Advancements in Novel Live Biotherapeutic Products for Clostridioides difficile Infection Prevention.

The profound impact of the human microbiome on health and disease has captivated the interest of clinical and scientific communities. The human body hosts a vast array of microorganisms collectively forming the human microbiome, which significantly influences various physiological processes and profoundly shapes overall well-being. Notably, the gut stands out as an exceptional reservoir, harboring the most significant concentration of microorganisms, akin to an organ in itself. The gut microbiome's composition and function are influenced by genetics, environment, age, underlying conditions, and antibiotic usage, leading to dysbiosis and pathogenesis, such as Clostridioides difficile infection (CDI). Conventional CDI treatment, involving antibiotics like oral vancomycin and fidaxomicin, fails to address dysbiosis and may further disrupt gut microbial communities. Consequently, emerging therapeutic strategies are focused on targeting dysbiosis and restoring gut microbiota to advance CDI therapeutics. Fecal microbiota transplantation (FMT) has demonstrated remarkable efficacy in treating recurrent CDI by transferring processed stool from a healthy donor to a recipient, restoring gut dysbiosis and enhancing bacterial diversity. Moreover, 2 newer Food and Drug Administration (FDA)-approved live biotherapeutic products (LBP), namely, Fecal Microbiota Live-JSLM and Fecal Microbiota Spores Live-BRPK, have shown promise in preventing CDI recurrence. This review explores the role of the gut microbiota in preventing and treating CDI, with an emphasis on gut-based interventions like FMT and fecal microbiota-based products that hold potential for gut restoration and prevention of CDI recurrence. Understanding the microbiome's impact on CDI prevention and treatment offers valuable insights for advancing future CDI therapeutics.

High yield soluble bacterial expression and streamlined purification of recombinant human interferon α-2a.

Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is clinically used to modulate hairy cell leukemia as well as hepatitis c. Historically, IFNA2 has been purified from human leukocytes as well as bacterial expression systems. In most cases, bacterial expression of IFNA2 resulted in inclusion body formation, or required numerous purification steps that decreased the protein yield. Here, we describe an expression and purification scheme for IFNA2 using a pET-SUMO bacterial expression system and a single purification step. Using the SUMO protein as the fusion tag achieved high soluble protein expression. The SUMO tag was cleaved with the Ulp1 protease leaving no additional amino acids on the fusion terminus following cleavage. Mass spectrometry, circular dichroism, 2D heteronuclear NMR, and analytical ultracentrifugation confirmed the amino acid sequence identity, secondary and tertiary protein structures, and the solution behavior of the purified IFNA2. The purified protein also had antiviral and anti-proliferative activities comparable to the WHO International Standard, NIBSC 95/650, and the IFNA2 standard available from PBL Assay Science. Combining the expression and purification protocols developed here to produce IFNA2 on a laboratory scale with the commercial fermenter technology commonly used in pharmaceutical industry may further enhance IFNA2 yields, which will promote the development of interferon-based protein drugs to treat various disorders.

Exploring variations in recommended first-choice therapy for complicated urinary tract infections in males: Insights from outpatient settings across age, race, and ethnicity.

INTRODUCTION: There are known disparities in the treatment of infectious diseases. However, disparities in treatment of complicated urinary tract infections (UTIs) are largely uninvestigated. OBJECTIVES: We characterized UTI treatment among males in Veterans Affairs (VA) outpatient settings by age, race, and ethnicity and identified demographic characteristics predictive of recommended first-choice antibiotic therapy. METHODS: We conducted a national, retrospective cohort study of male VA patients diagnosed with a UTI and dispensed an outpatient antibiotic from January 2010 through December 2020. Recommended first-choice therapy for complicated UTI was defined as use of a recommended first-line antibiotic drug choice regardless of area of involvement (ciprofloxacin, levofloxacin, or sulfamethoxazole/trimethoprim) and a recommended duration of 7 to 10 days of therapy. Multivariable models were used to identify demographic predictors of recommended first-choice therapy (adjusted odds ratio [aOR] > 1). RESULTS: We identified a total of 157,898 males diagnosed and treated for a UTI in the outpatient setting. The average antibiotic duration was 9.4 days (±standard deviation [SD] 4.6), and 47.6% of patients were treated with ciprofloxacin, 25.1% with sulfamethoxazole/trimethoprim, 7.6% with nitrofurantoin, and 6.6% with levofloxacin. Only half of the male patients (50.6%, n = 79,928) were treated with recommended first-choice therapy (first-line drug choice and appropriate duration); 77.6% (n = 122,590) were treated with a recommended antibiotic choice and 65.9% (n = 104,070) with a recommended duration. Age 18-49 years (aOR 1.07, 95% confidence interval [CI] 1.03-1.11) versus age ≥65 years was the only demographic factor predictive of recommended first-choice therapy. CONCLUSIONS: Nearly half of the patients included in this study did not receive recommended first-choice therapies; however, racial and ethnic disparities were not identified. Underutilization of recommended first-choice antibiotic therapy in complicated UTIs continues to be an area of focus for antimicrobial stewardship programs.

Characterization of Monoclonal Antibodies to Measure Cell Surface Protein Levels of Human Interferon-Lambda Receptor 1.

Type III interferons (IFN-lambdas, IFN-λs) are important antiviral cytokines that can also modulate immune responses by acting through a heterodimeric receptor composed of the specific and limited expressed IFN-λR1 chain and the ubiquitous IL-10R2 chain, which is shared with IL-10 family cytokines. Conflicting data have been reported regarding which cells express the IFN-λR1 subunit and directly respond to IFN-λs. This is, in part, owing to transcript levels of the IFN-λR1 gene, IFNLR1, not always correlating with cell surface protein levels. In this study, we tested a panel of novel monoclonal antibodies (mAbs) that specifically recognize human IFN-λR1. Initially, antigen specificity was confirmed by enzyme-linked immunosorbent assay (ELISA), from which a subset of antibodies was selected for additional flow cytometry and neutralization assays. We further characterized two antibodies based on their strong ELISA binding activity (HLR1 and HLR14) and found only HLR14 could reliably detect cell surface IFN-λR1 protein on a variety of cell lines by flow cytometry. HLR14 could also detect IFN-λR1 protein on certain primary human blood cells, including plasmacytoid dendritic cells and B cells from peripheral blood. Availability of the HLR14 mAb will enable the quantification of IFN-λR1 protein levels on cells and better characterization of the cell specificity of the IFN-λ response.

The differential activity of interferon-α subtypes is consistent among distinct target genes and cell types.

IFN-α proteins have been described to originate from 14 individual genes and allelic variants. However, the exceptional diversity of IFN-α and its functional impact are still poorly understood. To characterize the biological activity of IFN-α subtypes in relation to the cellular background, we investigated the effect of IFN-α treatment in primary fibroblasts and endothelial cells of vascular or lymphatic origin. The cellular response was evaluated for 13 distinct IFN-α proteins with respect to transcript regulation of the IFN-stimulated genes (ISGs) IFIT1, ISG15, CXCL10, CXCL11 and CCL8. The IFN-α proteins displayed a remarkably consistent potency in gene induction irrespective of target gene and cellular background which led to the classification of IFN-α subtypes with low (IFN-α1), intermediate (IFN-α2a, -4a, -4b, -5, -16, -21) and high (IFN-α2b, -6, -7, -8, -10, -14) activity. The differential potency of IFN-α classes was confirmed at the ISG protein level and the functional protection of cells against influenza virus infection. Differences in IFN activity were only observed at subsaturating levels of IFN-α proteins and did not affect the time course of ISG regulation. Cell-type specific responses were apparent for distinct target genes independent of IFN-α subtype and were based on different levels of basal versus inducible gene expression. While fibroblasts presented with a high constitutive level of IFIT1, the expression in endothelial cells was strongly induced by IFN-α. In contrast, CXCL10 and CXCL11 transcript levels were generally higher in endothelial cells despite a pronounced induction by IFN-α in fibroblasts. In summary, the divergent potency of IFN-α proteins and the cell-type specific regulation of individual IFN target genes may allow for the fine tuning of cellular responses to pathogen defense.

Binding and activity of all human alpha interferon subtypes.

Vertebrates have multiple genes encoding Type I interferons (IFN), for reasons that are not fully understood. The Type I IFN appear to bind to the same heterodimeric receptor and the subtypes have been shown to have different potencies in various experimental systems. To put this concept on a quantitative basis, we have determined the binding affinities and rate constants of 12 human Alpha-IFN subtypes to isolated interferon receptor chains 1 and 2. Alpha-IFNs bind IFNAR1 and IFNAR2 at affinities of 0.5-5 µM and 0.4-5 nM respectively (except for IFN-alpha1 - 220 nM). Additionally we have examined the biological activity of these molecules in several antiviral and antiproliferative models. Particularly for antiproliferative potency, the binding affinity and activity correlate. However, the EC50 values differ significantly (1.5 nM versus 0.1 nM for IFN-alpha2 in WISH versus OVCAR cells). For antiviral potency, there are several instances where the relationship appears to be more complicated than simple binding. These results will serve as a point of reference for further understanding of this multiple ligand/receptor system.

Design and construction of a phosphorylatable chimeric monoclonal antibody with a highly stable phosphate.

A recognition site for the cAMP-dependent protein kinase was introduced into the MAb-chCC49 by site-directed mutation of the coding sequence to make a variant of MAb-chCC49 containing a highly stable phosphate. To design this monoclonal antibody (MAb) without changing its immunoreactivity or biological properties, molecular modeling was used to locate appropriate regions for introduction of the cAMP-dependent phosphorylation site with desirable properties. We selected one position to mutate on the heavy chain based on molecular dynamics study of the solvated antibody. A vector expressing the mutant was constructed and transfected into mouse myeloma NS0 cells that expressed a high level of the resultant MAb-WW5. MAb-WW5 contained the cAMP-dependent phosphorylation site at the hinge region of the heavy chain, could be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity, and retained the phosphate stably. Compared with MAb-chCC49K1, another phosphorylatable variant of MAb-chCC49, the phosphate attached to MAb-WW5 showed much improved stability: about a 10-fold increase in resistance to hydrolysis. MAb-WW5 exhibited the same binding specificity to the TAG-72 antigen on MCF-7 4C10 breast cancer cells as we observed with MAb-chCC49K1. The improved stability of the attached phosphate provides a MAb with potential to be used in diagnosis and therapy of adenocarcinomas.

Potent and selective biphenyl azole inhibitors of adipocyte fatty acid binding protein (aFABP).

Herein we report the first disclosure of biphenyl azoles that are nanomolar binders of adipocyte fatty acid binding protein (aFABP or aP2) with up to thousand-fold selectivity against muscle fatty acid binding protein and epidermal fatty acid binding protein. In addition a new radio-ligand to determine binding against the three fatty acid binding proteins was also synthesized.