny. We induced the mouse model by hydrodynamic injection of adeno-associated virus plasmid vector (pAAV) and minicircle plasmid (pMC) constructs into C57Bl/6 and C3H/HeN mouse strains, monitoring HBV antigens and antibodies in blood by enzyme-linked immunosorbent assay and analyzing liver expression of HBV core antigen by immunohistology. Persisting expression of viral antigens over 140 days (study endpoint) was observed only in the C3H/HeN mouse strain when using pAAV/1.2HBV-A and pMC/1.0HBV-D with pre-C and pre-S recombination sites. In addition, pAAV/1.2HBV-A in C3H/HeN sustained HBV core antigen positivity up to the study endpoint in C3H/HeN mice. Moreover, introducing the point mutation in the START codon of polymerase effectively prevented the formation of viral progeny. Our study establishes an accessible and affordable experimental paradigm for developing a robust mouse model reflecting CHB suitable for preclinical testing of anti-HBV therapeutics in a BSL2 animal facility.</jats:p

Enzymatic Preparation of 2 5 ,3 5 Cyclic Dinucleotides, Their Binding Properties to Stimulator of Interferon Genes Adaptor Protein, and Structure Activity Correlations

Cyclic dinucleotides are second messengers in the cyclic GMP AMP synthase cGAS stimulator of interferon genes STING pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 TBK1 interferon regulatory factor 3 IRF3 and inhibitor of nuclear factor amp; 954;B I amp; 954;B kinase IKK nuclear factor amp; 954;B NF amp; 954;B signaling cascades. In this work, we describe an enzymatic preparation of 2 amp; 8242; 5 amp; 8242;,3 amp; 8242; 5 amp; 8242; cyclic dinucleotides 2 amp; 8242;3 amp; 8242;CDNs with use of cyclic GMP AMP synthases cGAS from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide 5 amp; 8242; triphosphate NTP analogues and use them to prepare 33 2 amp; 8242;3 amp; 8242;CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells PBMCs and evaluate their cytotoxic effect on monocytes. Additionally, we report X ray crystal structures of two new CDNs bound to STING protein and discuss structure activity relationship by using quantum and molecular mechanical QM MM computational modelin