Identification, Characterization, and Formulation of a Novel Carbapenemase Intended to Prevent Antibiotic-Mediated Gut Dysbiosis.

Antibiotics can damage the gut microbiome leading to opportunistic infections and the emergence of antibiotic resistance. Microbiome protection via antibiotic inactivation in the gastrointestinal (GI) tract represents a strategy to limit antibiotic exposure of the colonic microbiota. Proof of concept for this approach was achieved with an orally-administered beta-lactamase enzyme, SYN-004 (ribaxamase), that was demonstrated to degrade ceftriaxone excreted into the GI tract and protect the gut microbiome from antibiotic-mediated dysbiosis. Ribaxamase efficiently degrades penicillin and cephalosporin beta-lactam antibiotics, but is not active against carbapenems. To expand this microbiome protection strategy to include all classes of beta-lactams, three distinct carbapenemases were evaluated for manufacturability, antibiotic degradation spectrum, and stability in human intestinal fluid. E. coli production strains were generated for P2A, a novel metallo-enzyme isolated from B. cereus, New Delhi metallo-beta-lactamase (NDM), and Klebsiella pneumoniae carbapenemase (KPC). While all three enzymes effectively inactivated a broad range of antibiotics, including penicillins, most cephalosporins, and carbapenems in vitro, only P2A retained biological activity when incubated with human chyme. As functional stability in the intestinal tract is a key requirement for an orally-delivered enzyme, P2A was chosen as a potential clinical candidate. An enteric formulation of P2A was developed, called SYN-006, that was inert under high acid conditions, with enzyme dissolution occurring at pH > 5.5. SYN-006 has the potential to expand microbiome protection via antibiotic inactivation to include all classes of beta-lactam antibiotics.

Development of SYN-004, an oral beta-lactamase treatment to protect the gut microbiome from antibiotic-mediated damage and prevent Clostridium difficile infection.

The gut microbiome, composed of the microflora that inhabit the gastrointestinal tract and their genomes, make up a complex ecosystem that can be disrupted by antibiotic use. The ensuing dysbiosis is conducive to the emergence of opportunistic pathogens such as Clostridium difficile. A novel approach to protect the microbiome from antibiotic-mediated dysbiosis is the use of beta-lactamase enzymes to degrade residual antibiotics in the gastrointestinal tract before the microflora are harmed. Here we present the preclinical development and early clinical studies of the beta-lactamase enzymes, P3A, currently referred to as SYN-004, and its precursor, P1A. Both P1A and SYN-004 were designed as orally-delivered, non-systemically available therapeutics for use with intravenous beta-lactam antibiotics. SYN-004 was engineered from P1A, a beta-lactamase isolated from Bacillus licheniformis, to broaden its antibiotic degradation profile. SYN-004 efficiently hydrolyses penicillins and cephalosporins, the most widely used IV beta-lactam antibiotics. In animal studies, SYN-004 degraded ceftriaxone in the GI tract of dogs and protected the microbiome of pigs from ceftriaxone-induced changes. Phase I clinical studies demonstrated SYN-004 safety and tolerability. Phase 2 studies are in progress to assess the utility of SYN-004 for the prevention of antibiotic-associated diarrhea and Clostridium difficile disease.

In vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor miR-122.

Miravirsen is a ß-D-oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC50) of 0.67 µM. No cytotoxicity was observed up to the highest concentration tested (>320 µM) in different cell culture models, yielding a therapeutic index of ≥ 297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2'-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5' untranslated region (UTR) from cells passaged in the presence of up to 20 µM (40-fold the miravirsen EC50 concentration) at day 72 of passage but not at earlier time points (up to 39 days of passage). Likewise, a C3U nucleotide change was observed in the HCV 5'UTR from subjects with viral rebound after the completion of therapy in a miravirsen phase 2 clinical trial. An HCV variant constructed to contain the A4C change was fully susceptible to miravirsen. A C3U HCV variant demonstrated overall reductions in susceptibility to miravirsen but was fully susceptible to all other anti-HCV agents tested. In summary, miravirsen has demonstrated broad antiviral activity and a relatively high genetic barrier to resistance. The identification of nucleotide changes associated with miravirsen resistance should help further elucidate the biology of miR-122 interactions with HCV. (The clinical trial study has been registered at ClinicalTrials.gov under registration no. NCT01200420).

Genome sequence, full-length infectious cDNA clone, and mapping of viral double-stranded RNA accumulation determinant of hypovirus CHV1-EP721.

Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.

Analysis of altered G-protein subunit accumulation in Cryphonectria parasitica reveals a third Galpha homologue.

Heterotrimeric G-proteins mediate many responses of eukaryotic cells to external stimuli and have been shown to be important for fungal pathogenicity. In this study, we explored the accumulation of G-protein subunits of the chestnut blight fungus, Cryphonectria parasitica, in mutant strains deleted for one or more putative partner subunits. Using a series of extraction buffers and immunoblot end-point dilution analysis, we established a convenient method to assess the relative abundance of these membrane-associated proteins. Disruption of either cpg-1, which encodes the Galpha subunit CPG-1, or cpgb-1, the Gbeta subunit CPGB-1, consistently reduced the level of its presumptive partner protein. This was not observed in the case of a second Galpha subunit, CPG-2, suggesting that CPG-1 and CPGB-1 regulate each other's stability. Further, analysis of transcript levels indicated that the Galpha and Gbeta protein turnover rates were increased in the mutant strains. Additionally, a previously unidentified protein that was cross-reactive with anti-CPG-1 antiserum was found to be enhanced in liquid culture. We describe the sequence of a new Galpha subunit, CPG-3, that is most similar to three other filamentous fungal Galpha proteins that form a phylogenetically distinct grouping.

Distinct poly(rC) binding protein KH domain determinants for poliovirus translation initiation and viral RNA replication.

The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5'-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

Differential modulation of cellular signaling pathways by mild and severe hypovirus strains.

Hypoviruses persistently alter multiple phenotypic traits, stably modify gene expression, and attenuate virulence (hypovirulence) of their pathogenic fungal host, the chestnut blight fungus Cryphonectria parasitica. The pleiotropic nature of these changes is consistent with hypovirus-mediated perturbation of one or more cellular signal transduction pathways. We now report that two hypoviruses that differ in the severity of symptom expression differentially perturb specific cellular signaling pathways. The C. parasitica 13-1 gene, originally identified as a hypovirus-inducible and cyclic AMP (cAMP)-regulated gene, was used to design a promoter-GFP reporter construct with which to monitor perturbation of cAMP-mediated signaling. Virus-mediated modulation of calcium/calmodulin/inositol trisphosphate-dependent signaling was monitored by measuring transcript accumulation from the C. parasitica laccase gene, lac-1. Infection by the severe hypovirus strain CHV1-EP713 caused a substantial induction of 13-1 promoter activity and a reduction of total extracellular laccase enzymatic activity (LAC-1 and LAC-3). In contrast, 13-1 promoter activity and total laccase activity were only marginally altered upon infection with the mild hypovirus strain CHV1-Euro7. However, examination of lac-1-specific transcript accumulation under previously defined culture conditions revealed that both CHV1-EP713 and CHV1-Euro7 perturbed calcium/calmodulin/inositol trisphosphate-dependent signaling. CHV1-EP713/CHV1-Euro7 chimeric viruses were used to map viral determinants responsible for modulation of cAMP-dependent signaling to domains within the central portion of the second open reading frame.