Meningococcal polysaccharide vaccines: A review

Polysaccharides produced by Neisseria meningitidis are pharmaceutically important molecules, and are the active components of vaccines against N. meningitidis serogroups A, C, W135 and Y. Effective vaccines based on capsular polysaccharide, polysaccharide conjugates and outer membrane vesicles have been developed for strains expressing capsular polysaccharides that define the sero groups A, C, Y and W135. However, conventional approaches to develop a vaccine for group B strains have been largely unsuccessful. This review focuses on the various aspects of fermentative production of meningococcal polysaccharide from N. meningitidis, methods of conjugation for improving the immunogenicity of polysaccharide vaccine, and efficient and cost effective methods for the purification of N. meningitidis capsular polysaccharide and outer membrane vesicles. In addition, different analytical techniques for the quantitative determination of polysaccharide vaccine and evaluation of structural integrity of conjugate vaccine have been described.     

Glucogalactan: A polysaccharide isolated from the cell-wall of Verticillium Lecanii

A water-soluble polysaccharide was extracted with alkali from the cell wall of Verticillium lecanii (also called Lecanicillium lecanii). After freezing and thawing, the water-soluble fraction was purified by gel filtration chromatography on Sepharose CL-6B and eluted as one peak by HPSEC/RID. Monosaccharide analysis showed galactose and glucose (1.1:1), with traces of mannose (<1%). The structural characteristics were determined by spectroscopic analysis, FT-IR and 1D and 2D ¹H and ¹³C NMR, and methylation results. On the basis of the data obtained, the following structure of the polysaccharide (E₃S_IV fraction) was established:     

Purification of a conjugated polysaccharide vaccine using tangential flow diafiltration

Conjugated vaccines prepared from the capsular polysaccharide of Streptococcus pneumoniae can provide immunization against invasive pneumococcal disease, meningitis, and otitis media. One of the critical steps in the production of these vaccines is the removal of free (unreacted) polysaccharides from the protein-polysaccharide conjugate. Experimental studies were performed to evaluate the effects of membrane pore size, filtrate flux, and solution conditions on the transmission of both the conjugate and free polysaccharide through different ultrafiltration membranes. Conjugate purification was done using diafiltration performed in a linearly-scalable tangential flow filtration cassette. More than 98% of the free polysaccharide was removed within a 5-diavolume diafiltration process, which is a significant improvement over previously reported results for purification of similar conjugated vaccines. These results clearly demonstrate the opportunities for using ultrafiltration/diafiltration for the final purification of conjugated vaccine products.     

A novel water-soluble β-(1→6)-d-glucan isolated from the fruit bodies of Bulgaria inquinans (Fries)

A low molecular-weight polysaccharide named BIWP2 was purified from the fruit bodies of Bulgaria Inquinans (Fries) via hot-water extraction, followed by freeze-thawing and gel filtration chromatography on Sephadex G-75. Monosaccharide composition analysis revealed that BIWP2 contained exclusively glucose. High performance size exclusion chromatography (HPSEC) showed that it was a homogeneous polysaccharide fraction. Its molecular weight was estimated to be 2.6 KD and the polydispersity index (M_w/M_n) was calculated to be 1.4. Periodate oxidation, methylation, and NMR analyses indicated that BIWP2 was a linear β-(1→6)-d-glucan without side chains. This is the first time to report a linear β-(1→6)-d-glucan with low molecular weight in non-lichenized ascomycete.     

Increased efficiency of Campylobacter jejuni N-oligosaccharyltransferase PglB by structure-guided engineering

Conjugate vaccines belong to the most efficient preventive measures against life-threatening bacterial infections. Functional expression of N-oligosaccharyltransferase (N-OST) PglB of Campylobacter jejuni in Escherichia coli enables a simplified production of glycoconjugate vaccines in prokaryotic cells. Polysaccharide antigens of pathogenic bacteria can be covalently coupled to immunogenic acceptor proteins bearing engineered glycosylation sites. Transfer efficiency of PglB_Cj is low for certain heterologous polysaccharide substrates. In this study, we increased glycosylation rates for Salmonella enterica sv. Typhimurium LT2 O antigen (which lacks N-acetyl sugars) and Staphylococcus aureus CP5 polysaccharides by structure-guided engineering of PglB. A three-dimensional homology model of membrane-associated PglB_Cj, docked to the natural C. jejuni N-glycan attached to the acceptor peptide, was used to identify potential sugar-interacting residues as targets for mutagenesis. Saturation mutagenesis of an active site residue yielded the enhancing mutation N311V, which facilitated fivefold to 11-fold increased in vivo glycosylation rates as determined by glycoprotein-specific ELISA. Further rounds of in vitro evolution led to a triple mutant S80R-Q287P-N311V enabling a yield improvement of S. enterica LT2 glycoconjugates by a factor of 16. Our results demonstrate that bacterial N-OST can be tailored to specific polysaccharide substrates by structure-guided protein engineering.     

Effect of extraction temperature on the diffusion coefficient of polysaccharides from Spirulina and the optimal separation method

The extraction temperature had a significant impact on the concentration of polysaccharides derived from solid-liquid extraction of Spirulina. The polysaccharide concentration was significantly higher when the extraction was performed at 90°C than when it was performed at 80, 70, and 50°C. This result is related to the diffusion coefficients of the polysaccharides, which increased from 1.07 × 10^?12 at 50°C to 3.02 × 10^?12 m²/sec at 90°C. Using the Arrhenius equation, the pre-exponential factor (D ₀ ) and the activation energy (E _a ) for Spirulina polysaccharide extraction were calculated as 7.958 × 10^?9 m²/sec and 24.0 kJ/mol, respectively. Among the methods used for the separation of Spirulina polysaccharides, cetyltrimethylammonium bromide (CTAB, method I) and organic solvent (ethanol, in methods II and III) provided similar yields of polysaccharides. However, the separation of polysaccharides using an ultrafiltration (UF) process (method III) and ethanol precipitation was superior to separation via CTAB or vacuum rotary evaporation (method II). The use of a membrane with a molecular weight cut-off (MWCO) of 30 kDa and an area of 0.01 m² at a feed pressure of 103 kPa with a mean permeate flux of 39.3 L/m²/h and a retention rate of 95% was optimal for the UF process. The addition of two volumes (v/v) of ethanol, which gave a total polysaccharide content of approximately 4% dry weight, was found to be most suitable for polysaccharide precipitation. The results of a Sepharose 6B column separation showed that the molecular weights of the polysaccharides in fractions I and II were 212 and 12.6 kDa, respectively.     

Alignment of absolute and relative molecular size specifications for a polyvalent pneumococcal polysaccharide vaccine (PNEUMOVAX 23).

An approach was developed to align release and end-expiry specifications for molecular size for the polyvalent pneumococcal polysaccharide vaccine (PNEUMOVAX 23). Each of the 23 polysaccharide components of the vaccine was separately subjected to ultrasonication to produce a series of preparations of decreasing weight-average molecular mass (Mw). These size-reduced polysaccharides were analysed as monovalent solutions by high-performance size exclusion chromatography (HPSEC) with multi-angle laser light scattering (MALLS) and refractive index (RI) detection to measure their Mw. These samples were also analysed by HPSEC with rate nephelometry (RN) detection to measure their relative molecular size (r-MS). The data from the two molecular size measurements established a correlation between Mw and r-MS. For each polysaccharide component of the vaccine, this correlation permits the direct alignment of the r-MS specification in the final formulated product with the Mw specification for the monovalent polysaccharide preparation. The alignment of specifications provides a high level of assurance that the quality control of the final vaccine product is consistent with that of the polysaccharide starting materials.     

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.     

Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis

The global spread of enteric disease, the increasingly limited options for antimicrobial treatment and the need for effective eradication programs have resulted in an increased demand for glycoconjugate enteric vaccines, made with carbohydrate-based membrane components of the pathogen, and their precise characterisation. A set of physico-chemical and immunological tests are employed for complete vaccine characterisation and to ensure their consistency, potency, safety and stability, following the relevant World Health Organization and Pharmacopoeia guidelines. Variable requirements for analytical methods are linked to conjugate structure, carrier protein nature and size and O-acetyl content of polysaccharide. We investigated a key stability-indicating method which measures the percent free saccharide of Salmonella enterica subspecies enterica serovar Typhi capsular polysaccharide, by detergent precipitation, depolymerisation and HPAEC-PAD quantitation. Together with modern computational approaches, a more precise design of glycoconjugates is possible, allowing for improvements in solubility, structural conformation and stability, and immunogenicity of antigens, which may be applicable to a broad spectrum of vaccines. More validation experiments are required to establish the most effective and suitable methods for glycoconjugate analysis to bring uniformity to the existing protocols, although the need for product-specific approaches will apply, especially for the more complex vaccines. An overview of current and emerging analytical approaches for the characterisation of vaccines against Salmonella Typhi and Shigella species is described in this paper. This study should aid the development and licensing of new glycoconjugate vaccines aimed at the prevention of enteric diseases.     

The impact of protein-conjugate polysaccharide vaccines: an endgame for meningitis?

The development and implementation of conjugate polysaccharide vaccines against invasive bacterial diseases, specifically those caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, has been one of the most effective public health innovations of the last 25 years. These vaccines have resulted in significant reductions in childhood morbidity and mortality worldwide, with their effectiveness due in large part to their ability to induce long-lasting immunity in a range of age groups. At the population level this immunity reduces carriage and interrupts transmission resulting in herd immunity; however, these beneficial effects can be counterbalanced by the selection pressures that immunity against carriage can impose, potentially promoting the emergence and spread of virulent vaccine escape variants. Studies following the implementation of meningococcal serogroup C vaccines improved our understanding of these effects in relation to the biology of accidental pathogens such as the meningococcus. This understanding has enabled the refinement of the implementation of conjugate polysaccharide vaccines against meningitis-associated bacteria, and will be crucial in maintaining and improving vaccine control of these infections. To date there is little evidence for the spread of virulent vaccine escape variants of the meningococcus and H. influenzae, although this has been reported in pneumococci.     

Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations

For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa.     

Using pulse field gradient NMR diffusion measurements to define molecular size distributions in glycan preparations

Glycans comprise perhaps the largest biomass in nature, and more and more glycans are used in a number of applications, including those as pharmaceutical agents in the clinic. However, defining glycan molecular weight distributions during and after their preparation is not always straightforward. Here, we use pulse field gradient (PFG) ¹H NMR self-diffusion measurements to assess molecular weight distributions in various glycan preparations. Initially, we derived diffusion coefficients, D, on a series of dextrans with reported weight-average molecular weights from about 5 kDa to 150 kDa. For each dextran sample, we analyzed 15 diffusion decay curves, one from each of the 15 major ¹H resonance envelopes, to provide diffusion coefficients. By measuring D as a function of dextran concentration, we determined D at infinite dilution, D_inf, which allowed estimation of the hydrodynamic radius, R_h, using the Stokes-Einstein relationship. A plot of log D_inf versus log R_h was linear and provided a standard calibration curve from which R_h is estimated for other glycans. We then applied this methodology to investigate two other glycans, an α-(1→2)-l-rhamnosyl-α-(1→4)-d-galacturonosyl with quasi-randomly distributed, mostly terminal β(1→4)-linked galactose side-chains (GRG) and an α(1→6)-d-galacto-β(1→4)-d-mannan (Davanat), which is presently being tested against cancer in the clinic. Using the dextran-derived calibration curve, we find that average R_h values for GRG and Davanat are 76 ± 6 × 10⁻¹⁰ m and 56 ± 3 × 10⁻¹⁰ m, with GRG being more polydispersed than Davanat. Results from this study will be useful to investigators requiring knowledge of polysaccharide dispersity, needing to study polysaccharides under various solution conditions, or wanting to follow degradation of polysaccharides during production.     

Implications of the progressive self-association of wild-type human factor H for complement regulation and disease

Factor H (FH) is a major regulator of complement alternative pathway activation. It is composed of 20 short complement regulator (SCR) domains and is genetically associated as a risk factor for age-related macular degeneration. Previous studies on FH suggested that it existed in monomeric and dimeric forms. Improved X-ray scattering and analytical ultracentrifugation methodology for wild-type FH permitted a clarification of these oligomeric properties. Data at lower concentrations revealed a dependence of the X-ray radius of gyration values on concentration that corresponded to the weak self-association of FH. Global sedimentation equilibrium fits indicated that a monomer-dimer equilibrium best described the data up to 1.3 mg/ml with a fitted dissociation constant K_D of 28 μM and that higher oligomers formed at increased concentrations. The K_D showed that about 85-95% of serum FH will be monomeric in the absence of other factors. Size-distribution analyses in sedimentation velocity experiments showed that monomeric FH was the major species but that as many as six oligomeric forms co-existed with it. The data were explained in terms of two weak dimerisation sites recently identified in the SCR-6/8 and SCR-16/20 fragments of FH with similar K_D values. These observations indicate a mechanism for the progressive self-association of FH and may be relevant for complement regulation and the formation of drusen deposits that are associated with age-related macular degeneration.     

Dodonaea viscosa var. angustifolia leaf: New source of polysaccharide and its anti-oxidant activity

Ultrasonic technology was applied for polysaccharide extraction from the leaves of Dodonaea viscosa and response surface methodology (RSM) was used to optimize the effects of processing parameters on polysaccharide extraction yield. Three independent variables were extraction time (X₁), extraction temperature (X₂) and ultrasonic power (X₃), respectively. The statistical analysis indicated the independent variables (X₁, X₂, X₃), the quadratic terms (X₁₁ and X₃₃) and the interaction terms (X₁X₂, X₁X₃, X₂X₃) had significant effects on the yield of polysaccharides (P < 0.05). The optimal extraction conditions of D. viscosa leaf were determined as follows: extraction time 50.54 min, extraction temperature 85 °C and ultrasonic power 400 W. Under these conditions, the experimental yield of polysaccharides was 9.455 ± 0.24%, which was agreed closely with the predicted value (9.398%). The evaluation of anti-oxidant activity suggested that the polysaccharide exhibited significant protection against DPPH and hydroxyl radicals and could be explored as a nutraceutical agent.     

Synthesis of Staphylococcus aureus type 5 capsular polysaccharide repeating unit using novel l-FucNAc and d-FucNAc synthons and immunochemical evaluation

Staphylococcus aureus is a major cause of nosocomial infections. Glycoconjugates of type 5 and 8 capsular polysaccharides have been investigated for vaccine application. The proposed structure of type 5 polysaccharide is: →4-β-d-ManNAcA-(1→4)-α-l-FucNAc(3OAc)-(1→3)-β-d-FucNAc-(1→. The stereocontrolled insertion of these three glycosydic bonds is a real synthetic challenge. In the present paper we report the preparation of two novel versatile l- and d-fucosamine synthons from commercially available starting materials. In addition we applied the two building blocks to the synthesis of type 5 trisaccharide repeating unit. The immunochemical properties of the synthesized trisaccharide were assessed by competitive ELISA and by immunodot blot analysis using sera of mice immunized with type 5 polysaccharide conjugated to CRM₁₉₇. The results suggest that although the type 5 S. aureus trisaccharide is recognized by specific anti polysaccharide antibodies in dot blot, structures longer than the trisaccharide may be needed in order to significantly compete with the native type 5 polymer in the binding with sera from mice immunized with S. aureus type 5 polysaccharide–CRM₁₉₇ conjugate.     

Effect of acetylation on antioxidant and cytoprotective activity of polysaccharides isolated from pumpkin (Cucurbita pepo, lady godiva)

Acetylation of pumpkin (Cucurbita pepo, lady godiva variety) polysaccharide using acetic anhydride with pyridines as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale. Furthermore, antioxidant activities and cytoprotective effects of pumpkin polysaccharide and its acetylated derivatives were investigated employing various established in vitro systems. Results showed that addition of pyridine as catalyst could increase the degree of substitution, whereas volume of acetic anhydride had little effect. The acetylated polysaccharides in DPPH scavenging radical activity assay, superoxide anion radical activity assay and reducing power assay exhibited higher antioxidant activity than that of unmodified polysaccharide. H₂O₂-induced oxidative damages on rat thymic lymphocyte were also prevented by pumpkin polysaccharide and its acetylated derivatives and the derivatives presented higher protective effects. On the whole, acetylated polysaccharide showed relevant antioxidant activity both in vitro and in a cell system.     

Extraction, preliminary structural characterization, and antioxidant activities of polysaccharides from Salvia miltiorrhiza Bunge

Four polysaccharides were extracted from Salvia miltiorrhiza Bunge using hot water, ultrasonic, alkali, and enzyme methods. Preliminary structural characterization was conducted by physicochemical property, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) analyses. Antioxidant activities against 2,2-diphenyl-1-picryl-hydrazyl (DPPH), hydroxyl, and superoxide radicals were also evaluated. The physicochemical property analysis indicated the identicalness of the polysaccharide indices obtained by the hot water and ultrasonic methods. The indices obtained by the alkali and enzyme methods were significantly different. The FTIR spectra revealed the general characteristic absorption peaks of the four polysaccharides. The SEM images demonstrated significant differences in the surface features of the different polysaccharides. The antioxidant activity assay revealed the significant antioxidant activities of three polysaccharides. Overall, the polysaccharides from S. miltiorrhiza Bunge may have potential applications in the medical and food industries.     

EmrE dimerization depends on membrane environment

The small multi-drug resistant (SMR) transporter EmrE functions as a homodimer. Although the small size of EmrE would seem to make it an ideal model system, it can also make it challenging to work with. As a result, a great deal of controversy has surrounded even such basic questions as the oligomeric state. Here we show that the purified protein is a homodimer in isotropic bicelles with a monomer–dimer equilibrium constant (K_MD^2D) of 0.002–0.009 mol% for both the substrate-free and substrate-bound states. Thus, the dimer is stabilized in bicelles relative to detergent micelles where the K_MD^2D is only 0.8–0.95 mol% (Butler et al. 2004). In dilauroylphosphatidylcholine (DLPC) liposomes K_MD^2D is 0.0005–0.0008 mol% based on Förster resonance energy transfer (FRET) measurements, slightly tighter than bicelles. These results emphasize the importance of the lipid membrane in influencing dimer affinity.     

Development of approaches to creation of experimental test system for evaluation of antigenic activity of synthetic oligosaccharide ligands related to fragments of the Streptococcus pneumoniae type 14 capsular polysaccharide

A conjugate of a synthetic hexasaccharide fragment of the Streptococcus pneumoniae type 14 capsular polysaccharide with bovine serum albumin (BSA) has been prepared. The antigenic activity and specificity of this conjugate are comparable with those of natural antigens of S. pneumoniae type 14. The data suggest that the resulting synthetic conjugate can be used as a coating antigen in an experimental test system (based on enzyme immunoassay) for evaluating the antigenic activity and specificity of synthetic oligosaccharide ligands and for testing specimens of natural capsular polysaccharides and immune sera.     

Interpolation method for accurate affinity ranking of arrayed ligand–analyte interactions

The values of the affinity constants (k_d, k_a, and K_D) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (k_d and k_a) and dissociation equilibrium (K_D) constants for various ligand densities and analyte concentrations are exponentially interpolated to the K_D at R_max = 100 RU response level (K_D^R100).