Browsing by Author "Ravenscroft, Neil"

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    The application of physicochemical methods for the analysis of small and complex pharmaceutical drugs
    (2003) Mabotha, Tebogo E; Ravenscroft, Neil; Jackson, Graham Ellis
    The application of physicochemical methods for the analysis of small and complex pharmaceutical drugs was investigated. The methods were applied to small chiral molecules and further extended to analysis of complex glycoconjugate vaccines.
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    Capsular polysaccharide conformations in pneumococcal serotypes 19F and 19A
    (Elsevier, 2015) Kuttel, Michelle M; Jackson, Graham E; Mafata, Mpho; Ravenscroft, Neil
    Streptococcus pneumoniae is a significant pathogen in children. Although the PCV7 pneumococcal conjugate vaccine has reduced pneumococcal disease, non-vaccine serotype 19A infection has increased, despite expectations of cross-protection from vaccine serotype 19F. Serotype 19A is included in the new PCV13 vaccine, but not in PCV10. In the solution simulations of 19F and 19A oligosaccharide chains reported here, both polysaccharides form unstructured random coils, with inflexible repeat units linked by mobile phosphodiester linkages. However, there are clear conformational differences. In the 19F repeat unit, the rhamnose residue is nearly orthogonal to the other residues, whereas 19A has residues in similar orientations. This finding is corroborated by key inter-residue distances calculated from NMR NOESY experiments. Further, 19F is predominantly in extended conformations, whereas 19A exhibits a high prevalence of tight hairpin bends. These conformational differences may account for a lack of antibody cross-protection between serotypes 19F and 19A.
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    Chemical and spectroscopic studies of the capsular polysaccharides of some Klebsiella serotypes
    (1988) Ravenscroft, Neil; Stephen, A M
    As part of an international collaborative programme concerned with the elucidation of the molecular structures of capsular polysaccharides (the K-antigen) produced by strains of the bacterial genus Klebsiella, the capsular material of serotype K71 has been investigated, and that of serotypes K36 and K64 re-examined, by novel enzymic and spectroscopic methods. The cultivation and employment of bacteriophages which are capable of cleaving (by specific glycanase action) the isolated, cognate bacterial polysaccharide in vitro has yielded highly significant oligosaccharides. These may represent the repeating unit in the polysaccharide or be derivatives resulting from conversion of uronic acid to the 4,5-unsaturated analogue where, as found for serotype K64, the mode of cleavage is β-elimination not hydrolysis. The oligosaccharides thus generated have proved to be far more amenable to chemical and spectroscopic studies than their parent polymers, thereby facilitating complete characterisation of the molecular structures of the original polysaccharides. Chemical methods applied to these oligosaccharides included specific degradations by periodate oxidation and acid-, alkali- or enzyme- catalysed hydrolysis, products being identified by methylation analysis (involving the extensive use of gas-liquid chromatography coupled to mass spectrometry) and spectroscopic studies (mass and n.m.r.). During the course of these investigations it became apparent that the structures of the intact oligosaccharides (containing six or seven sugar residues) could be determined almost entirely from spectroscopic analysis, chiefly by detailed two-dimensional n.m.r. studies involving the use of high field spectrometers and the application of homo- and heteronuclear shift correlated spectroscopy, the sequence of sugar units being confirmed by mass spectrometric analysis of the permethylated derivatives. Methylation analysis of the oligosaccharides derived from Klebsiella serotype K36 proved that the glucuronic acid residue is linked through 0-2, and not 0-4 as published by others; this finding was corroborated during characterisation of the monomeric oligosaccharide by mass- and n.m.r. spectroscopy. Bacteriophage φ64 was shown to cleave the cognate K64 exopolysaccharide by a β-elimination process; the resulting hex-4-enuronic acid, present as a terminal group in the derived oligosaccharide was fully characterised by hydrogenation and g.l.c.-m.s. of acetylated products, and by detailed n.m.r. studies including long-range heteronuclear experiments. Finally the structure of the heptasaccharide repeating unit of the Klebsiella K71 capsular polysaccharide was established by spectroscopic analysis of the oligosaccharides derived by bacteriophage φ71 cleavage of the polymer; features of the proposed structure were confirmed by chemical degradation studies performed on the native polysaccharide.
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    Comparative evaluation of two carbohydrate force fields for modelling polysaccharide conformation
    (2022) Lazar, Ryan; Kuttel, Michelle; Ravenscroft, Neil; Akher, Farideh
    Modern carbohydrate simulation models have reached a level of maturity whereby their accuracy is often assumed. However, concerning differences have been reported when comparing the conformational predictions of rhamnose-rich polysaccharides between GLYCAM06 and other widely used carbohydrate force fields. This thesis investigates the scope and origin of these differences. We compare Molecular Dynamics simulations of strategically selected saccharide chains, with both the GLYCAM06 and CHARMM36 carbohydrate force fields. We find significant differences in the conformational predictions of the two force fields. More specifically, collapsed, globular conformations occur in the GLYCAM06 simulations, but are absent in the equivalent CHARMM36 results. The collapsing phenomenon is brought about by a gradual folding process, facilitated by instabilities in the GLYCAM06 a-L-Rha(1®X)-a-L-Rha glycosidic linkage that are stabilised by strong intramolecular interactions. The reduced consideration for repulsive Coulombic forces in GLYCAM06, originating from a collective lack of partial aliphatic hydrogen charges, is likely the principle factor behind these differences. This work suggests critical areas for refinement in GLYCAM06 that will be required for the force field to accurately model rhamnose-rich polysaccharides. The insights gained in this work have the potential to assist in the development of more accurate force fields for modelling carbohydrates.
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    Conformation and cross-protection in Group B Streptococcus serotype III and Streptococcus pneumoniae serotype 14: a molecular modeling study.
    (2019-02-13) Kuttel, Michelle Mary; Ravenscroft, Neil
    Although the branched capsular polysaccharides of Streptococcus agalactiae serotype III (GBSIII PS) and Streptococcus pneumoniae serotype 14 (Pn14 PS) differ only in the addition of a terminal sialic acid on the GBSIII PS side chains, these very similar polysaccharides are immunogenically distinct. Our simulations of GBSIII PS, Pn14 PS and the unbranched backbone polysaccharide provide a conformational rationale for the different antigenic epitopes identified for these PS. We find that side chains stabilize the proximal β dGlc(1→6) β dGlcNAc backbone linkage, restricting rotation and creating a well-defined conformational epitope at the branch point. This agrees with the glycotope structure recognized by an anti-GBSIII PS functional monoclonal antibody. We find the same dominant solution conformation for GBSIII and Pn14 PS: aside from the branch point, the backbone is very flexible with a “zig-zag” conformational habit, rather than the helix previously proposed for GBSIII PS. This suggests a common strategy for bacterial evasion of the host immune system: a flexible backbone that is less perceptible to the immune system, combined with conformationally-defined branch points presenting human-mimic epitopes. This work demonstrates how small structural features such as side chains can alter the conformation of a polysaccharide by restricting rotation around backbone linkages.
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    Conformational and Immunogenicity Studies of the Shigella flexneri Serogroup 6 O-Antigen: The Effect of O-Acetylation
    (2021-04-27) Richardson, Nicole Inge; Ravenscroft, Neil; Arato, Vanessa; Oldrini, Davide; Micoli, Francesca; Kuttel, Michelle M
    The pathogenic bacterium Shigella is a leading cause of diarrheal disease and mortality, disproportionately affecting young children in low-income countries. The increasing prevalence of antibiotic resistance in Shigella necessitates an effective vaccine, for which the bacterial lipopolysaccharide O-antigen is the primary target. S. flexneri serotype 6 has been proposed as a multivalent vaccine component to ensure broad protection against Shigella. We have previously explored the conformations of S. flexneri O-antigens from serogroups Y, 2, 3, and 5 that share a common saccharide backbone (serotype Y). Here we consider serogroup 6, which is of particular interest because of an altered backbone repeat unit with non-stoichiometric O-acetylation, the antigenic and immunogenic importance of which have yet to be established. Our simulations show significant conformational changes in serogroup 6 relative to the serotype Y backbone. We further find that O-acetylation has little effect on conformation and hence may not be essential for the antigenicity of serotype 6. This is corroborated by an in vivo study in mice, using Generalized Modules for Membrane Antigens (GMMA) as O-antigen delivery systems, that shows that O-acetylation does not have an impact on the immune response elicited by the S. flexneri serotype 6 O-antigen.
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    Desiccation-induced ultrastructural and biochemical changes in the leaves of the resurrection plant Myrothamnus flabellifolia
    (CSIRO Publishing, 2007) Moore, John P; Hearshaw, Meredith; Ravenscroft, Neil; Lindsey, George G; Farrant, Jill M; Brandt, Wolf F
    Light microscopy and low-temperature scanning electron microscopy were used to systematically compare the surface and internal ultrastructures of hydrated and desiccated leaves of the resurrection plant Myrothamnus flabellifolia (Welw.). This revealed that leaf tissue underwent considerable shrinkage and collapse on desiccation but was supported by a framework of vascular and sclerenchymous tissue, which is responsible for the fan-like shape of the leaves. In addition, the leaf ribs were covered with wax and an internal wax cuticle was observed. Biochemical analysis showed that the cyanidin 3-glucoside content increased on desiccation as did the trehalose and sucrose contents. Salt deposits were observed at the apices of desiccated leaves in the proximity of hydathode-like structures. We propose that this might regulate the leaf salt content since decreased intracellular cation concentration was observed in desiccated leaves. We believe that these unique adaptations contribute to the remarkable desiccation-tolerance properties of this plant.
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    The development of a process and quality control methods for conjugate vaccine against streptococcus pneumoniae serotype 1
    (2013) Edmonds-Smith, Cesarina; Ravenscroft, Neil; Wilson, Seanette
    Pneumonia is the leading cause of death in children worldwide and is estimated to kill 1.6 million children every year. Pneumonia affects children and families everywhere, but is most prevalent in sub–Saharan Africa and South–east Asia. Serotype 1 is responsible for up to 20 % of invasive pneumococcal diseases (IPD) in developing countries and has been the cause of several outbreaks in the African meningitis belt. Conjugate vaccines are effective in young children, induce immunological memory and reduce carriage. A conjugate vaccine against 7 serotypes (PCV7) was licensed in 2000 which resulted in a dramatic reduction of IPD. An increase in the number of cases due to non–vaccine serotypes (serotype replacement) led to the recent development and licensure of 10– and 13– valent conjugate vaccines that provide broader coverage. This thesis describes the development of purification and conjugation processes and associated analytical methods for the preparation of a Streptococcus pneumoniae serotype 1 polysaccharide (Pn1) conjugate vaccine. The Pn1 polysaccharide was purified following a two–step process utilising a differential filtration with ethanol. Analytical tests including size analysis, uronic acid composition, O–acetylation and purity (nucleic acids and protein) were optimized and performed on Pn 1 lots. The purified polysaccharide was found to meet World Health Organisation (WHO) specifications.The purified polysaccharide is viscous with a rigid structure that hampers full conjugation reactions and detailed characterisation. Size–reduction was performed and shown to have no impact on the structural integrity of the generated saccharide. The O–acetylated size–reduced polysaccharide was amenable to full nuclear magnetic resonance (NMR) characterisation to confirm the structural identity of Pn1 and determine the percentage of cell wall polysaccharide (CWPS) and the degree and position of O–acetylation present.Composition analysis was performed using published hydrolysis methods, however, they resulted in low recoveries and therefore alternative microwave assisted conditions were investigated followed by chromatographic separation and analysis. The size–reduced polysaccharide was conjugated to hydrazide–derivatized protein carriers via the polysaccharide carboxyl groups. The conjugates prepared using different activators were evaluated in mice and the immunogenicity data showed that they were non–inferior to two commercially available conjugate vaccines.
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    Development of an alternative synthesis of 2-acetamido-2-deoxy-L-altruronic acid: an unusual sugar found in the O-specific polysaccharide of Shigella sonnei
    (2017) Anderson, Kirstin P C; Gammon, David W; Ravenscroft, Neil
    A new synthetic route has been explored for the preparation of derivatives of 2-acetamido-2-deoxy-L-altruronic acid (L-AltNAcA). This is a rare sugar found together with 2-acetamido-4-amino-2,4-dideoxy-D-fucose(D-FucNAc4N) in the repeating unit of Shigella sonnei. Derivatives are needed inter alia for chemical and spectroscopic calibration standards, and as building blocks for preparing oligomeric subunits of the O-polysaccharide antigen for possible incorporation into a synthetic glycoconjugate vaccine. Two synthetic routes were investigated. The first route successfully repeated a published four step sequence converting diacetone-D-glucose to 1,6-anhydro--L-idopyranose in a 38% yield overall, and a further selective benzylation at O-3. Attempts to discriminate between O-2, O-3 and O-4 using low temperature acylation or alkylation conditions were unsuccessful, but modest selectivity for the 4-benzoate was observed in a Bu₂SnO-mediated benzoylation, although this product could not be easily separated from other mono-benzoates. The second route started from N-acetyl-D-glucosamine which was successfully converted in the first step to 2-methyl-(1,2-dideoxyl-5,6-O-isopropylidene-α-D-glucofurano)-[2,1-d]-2-oxazoline. The oxazoline and dioxolane units could be selectively manipulated in a series of steps to afford 2-acetamido-2-deoxy-3-O-benzyl-6-O-t-butyldimethylsilyl-α-D-glucofuranosyl acetate in a 41% yield over four steps. This is a key synthetic intermediate in which the 5-OH is available for the required inversion step. During this study, an unusual minor side-product, 1,6-anhydro-2-acetamido-O-acetyl-2-deoxy-3-O-benzyl-α-D-glucofuranose, was isolated. While this was also a potentially useful intermediate, having only the 5-OH unprotected, it proved not possible to find conditions for optimizing this product. Inversion of configuration at C-5 in the 6-O-silylated glucofuranose was attempted via the 5-O-triflate and 5-O-mesylate: the triflate formed but was displaced in situ by the solvent pyridine to give an unusual 5-pyridinium derivative, while the mesylate was stable but unreactive towards subsequent SN2 inversion. These outcomes were attributed to the steric congestion imposed by the combination of the 3,4-cis-disubstitution of the furanose ring and the very bulky silyl substituent at O-6. While the goal of preparing L-AltNAcA was not achieved via these approaches, useful insights have been contributed towards the ongoing study.
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    The development of appropriate methods for drug analysis at the Forensic Chemistry Laboratory, Cape Town
    (2008) Adams, Deidré Ilana; Ravenscroft, Neil; Lourens, Denise; Baard, André
    The Forensic Chemistry Laboratory, Cape Town, analyses samples submitted by forensic pathologists in order to assist with determining the cause of death in cases of unnatural death. Many of these samples test positive for the presence of drugs and other toxic substances. Because of resource constraints, pathologists submit samples at their discretion and not on a routine basis. In this study, forensic and chemical aspects were combined and used as the motivation for the development of an improved extraction procedure for systematic toxicological analysis. The scope of the study was therefore twofold. Firstly, a study was undertaken of unnatural deaths in the greater Cape Town area for which samples would not normally have been submitted.
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    The development of physico-chemical quality control methods for Haemophilus influenzae type b vaccine production
    (2013) Behr,Heinrich Richard; Ravenscroft, Neil
    The development of a low cost Haemophilus influenzaetype b (Hib) manufacturing platform at The Biovac Institute (TBI) required analytical method development in parallel with the production process development. Technology transfer enabled TBI to develop Hib vaccine production which could lead to the development of vaccine manufacturing capacity in subSaharan Africa. Initial studies were conducted in the Research and Development (R&D) department from where the process was transferred to the Good Manufacturing Process (GMP) environments of the Production and Quality Control departments respectively. Scaling of the development process to a process commercially viable required the development of additional quality control test methods. The quality control of Hib is performed by characterisation of the manufactured batch using physico-chemical analysis. The data generated are compared against that of a successful clinical trial batch. Animal based models for the potency and safety tests of Hib are ineffective. Chromatographic methods of analysis are often used in the pharmaceutical and biotechnological industry. Gas chromatography with flame ionisation detection (GC-FID) is a conventional technique used for the analysis of volatile analytes. The analysis of process residuals ethanol and ethylene glycol were performed using headspace or direct injection GC-FID analysis. Ethylene glycol, a non-volatile solvent, was chemically dried after which it was derivatised with a trimethylsilylating reagent. In addition, a method was developed to determine polyribosylribitolphosphate. Samples were dried by means of lyophilisation and then subjected to methanolysis to yield methyl glycosides. A trimethylsilylating reagent was used to volatilise the analyte and analysis was performed using GC-FID with direct injection. The use of internal standards throughout the sample preparation processes minimised errors due to sample handling, processing or injector reproducibility. Analytical method validation parameters were applied to the developed methods.
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    Evaluation of Critical Quality Attributes of a Pentavalent (A, C, Y, W, X) Meningococcal Conjugate Vaccine for Global Use
    (2021-07-23) Bolgiano, Barbara; Moran, Eilís; Beresford, Nicola J.; Gao, Fang; Care, Rory; Desai, Trusha; Nordgren, Ida Karin; Rudd, Timothy R.; Feavers, Ian M.; Bore, Prashant; Patni, Sushil; Gavade, Vinay; Mallya, Asha; Kale, Sameer; Sharma, Pankaj; Goel, Sunil K; Gairola, Sunil; Hattarki, Suhas; Avalaskar, Nikhil; Sarma, Annamraju D; LaForce, Marc; Ravenscroft, Neil; Khandke, Lakshmi; Alderson, Mark R; Dhere, Rajeev M; Pisal, Sambhaji S
    Towards achieving the goal of eliminating epidemic outbreaks of meningococcal disease in the African meningitis belt, a pentavalent glycoconjugate vaccine (NmCV-5) has been developed to protect against Neisseria meningitidis serogroups A, C, Y, W and X. MenA and X polysaccharides are conjugated to tetanus toxoid (TT) while MenC, Y and W polysaccharides are conjugated to recombinant cross reactive material 197 (rCRM197), a non-toxic genetic variant of diphtheria toxin. This study describes quality control testing performed by the manufacturer, Serum Institute of India Private Limited (SIIPL), and the independent control laboratory of the U.K. (NIBSC) on seven clinical lots of the vaccine to ensure its potency, purity, safety and consistency of its manufacturing. In addition to monitoring upstream-manufactured components, samples of drug substance, final drug product and stability samples were evaluated. This paper focuses on the comparison of the vaccine’s critical quality attributes and reviews key indicators of its stability and immunogenicity. Comparable results were obtained by the two laboratories demonstrating sufficient levels of polysaccharide O-acetylation, consistency in size of the bulk conjugate molecules, integrity of the conjugated saccharides in the drug substance and drug product, and acceptable endotoxin content in the final drug product. The freeze-dried vaccine in 5-dose vials was stable based on molecular sizing and free saccharide assays. Lot-to-lot manufacturing consistency was also demonstrated in preclinical studies for polysaccharide-specific IgG and complement-dependent serum bactericidal activity for each serogroup. This study demonstrates the high quality and stability of NmCV-5, which is now undergoing Phase 3 clinical trials in Africa and India.
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    Investigation of the effect of hydroxycitric acid on urinary calcium oxalate risk factors for kidney stone formation in artificial urine: theoretical modelling and in vitro crystallisation experiments
    (2020) Ahmed, Amouna; Ravenscroft, Neil; Jackson, Graham; Rodgers, Allen
    Nephrolithiasis, or urolithiasis, commonly known as kidney stones, is a common medical problem. Kidney stones are composed of different mineral types. Calcium Oxalate is the most common kind of stone. The principal aim of this research was to establish whether hydroxycitrate (HCA) affects and/or inhibits calcium oxalate crystallisation. A three-pronged approach was adopted, involving the determination of thermodynamic binding constants for Ca, Mg and Zn-HCA complexes, theoretical modelling of HCA-complex formation in artificial urine and in vitro crystallisation experiments. A potentiometric analysis was conducted to determine thermodynamic binding constants. These were included in the database of the JESS computer program to model the effect of HCA on the urinary supersaturation of calcium salts. A 1 mM HCA concentrations successfully decreased the concentration of ionised calcium and hence the urinary supersaturation of calcium salts. The solution structures of H+ , Ca 2+, Mg 2+, and Zn 2+ -HCA complexes were investigated using 1H-NMR. For protonation, the results showed that the pKa values were too close to resolve and that several microstates were in rapid exchange. Similarly, for the metal complexes, several species were found to be in rapid exchange. Crystallisation experiments were conducted in artificial urine, to determine the effect of HCA on the thermodynamics and kinetics of crystallisation of calcium oxalate, which is the most common component of kidney stones. The effect of HCA on calcium oxalate metastable limit (MSL) and crystallisation kinetics were measured. The results confirmed those predicted by theoretical modelling. The MSL was significantly affected by 1 mM HCA. Also, 1 mM HCA increased the rate of CaOx crystallisation. Both these effects are favourable and decrease the risk of in vivo crystal and stone formation. This augurs well for the potential application of HCA as a therapeutic agent in the management of kidney stone disease. Such an outcome needs to be tested in human trials.
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    Investigation of the potential beneficial effects of supplemental polyunsaturated fatty acids and glycosaminoglycans on the risk factors for calcium oxalate kidney stone formation using theoretical, experimental and human models
    (2014) Gogwana, Pumeza Christine; Rodgers, Allen; Ravenscroft, Neil
    Introduction: Two hypotheses with regard to calcium oxalate (CaOx) renal stone formation were tested in this thesis. The first hypothesis is that fatty acid (FA) supplementation (n-6 and n-3) and chondroitin sulphate (CS) supplementation may reduce the plasma (FA) and urinary (FA and CS) risk factors for CaOx renal stone formation, and ultimately serve as therapeutic agents in the management of this disease. The notion that FAs may reduce plasma risk factors is based on previous studies which have shown that n-6 and n-3 FA supplementation reduces the concentrations of arachidonic acid, while the notion of an effect on urinary risk factors is based on reports of these supplements decreasing urinary calcium and/or oxalate excretion in animal and human studies. The notion of CS playing a role in reducing CaOx stone risk is based on its chemical structure which presents potential binding sites for calcium and magnesium. The second hypothesis is that black and white healthy South African subjects may respond differently to these dietary supplements and that these differences may provide insights which could account for the lower stone incidence in the former group compared to the latter. This hypothesis is based on the observation in many previous studies of different renal responses in the two race groups, to different dietary and supplemental challenges. FA’s and CS have not been previously investigated in this regard. Methods: These hypotheses were tested simultaneously by administering n-6 and n-3 FA supplements individually and in combination, and supplemental CS, to different groups of black and white healthy male subjects. For the FA studies, blood samples were analyzed for serum biomarkers (25-hydroxyvitamin 03 and triglycerides) for CaOx stone formation and FA profiles in plasma total phospholipids since arachidonic acid regulates calcium excretion. Urine samples were analyzed for individual CaOx stone risk factors, risk indices (Tiselius risk index and supersaturation (SS) of calcium oxalate, brushite and uric acid); crystallization experiments (metastable limit and crystal growth kinetics) were also conducted. For the CS studies, thermodynamic binding constants for calcium-CS and magnesium-CS complexes were determined by isothermal titration calorimetry. These constants were then used to model speciation in different urines using the computer program Joint Expert Speciation System (JESS), and to calculate supersaturation values under different urinary conditions. This was followed by in vitro crystallization experiments in which the effects of exogeneous CS on the CaOx metastable limit and CaOx crystallization kinetics were investigated in artificial and real urine samples. Finally, human studies were performed in which CS supplements were administered to subjects to test their efficacy on reducing the urinary risk factors for CaOx stone formation. Urines were analyzed and crystallization experiments were performed as described for the FA supplementation studies. Results: In the FA studies, favourable changes in the plasma CaOx stone risk factors were achieved by the supplementation of n-3 FA alone. Post-supplementation, the concentration of arachidonic acid in plasma total phospholipids was significantly reduced in both groups, thereby implying a reduction in urinary calcium excretion. However, FA supplementation had no positive effect on the urinary risk factors or on CaOx metastable limits and CaOx crystallization kinetics. In the CS studies, theoretical modelling showed that the reduction of ionized calcium concentrations can only be attainable at 100 times physiological concentrations of urinary CS and that the formation of the calcium-CS complex does not influence the urinary supersaturation of CaOx. The formation of the magnesium-CS complexes was unfavourable because it resulted in an increase in the concentrations of ionized oxalate, a risk factor for CaOx stone formation. The in vitro crystallization experiments showed that exogeneous CS at physiological and above physiological concentrations had no favourable effect on the metastable limit and crystal growth kinetics in all the tested urine samples. Finally, the human study showed that CS supplementation had no effect on urine chemistry and crystallization kinetics. Speciation calculations also showed that the SS values of CaOx and the concentrations of ionized calcium were not significantly changed by supplementation. Within groups, the effects of FA supplementation on the urinary risk factors were different. n-6 FA supplementation significantly increased magnesium and significantly decreased urate in the black group whereas in whites citrate, oxalate and potassium were significantly increased while ionized calcium was significantly reduced. In the n-3 FA study, magnesium was significantly increased and SS value of brushite was significantly decreased in whites. In the black group, there was no significant difference in the urinary risk factors after supplementation compared to baseline values. With regards to n-6 & n-3 FA supplementation, citrate was significantly increased while oxalate and SS CaOx were significantly decreased in the black group whereas in the white group, magnesium was significant increased. With regards to the CS study, SS values for brushite, tribasic calcium phosphate, hydroxylapatite and octacalcium phosphate decreased significantly in black subjects after CS supplementation, whereas the SS value for hydroxylapatite increased significantly in the white group. These effects could not be attributed to complexation of CS with calcium or magnesium. However, they are noteworthy because they are different in the two groups. Discussion: The results of these studies do not support the hypothesis that supplemental fatty acids or chondroitin sulfate have significant beneficial effects for reducing blood and urinary risk factors for calcium oxalate stone formation. Although the response to FA and CS supplementation was different between the two race groups, these findings did not provide new information to explain the difference in the incidence of calcium oxalate stone disease in the two population groups. The work described in this thesis provides a foundation for future studies in which CaOx stone patients, rather than healthy individuals are investigated.
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    Investigation of the role of dietary myo-inositol hexakisphosphate (phytate) on the relative risk of calcium oxalate kidney stone formation in black and white male South African subjects
    (2005) Lesotho, Ntlama; Rodgers, Allen; Ravenscroft, Neil
    Previous studies have shown that caJclum oxalate (CaOx) stone-formers have lower urinary concentrations of myo-inositol hexakisphosphate (phytate or IPe) than healthy individuals, that dietary intake of this substance leads to its increased urinary excretion and that it is an inhibitor of CaOx nucleation and growth In South Africa it has been reported that the black population has a higher dietary phytate intake than whites. The present study was undertaken to test the hypothesis that South African black subjects have higher urinary phytate levels than their white cOLlflterparts and that this contributes to the relative rarity of caOx kidney stone disease in this population group A modified indirect extraction/photometry method to measure urinary IPe was designed, developed and tested in the present study. This assay was then used to measure IPo in the urine of rural black and urban white subjects while on their free unrestricted diets In addition, urban black and white subjects each followed IPo-restricted followed by lPG-rich dietary protocols for a period of three days Urines were collected after administration of each protocol and were again analysed for IPe using the newly developed assay. Urines were then used in several crystallization experiments to measure the CaOx metastable limit, "C-oxalate deposition kinetics and inhiOition of CaOx crystal aggregation. The results showed that while on their free diets, rural blacks excreted significantly less IPs than urban whites despite their previously reported higher dietary intake of this substance This suggests that the renal handling of dietary IP
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    An investigation towards a conjugate vaccine against Streptococcus pneumoniae serotype 19A
    (2017) Trimmel, Astrid Joan; Ravenscroft, Neil; Wilson, Seanette
    Streptococcus pneumoniae is a human pathogen that causes invasive pneumococcal diseases (IPD) such as pneumonia, otitis media and sepsis particularly in children, the elderly, and patients with HIV, and other immunosuppressive conditions. Conjugate vaccines comprised of the bacterial surface polysaccharide conjugated to a carrier protein are very effective in protecting young children against disease by inducing immunological memory and reducing carriage of the bacteria. A pneumococcal conjugate vaccine against seven serotypes (PCV7) was licensed in 2000, which resulted in a dramatic reduction of IPD. However; there was a gradual increase in the number of cases due to non-vaccine serotypes (serotype replacement). Serotype 19A, not included in PCV7 as the structurally similar serotype 19F was assumed to crossprotect against 19A disease, emerged as the most prevalent serotype in several studies with significant presence in Sub-Saharan Africa and South-East Asia and is associated with multidrug resistance. As a result a 13-valent conjugate vaccine that includes serotype 19A was developed and licensed which provided broader coverage. The aim of the work presented in this thesis was to develop processes for the manufacture of 19A capsular polysaccharide (CPS) for the production of a conjugate vaccine based on work performed on the model Pn1. The cultivation process included clonal selection for the growth and isolation of a serotype 19A clone producing high levels of CPS and cultivation using disposable bag technology as an alternative to the traditional fermentor. The culture was inactivated at low temperatures using cold phenol to prevent CPS degradation and to improve the release of CPS from the bacteria. The culture was clarified using a scalable flow-through centrifugation process. The Pn19A polysaccharide was purified using a single step process utilizing differential filtration with ethanol. Analytical tests including identity, purity (from nucleic acid and protein) and size analysis were optimized and performed on Pn19A CPS lots. All purified batches of polysaccharide met World Health Organisation (WHO) specifications as defined in the Technical Report Series. Structural studies were performed on closely related CPS namely; 19F and 19A CPS, both of which contain a labile phosphodiester linkage. The composition of the polysaccharides determined by colorimetric assays was confirmed by hydrolysis and monomeric analysis using gas chromatography/mass spectroscopy (GC/MS) of the methyl glycoside derivatives. Use of 1H, 13C and 31P nuclear magnetic resonance (NMR) experiments confirmed the structure of the 19F and 19A CPS repeating units and permitted determination of the extent of the cell wall polysaccharide contamination. CPS was size-reduced by microfluidization prior to conjugation experiments using cyanylating chemistry and a model carrier protein bovine serum albumin (BSA) and tetanus toxoid (TT). Pn19F and Pn19A conjugates prepared using TT were subjected to thermal stability studies and demonstrated similar stability based on the free saccharide generated. This proof of concept study established small-scale processes that can be further optimized for the manufacture of a conjugate vaccine against Pn19A disease.
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    Molecular modeling of bacterial polysaccharide antigens to inform future vaccine development
    (2021) Hlozek, Jason; Ravenscroft, Neil; Kuttel, Michelle
    Polysaccharide conjugate vaccines have been pivotal in reducing the prevalence and severity of bacterial infectious diseases worldwide, preventing countless deaths. The effectiveness of a vaccine can be extended if the selected vaccine strains in a multivalent vaccine cross-protect against non-vaccine strains. Detailed knowledge of antigen structure and conformation is required for vaccine components to be rationally selected. However, experimental methods may not be able to ascertain the conformations of polysaccharide chains. To address this, molecular dynamics simulations can provide key theoretical insights on molecular conformation to rationalize cross-protection data and inform vaccine development. In this work, we use molecular dynamics to investigate the conformations of glycan antigens of Neisseria meningitidis and Shigella flexneri bacteria - causative agents of meningitis and diarrheal disease. For N. meningitidis, our modeling indicates that serogroup A is unlikely to cross-protect against serogroup X infection, justifying the inclusion of serogroup X in future multivalent meningococcal vaccines. We also find that a chemically-stable carba-analogue of serogroup A has significant conformational differences to the native serogroup A chain, which does not support its use as a suitable serogroup A vaccine replacement. Our simulations of S. flexneri glycan antigens (serogroups Y, 2, 3, and 5) identify heuristics for the effects of substitution on backbone conformation and supports a proposed vaccine containing serotypes 2a (with O-acetylation) and 3a that will provide broad crossprotection. These findings can guide the rational selection of vaccine components to result in next-generation vaccines with greater cost-effectiveness and improved disease coverage.
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    Molecular Modeling of the Shigella flexneri Serogroup 3 and 5 O-Antigens and Conformational Relationships for a Vaccine Containing Serotypes 2a and 3a
    (2020-11-02) Hlozek, Jason; Owen, Sara; Ravenscroft, Neil; Kuttel, Michelle M
    The pathogenic bacterium Shigella flexneri is a leading global cause of diarrheal disease. The O-antigen is the primary vaccine target and distinguishes the 30 serotypes reported. Except for serotype 6, all S. flexneri serotypes have a common backbone repeating unit (serotype Y), with variations in substitution creating the various serotypes. A quadrivalent vaccine containing serotypes 2a and 3a (as well as 6 and Shigella sonnei) is proposed to provide broad protection against non-vaccine S. flexneri serotypes through shared epitopes and conformations. Here we model the O-antigen (O-Ag) conformations of serogroups 3 and 5: a continuation of our ongoing systematic study of the S. flexneri O-antigens that began with serogroup 2. Our simulations show that S. flexneri serogroups 2, 3, and 5 all have flexible O-Ags, with substitutions of the backbone altering the chain conformations in different ways. Our analysis suggests three general heuristics for the effects of substitution on the Shigella O-Ag conformations: (1) substitution on rhamnose C reduces the extension of the O-Ag chain; (2) substitution at O-3 of rhamnose A restricts the O-Ags to predominantly helical conformations, (3) substitution at O-3 of rhamnose B has only a slight effect on conformation. The common O-Ag conformations across serotypes identified in this work support the assumption that a quadrivalent vaccine containing serotypes 2a and 3a could provide coverage against S. flexneri serotype 3b and serogroup 5.
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    Molecular Modeling of the Shigella flexneri Serogroup 3 and 5 O-Antigens and Conformational Relationships for a Vaccine Containing Serotypes 2a and 3a
    (2020-11-02) Hlozek, Jason; Owen, Sara; Ravenscroft, Neil; Kuttel, Michelle M.
    The pathogenic bacterium Shigella flexneri is a leading global cause of diarrheal disease. The O-antigen is the primary vaccine target and distinguishes the 30 serotypes reported. Except for serotype 6, all S. flexneri serotypes have a common backbone repeating unit (serotype Y), with variations in substitution creating the various serotypes. A quadrivalent vaccine containing serotypes 2a and 3a (as well as 6 and Shigella sonnei) is proposed to provide broad protection against non-vaccine S. flexneri serotypes through shared epitopes and conformations. Here we model the O-antigen (O-Ag) conformations of serogroups 3 and 5: a continuation of our ongoing systematic study of the S. flexneri O-antigens that began with serogroup 2. Our simulations show that S. flexneri serogroups 2, 3, and 5 all have flexible O-Ags, with substitutions of the backbone altering the chain conformations in different ways. Our analysis suggests three general heuristics for the effects of substitution on the Shigella O-Ag conformations: (1) substitution on rhamnose C reduces the extension of the O-Ag chain; (2) substitution at O-3 of rhamnose A restricts the O-Ags to predominantly helical conformations, (3) substitution at O-3 of rhamnose B has only a slight effect on conformation. The common O-Ag conformations across serotypes identified in this work support the assumption that a quadrivalent vaccine containing serotypes 2a and 3a could provide coverage against S. flexneri serotype 3b and serogroup 5.
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    Molecular modeling studies of carbohydrate vaccine antigens: informing the future of vaccine design
    (2024) Richardson, Nicole; Ravenscroft, Neil; Kuttel Michelle
    This thesis delves into the intricate world of carbohydrate-based vaccine antigens by employing molecular dynamics simulations to explore the link between their structure, conformation, and immune function. Through a series of four case studies focused on distinct antigen targets, this research aims to predict potential cross-reactivity and cross-protection, rationalize observed immunological reactivity, and provide valuable insights into key epitopes and mechanisms for antigen-antibody binding. The case studies encompass the following antigens: Haemophilus influenzae types a and b, Pasteurella multocida types B and E, Shigella flexneri serotype 6, and Streptococcus pneumoniae serogroup 10. Each case study investigates the conformational aspects of the target antigens and proposes mechanisms for observed immunological phenomena. The collective findings propose connections between structural features, conformation, and their functional implications in immune responses, drawing parallels across individual case studies to elucidate recurring motifs employed by pathogens such as antigen flexibility, structural modifications, and backbone shielding. By broadening the applicability of this molecular modeling methodology, this research extends its reach to new target antigens and pathogens, offering a complementary approach to establish structurefunction relationships and inform rational vaccine design. The continued application of this methodology to a diverse range of vaccine targets promises to expand the knowledge base in the field, potentially revealing additional features harnessed by pathogens to gain a competitive advantage and evade the immune system. As computational power continues to grow, the cost and time associated with modeling may decrease, further enhancing the accessibility of this methodology for future vaccine development endeavors.