1,721,094 research outputs found
Click Chemistry Applied to the Synthesis of Salmonella Typhimurium O-Antigen Glycoconjugate Vaccine on Solid Phase with Sugar Recycling
No full text: A solid-phase conjugation method was developed and applied to the synthesis of an O-antigen based glycoconjugate vaccine against Salmonella Typhimurium, with CRM197 as the carrier protein. Copper-free click chemistry was used as the conjugation chemistry, after derivatizing the sugar and the protein components with alkyne and azido linkers, respectively. This chemistry has the advantage of not deactivating functional groups during the conjugation step, thereby allowing the recycling of unreacted components. The activated carrier protein was adsorbed to an anion exchange matrix and quantitatively conjugated to the O-antigen. The resulting conjugate was eluted from the resin free of unconjugated sugar which was previously removed by simple washing steps. Unreacted O-antigen was recycled by addition to a new batch of resin-CRM197 resulting in further quantitative protein conjugation. This process has advantages in relation to reduction of costs for production of conjugate vaccines, allowing unreacted sugar recovery and simplifying the purification of the glycoconjugate
Exploring the variables influencing the immune response of traditional and innovative glycoconjugate vaccines
Full text linkVaccines are cost-effective tools for reducing morbidity and mortality caused by infectious diseases. The rapid evolution of pneumococcal conjugate vaccines, the introduction of tetravalent meningococcal conjugate vaccines, mass vaccination campaigns in Africa with a meningococcal A conjugate vaccine, and the recent licensure and introduction of glycoconjugates against S. Typhi underlie the continued importance of research on glycoconjugate vaccines. More innovative ways to produce carbohydrate-based vaccines have been developed over the years, including bioconjugation, Outer Membrane Vesicles (OMV) and the Multiple antigen-presenting system (MAPS). Several variables in the design of these vaccines can affect the induced immune responses. We review immunogenicity studies comparing conjugate vaccines that differ in design variables, such as saccharide chain length and conjugation chemistry, as well as carrier protein and saccharide to protein ratio. We evaluate how a better understanding of the effects of these different parameters is key to designing improved glycoconjugate vaccines
Impact and Control of Sugar Size in Glycoconjugate Vaccines
Full text linkGlycoconjugate vaccines have contributed enormously to reducing and controlling encapsulated bacterial infections for over thirty years. Glycoconjugate vaccines are based on a carbohydrate antigen that is covalently linked to a carrier protein; this is necessary to cause T cell responses for optimal immunogenicity, and to protect young children. Many interdependent parameters affect the immunogenicity of glycoconjugate vaccines, including the size of the saccharide antigen. Here, we examine and discuss the impact of glycan chain length on the efficacy of glycoconjugate vaccines and report the methods employed to size polysaccharide antigens, while highlighting the underlying reaction mechanisms. A better understanding of the impact of key parameters on the immunogenicity of glycoconjugates is critical to developing a new generation of highly effective vaccines
Strain selection for generation of O-antigen-based glycoconjugate vaccines against invasive nontyphoidal salmonella disease
Full text linkNontyphoidal Salmonellae, principally S. Typhimurium and S. Enteritidis, are a major cause of invasive bloodstream infections in sub-Saharan Africa with no vaccine currently available. Conjugation of lipopolysaccharide O-antigen to a carrier protein constitutes a promising vaccination strategy. Here we describe a rational process to select the most appropriate isolates of Salmonella as source of O-antigen for developing a bivalent glycoconjugate vaccine. We screened a library of 30 S. Typhimurium and 21 S. Enteritidis in order to identify the most suitable strains for large scale O-antigen production and generation of conjugate vaccines. Initial screening was based on growth characteristics, safety profile of the isolates, O-antigen production, and O-antigen characteristics in terms of molecular size, O-acetylation and glucosylation level and position, as determined by phenol sulfuric assay, NMR, HPLC-SEC and HPAEC-PAD. Three animal isolates for each serovar were identified and used to synthesize candidate glycoconjugate vaccines, using CRM197 as carrier protein. The immunogenicity of these conjugates and the functional activity of the induced antibodies was investigated by ELISA, serum bactericidal assay and flow cytometry. S. Typhimurium O-antigen showed high structural diversity, including O-acetylation of rhamnose in a Malawian invasive strain generating a specific immunodominant epitope. S. Typhimurium conjugates provoked an anti-O-antigen response primarily against the O:5 determinant. O-antigen from S. Enteritidis was structurally more homogeneous than from S. Typhimurium, and no idiosyncratic antibody responses were detected for the S. Enteritidis conjugates. Of the three initially selected isolates, two S. Typhimurium (1418 and 2189) and two S. Enteritidis (502 and 618) strains generated glycoconjugates able to induce high specific antibody levels with high breadth of serovar-specific strain coverage, and were selected for use in vaccine production. The strain selection approach described is potentially applicable to the development of glycoconjugate vaccines against other bacterial pathogens
Genetic susceptibility to invasive nontyphoidal Salmonella disease in African children
Full text linkNontyphoidal Salmonella (NTS) causes invasive, and frequently fatal, disease in African children. The burden of disease secondary to NTS reflects inadequacy of Salmonella-control strategies in Africa, with expanding antibiotic resistance, and no licensed anti-NTS vaccine. The delivery of improved interventions to prevent, diagnose, and treat invasive NTS (iNTS) infection, will be facilitated by an improved understanding of the biological determinants of susceptibility to iNTS, including host genetic factors.
To identify host genetic determinants of iNTS disease, we performed a GWAS and replication analysis of NTS bacteraemia in African children. This analysis identified and validated a common genetic variant in STAT4 associated with increased iNTS risk.
To characterise the function of the NTS-associated STAT4 variant, we utilised a genotype-selectable bioresource of healthy European adults and samples from African children with iNTS disease. In these experiments, the risk genotype at STAT4 is associated with reduced STAT4 RNA expression in stimulated leukocytes, and reduced IFNγ production in both ex vivo stimulated natural killer cells and in the serum of African children with acute NTS bacteraemia.
To validate genetic variation suggestively associated with NTS bacteraemia in the GWAS, NTS-associated loci with evidence of regulatory function were prioritised for functional characterisation. Using in vitro models of intracellular Salmonella infection and RNA interference, I characterise the role of a candidate NTS-susceptibility determinant, EVI5L, in Salmonella infections.
Finally, applying a pathway enrichment analysis to the NTS bacteraemia GWAS demonstrated that NTS-associated genetic variation in African children is enriched for methionine salvage enzymes. I further investigate the potential for host-pathogen interaction in this pathway, generating and characterising Salmonella mutants deficient in methionine metabolism.
Taken together, these data represent the first unbiased assessment of genetic susceptibility to iNTS disease in unselected populations. These results have important implications for the design of Salmonella-control strategies for use in Africa.</p
Understanding immunity to Neisseria gonorrhoeae and gonococcal outer membrane vesicles for the development of a vaccine against gonorrhoea
No full textGonorrhoea, the second commonest sexually transmitted infection after chlamydia, can lead to serious long-term consequences for reproductive health, including pelvic inflammatory disease and infertility. The high prevalence of multidrug-resistant gonococcal strains highlights the need for prevention strategies. The Neisseria meningitidis serogroup B vaccine, 4CMenB, has been shown to protect against gonorrhoea, suggesting the feasibility of developing an effective gonococcal vaccine. This thesis examines the immunogenicity of a novel outer membrane vesicle- (OMV)-based gonococcal vaccine candidate, GonoVac, in mice.
The immunogenicity of GonoVac, produced in shake flasks and fermenters, was compared to 4CMenB in dose-escalation studies. Systemic and mucosal antibody responses and cellular responses were characterised using ELISAs, serum bactericidal assays (SBA), and FluoroSpot assays. Persistence of the serological response to GonoVac was assessed in genetically distinct strains of mice (BALB/c, C57BL/6, and CD1). The ability of a range of adjuvants (Al(OH)₃, AlPO4, CpG-C ODN 2395, SMNP) to improve the immunogenicity of GonoVac was investigated.
GonoVac formulated with or without aluminium hydroxide induced stronger serum IgG responses and bactericidal activity against multiple global gonococcal strains compared with 4CMenB. GonoVac produced in shake flasks and fermenters induced equivalent immunogenicity, supporting scalability of production. Both GonoVac and 4CMenB induced serum anti-gonococcal IgG and SBA in different strains of mice that persisted for up to a year. Formulation of GonoVac with aluminium hydroxide increased gonococcal-specific IgG levels, characterised by a high IgG1:IgG2a ratio, but not ability to kill gonococcus, and induced IL-17A-producing splenocytes. In contrast, investigating other adjuvants, formulation with SMNP induced the highest levels of serum anti-gonococcal IgG, with lower IgG1:IgG2a and greater ability to kill gonococcus compared with aluminium hydroxide. SMNP was the only adjuvant to significantly increase cellular responses to GonoVac compared with naïve mice.
From these studies, GonoVac is highly immunogenic and a promising gonococcal vaccine candidate, which should now be assessed in clinical studies
Structural analysis of O-polysaccharide chains extracted from different Salmonella Typhimurium strains
No full textSalmonella Typhimurium is the major cause of invasive nontyphoidal Salmonella disease in Africa, with high mortality among children and HIV-infected individuals. Currently, no vaccine is available for use in humans. Antibodies directed against the O-polysaccharide of the lipopolysaccharide molecule of Salmonella mediate bacterial killing and are protective, and conjugation of the O-polysaccharide to a carrier protein represents a possible strategy for vaccine development.
Here we have purified the O-polysaccharide from six different strains of S. Typhimurium and fully characterized them using analytical methods including HPLC–SEC, HPAEC-PAD, GC, GC–MS, 1D and 2D NMR spectroscopy. All the O-polysaccharide samples showed a similar bimodal molecular mass distribution,
but differed with respect to the amount and position of O-acetylation and glucosylation. For some strains,
O-acetyl groups were found not only on C-2 of abequose (factor 5 specificity), but also on C-2 and C-3 of rhamnose; glucose was found to be linked 1-4 or 1-6 to galactose in different amounts according to the strain of origin.
This structural variability could have an impact on the immunogenicity of corresponding glycoconjugate vaccines and different strains need to be evaluated in order to identify the appropriate source of O-polysaccharide to use for the development of a candidate conjugate vaccine with broad coverage against S. Typhimurium
Vaccines for low-income countries
No full textAbstractLow-income countries typically lag behind industrialised nations, where the introduction of new vaccines is commonly tailored to the pressures of the commercial market. Happily in recent years this paradigm has started to change with the introduction of a univalent meningococcal A conjugate vaccine that is specifically targeted for the prevention of epidemic meningitis in Africa. The declaration of the 2010s as a New Decade of Vaccines, together with Millennium Development Goals 4 and 5, provide a strong mandate for a new approach to the development of vaccines for low-income countries, so that there has never been a more exciting time to work in this field. This review considers the opportunities and challenges of developing these new vaccines in the context of innovations in vaccinology, the need to induce protective immunity in the populations at risk and the requirement for strong partnership between the countries that will use these vaccines and different elements of the vaccine industry
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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