Evaluation of a standardised Vi poly-l-lysine ELISA for serology of Vi capsular polysaccharide antibodies.

Typhoid vaccines based on protein-conjugated capsular Vi polysaccharide (TCVs) prevent typhoid in infants and young children. Analysis of the serum anti-Vi IgG response following immunisation against typhoid confirms the immunogenicity of TCVs and forms an important part of the pathway to licensing. Comparative studies could expedite the licencing process, and the availability of a standardised ELISA method alongside the 1st International Standard (IS) 16/138 for anti-typhoid capsular Vi polysaccharide IgG (human) will facilitate this process. To this end, a non-commercial ELISA based on a coat of Vi and poly-l-lysine (Vi-PLL ELISA) was evaluated by 10 laboratories. Eight serum samples, including IS 16/138, were tested in the standardised Vi-PLL ELISA (n = 10), a commercial Vi ELISA (n = 3) and a biotinylated Vi ELISA (n = 1). Valid estimates of potencies relative to IS 16/138 were obtained for all samples in the Vi-PLL ELISA and the commercial ELISA, with good repeatability and reproducibility evident from the study results and concordant estimates obtained by the two ELISA methods. The study demonstrates that the Vi-PLL ELISA can be used in clinical trial studies to determine the immunogenicity of TCVs.

Evaluation of two WHO First International Standards for Vi polysaccharide from Citrobacter freundii and Salmonella enterica subspecies enterica serovar Typhi.

Numerous Vi capsular polysaccharide (Vi PS) conjugate vaccines to protect young children and infants from Typhoid are either licensed or under development. These vaccines are evaluated by laboratory methods to ensure their potency and that quality requirement are met. International Standard (IS) preparations of Vi PS are needed to calibrate and harmonise these assays. Twenty laboratories from 12 countries participated in a collaborative study to evaluate two candidate ISs: Citrobacter freundii Vi PS (NIBSC code 12/244) and Salmonella enterica serovar Typhi Vi PS (16/126). On the basis of returned results and stability profiles, these standards were established by the WHO Expert Committee on Biological Standardization in Oct 2017 as the First WHO IS for C. freundii Vi PS with a content of 1.94 ±â€¯0.12 mg Vi PS per ampoule (expanded uncertainty with coverage factor of k = 2.11 corresponding to a 95% level of confidence) and the First WHO IS for S. Typhi Vi PS with a content of 2.03 ±â€¯0.10 mg Vi PS per ampoule (expanded uncertainty with coverage factor of k = 2.11), as determined by quantitative NMR. The study also showed the ISs are suitable for physicochemical and immuno assays used for the quantitation of the Vi PS component in Vi PS and conjugate vaccines.

Establishment of the first International Standard for human anti-typhoid capsular Vi polysaccharide IgG.

Vi capsular polysaccharide (Vi) conjugate vaccines, which can prevent typhoid in infants and young children, are being developed. Comparative immunogenicity studies are facilitated by an International Standard (IS) for human anti-Vi IgG. 16/138, a pool of sera from volunteers which received either Vi conjugate vaccine or plain Vi vaccine, was assessed as an IS alongside U.S. reference reagent Vi-IgGR1, 2011. Samples were tested in a commercial ELISA (n = 7), a standardised ELISA based on biotinylated Vi (n = 7) and in-house ELISAs (n = 7). Valid estimates were obtained for the potency of all samples in the commercial ELISA, and the commutability of 16/138 and Vi-IgGR1, 2011 was evident for the commercial ELISA and in-house ELISAs based on a coating of Vi and protein. The WHO Expert Committee on Biological Standardization established 16/138 as the first IS for anti-Vi IgG with 100 IU per ampoule and assigned 163 IU per vial of Vi-IgGR1, 2011.

Discovery and in vivo evaluation of alcohol-containing benzothiazoles as potent dual-targeting bacterial DNA supercoiling inhibitors.

A series of dual-targeting, alcohol-containing benzothiazoles has been identified with superior antibacterial activity and drug-like properties. Early lead benzothiazoles containing carboxylic acid moieties showed efficacy in a well-established in vivo model, but inferior drug-like properties demanded modifications of functionality capable of demonstrating superior efficacy. Eliminating the acid group in favor of hydrophilic alcohol moieties at C(5), as well as incorporating solubilizing groups at the C(7) position of the core ring provided potent, broad-spectrum Gram-positive antibacterial activity, lower protein binding, and markedly improved efficacy in vivo.

An improved small-molecule inhibitor of FtsZ with superior in vitro potency, drug-like properties, and in vivo efficacy.

The bacterial cell division protein FtsZ is an attractive target for small-molecule antibacterial drug discovery. Derivatives of 3-methoxybenzamide, including compound PC190723, have been reported to be potent and selective antistaphylococcal agents which exert their effects through the disruption of intracellular FtsZ function. Here, we report the further optimization of 3-methoxybenzamide derivatives towards a drug candidate. The in vitro and in vivo characterization of a more advanced lead compound, designated compound 1, is described. Compound 1 was potently antibacterial, with an average MIC of 0.12 µg/ml against all staphylococcal species, including methicillin- and multidrug-resistant Staphylococcus aureus and Staphylococcus epidermidis. Compound 1 inhibited an S. aureus strain carrying the G196A mutation in FtsZ, which confers resistance to PC190723. Like PC190723, compound 1 acted on whole bacterial cells by blocking cytokinesis. No interactions between compound 1 and a diverse panel of antibiotics were measured in checkerboard experiments. Compound 1 displayed suitable in vitro pharmaceutical properties and a favorable in vivo pharmacokinetic profile following intravenous and oral administration, with a calculated bioavailability of 82.0% in mice. Compound 1 demonstrated efficacy in a murine model of systemic S. aureus infection and caused a significant decrease in the bacterial load in the thigh infection model. A greater reduction in the number of S. aureus cells recovered from infected thighs, equivalent to 3.68 log units, than in those recovered from controls was achieved using a succinate prodrug of compound 1, which was designated compound 2. In summary, optimized derivatives of 3-methoxybenzamide may yield a first-in-class FtsZ inhibitor for the treatment of antibiotic-resistant staphylococcal infections.

Biological evaluation of benzothiazole ethyl urea inhibitors of bacterial type II topoisomerases.

The type II topoisomerases DNA gyrase (GyrA/GyrB) and topoisomerase IV (ParC/ParE) are well-validated targets for antibacterial drug discovery. Because of their structural and functional homology, these enzymes are amenable to dual targeting by a single ligand. In this study, two novel benzothiazole ethyl urea-based small molecules, designated compound A and compound B, were evaluated for their biochemical, antibacterial, and pharmacokinetic properties. The two compounds inhibited the ATPase activity of GyrB and ParE with 50% inhibitory concentrations of <0.1 µg/ml. Prevention of DNA supercoiling by DNA gyrase was also observed. Both compounds potently inhibited the growth of a range of bacterial organisms, including staphylococci, streptococci, enterococci, Clostridium difficile, and selected Gram-negative respiratory pathogens. MIC90s against clinical isolates ranged from 0.015 µg/ml for Streptococcus pneumoniae to 0.25 µg/ml for Staphylococcus aureus. No cross-resistance with common drug resistance phenotypes was observed. In addition, no synergistic or antagonistic interactions between compound A or compound B and other antibiotics, including the topoisomerase inhibitors novobiocin and levofloxacin, were detected in checkerboard experiments. The frequencies of spontaneous resistance for S. aureus were <2.3 × 10(-10) with compound A and <5.8 × 10(-11) with compound B at concentrations equivalent to 8× the MICs. These values indicate a multitargeting mechanism of action. The pharmacokinetic properties of both compounds were profiled in rats. Following intravenous administration, compound B showed approximately 3-fold improvement over compound A in terms of both clearance and the area under the concentration-time curve. The measured oral bioavailability of compound B was 47.7%.

Design, synthesis and biological evaluation of α-substituted isonipecotic acid benzothiazole analogues as potent bacterial type II topoisomerase inhibitors.

The discovery and optimisation of a new class of benzothiazole small molecules that inhibit bacterial DNA gyrase and topoisomerase IV are described. Antibacterial properties have been demonstrated by activity against DNA gyrase ATPase and potent activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes and Haemophilus influenzae. Further refinements to the scaffold designed to enhance drug-likeness included analogues bearing an α-substituent to the carboxylic acid group, resulting in excellent solubility and favourable pharmacokinetic properties.

Developing whole cell standards for the microbiome field.

BACKGROUND: Effective standardisation of the microbiome field is essential to facilitate global translational research and increase the reproducibility of microbiome studies. In this study, we describe the development and validation of a whole cell reference reagent specific to the gut microbiome by the UK National Institute for Biological Standards and Control. We also provide and test a two-step reporting framework to allow microbiome researchers to quickly and accurately validate choices of DNA extraction, sequencing, and bioinformatic pipelines. RESULTS: Using 20 strains that are commonly found in the gut, we developed a whole cell reference reagent (WC-Gut RR) for the evaluation of the DNA extraction protocols commonly used in microbiome pipelines. DNA was first analysed using the physicochemical measures of yield, integrity, and purity, which demonstrated kits widely differed in the quality of the DNA they produced. Importantly, the combination of the WC-Gut RR and the three physicochemical measures allowed us to differentiate clearly between kit performance. We next assessed the ability of WC-Gut RR to evaluate kit performance in the reconstitution of accurate taxonomic profiles. We applied a four-measure framework consisting of Sensitivity, false-positive relative abundance (FPRA), Diversity, and Similarity as previously described for DNA reagents. Using the WC-Gut RR and these four measures, we could reliably identify the DNA extraction kits' biases when using with both 16S rRNA sequencing and shotgun sequencing. Moreover, when combining this with complementary DNA standards, we could estimate the relative bias contributions of DNA extraction kits vs bioinformatic analysis. Finally, we assessed WC-Gut RR alongside other commercially available reagents. The analysis here clearly demonstrates that reagents of lower complexity, not composed of anaerobic and hard-to-lyse strains from the gut, can artificially inflate the performance of microbiome DNA extraction kits and bioinformatic pipelines. CONCLUSIONS: We produced a complex whole cell reagent that is specific for the gut microbiome and can be used to evaluate and benchmark DNA extractions in microbiome studies. Used alongside a DNA standard, the NIBSC DNA-Gut-Mix RR helps estimating where biases occur in microbiome pipelines. In the future, we aim to establish minimum thresholds for data quality through an interlaboratory collaborative study. Video Abstract.

Exploring how microbiome signatures change across inflammatory bowel disease conditions and disease locations.

Understanding the variables that influence microbiome studies is critical for successful translational research. Inflammatory bowel disease (IBD) is a complex group of diseases that can present at multiple locations within the Gastrointestinal tract. Here, using the FAMISHED study cohort, we aimed to investigate the relationship between IBD condition, IBD disease location, and the microbiome. Signatures of the microbiome, including measures of diversity, taxonomy, and functionality, all significantly differed across the three different IBD conditions, Crohn's disease (CD), ulcerative colitis (UC), and microscopic colitis (MC). Notably, when stratifying by disease location, patients with CD in the terminal ileum were more similar to healthy controls than patients with CD in the small bowel or colon, however no differences were observed at different disease locations across patients with UC. Change in taxonomic composition resulted in changes in function, with CD at each disease location, UC and MC all having unique functional dysbioses. CD patients in particular had deficiencies in Short-Chain Fatty Acid (SCFA) pathways. Our results demonstrate the complex relationship between IBD and the microbiome and highlight the need for consistent strategies for the stratification of clinical cohorts and downstream analysis to ensure results across microbiome studies and clinical trials are comparable.

Evidence of Extended Thermo-Stability of Typhoid Polysaccharide Conjugate Vaccines.

Typhoid conjugate vaccines (TCV) are effective in preventing enteric fever caused by Salmonella enterica serovar Typhi in Southeast Asia and Africa. To facilitate vaccination with the Vi capsular polysaccharide-tetanus toxoid conjugate vaccine, Typbar TCV, and allow it to be transported and stored outside a cold chain just prior to administration, an extended controlled-temperature conditions (ECTC) study was performed to confirm the quality of the vaccine at 40 °C for 3 days at the end of its shelf-life (36 months at 2-8 °C). Studies performed in parallel by the vaccine manufacturer, Bharat Biotech International Limited, and an independent national control laboratory (NIBSC) monitored its stability-indicating parameters: O-acetylation of the Vi polysaccharide, integrity of the polysaccharide-protein conjugate, and its molecular size and pH. ECTC samples stored at 40 °C and 45 °C in comparison with control samples stored at 4 °C and 55 or 56 °C, were shown to have stable O-acetylation and pH; only very slight increases in the percentage of free saccharide and corresponding decreases in molecular size were observed. The deoxycholate method for precipitating conjugated polysaccharide was very sensitive to small incremental increases in percentage of free saccharide, in line with storage temperature and duration. This extended ECTC study demonstrated minimal structural changes to the Vi polysaccharide and conjugate vaccine and a stable formulation following extended exposure to elevated temperatures for the desired durations. This outcome supports the manufacturer's ECTC claim for the vaccine to be allowed to be taken outside the cold chain before its administration.

Developing standards for the microbiome field.

BACKGROUND: Effective standardisation of methodologies to analyse the microbiome is essential to the entire microbiome community. Despite the microbiome field being established for over a decade, there are no accredited or certified reference materials available to the wider community. In this study, we describe the development of the first reference reagents produced by the National Institute for Biological Standards and Control (NIBSC) for microbiome analysis by next-generation sequencing. These can act as global working standards and will be evaluated as candidate World Health Organization International Reference Reagents. RESULTS: We developed the NIBSC DNA reference reagents Gut-Mix-RR and Gut-HiLo-RR and a four-measure framework for evaluation of bioinformatics tool and pipeline bias. Using these reagents and reporting system, we performed an independent evaluation of a variety of bioinformatics tools by analysing shotgun sequencing and 16S rRNA sequencing data generated from the Gut-Mix-RR and Gut-HiLo-RR. We demonstrate that key measures of microbiome health, such as diversity estimates, are largely inflated by the majority of bioinformatics tools. Across all tested tools, biases were present, with a clear trade-off occurring between sensitivity and the relative abundance of false positives in the final dataset. Using commercially available mock communities, we investigated how the composition of reference reagents may impact benchmarking studies. Reporting measures consistently changed when the same bioinformatics tools were used on different community compositions. This was influenced by both community complexity and taxonomy of species present. Both NIBSC reference reagents, which consisted of gut commensal species, proved to be the most challenging for the majority of bioinformatics tools tested. Going forward, we recommend the field uses site-specific reagents of a high complexity to ensure pipeline benchmarking is fit for purpose. CONCLUSIONS: If a consensus of acceptable levels of error can be agreed on, widespread adoption of these reference reagents will standardise downstream gut microbiome analyses. We propose to do this through a large open-invite collaborative study for multiple laboratories in 2020. Video Abstract.