A stabilized glycomimetic conjugate vaccine inducing protective antibodies against Neisseria meningitidis serogroup A.

Neisseria meningitidis serogroup A capsular polysaccharide (MenA CPS) consists of (1 → 6)-2-acetamido-2-deoxy-α-D-mannopyranosyl phosphate repeating units, O-acetylated at position C3 or C4. Glycomimetics appear attractive to overcome the CPS intrinsic lability in physiological media, due to cleavage of the phosphodiester bridge, and to develop a stable vaccine with longer shelf life in liquid formulation. Here, we generate a series of non-acetylated carbaMenA oligomers which are proven more stable than the CPS. An octamer (DP8) inhibits the binding of a MenA specific bactericidal mAb and polyclonal serum to the CPS, and is selected for further in vivo testing. However, its CRM197 conjugate raises murine antibodies towards the non-acetylated CPS backbone, but not the natural acetylated form. Accordingly, random O-acetylation of the DP8 is performed, resulting in a structure (Ac-carbaMenA) showing improved inhibition of anti-MenA CPS antibody binding and, after conjugation to CRM197, eliciting anti-MenA protective murine antibodies, comparably to the vaccine benchmark.

Use of NMR as an analytical tool in the process development of conjugate vaccines against Haemophilus influenzae type b (Hib) and meningococcal serogroup A (MenA).

The native structure of the bacterial polysaccharide is the key immunogenic component of conjugate vaccines and antibodies raised against the polysaccharide structure are responsible for providing protection against the corresponding pathogen. The manufacturing process of conjugate vaccines is very complex and has various biological and chemical steps. It is important to monitor the process to ensure that the structural identity of the polysaccharide is maintained throughout the process. NMR spectroscopy can be used as a versatile analytical tool to monitor the structural integrity of the polysaccharide component from isolated polysaccharide to conjugate vaccine and for identifying different impurities generated during the process.

Spontaneous point mutations in the capsule synthesis locus leading to structural and functional changes of the capsule in serogroup A meningococcal populations.

Whole genome sequencing analysis of 100 Neisseria meningitidis serogroup A isolates has revealed that the csaABCD-ctrABCD-ctrEF capsule polysaccharide synthesis locus represents a spontaneous point mutation hotspot. Structural and functional properties of the capsule of 11 carriage and two disease isolates with non-synonymous point mutations or stop codons in capsule synthesis genes were analyzed for their capsular polysaccharide expression, recognition by antibodies and sensitivity to bactericidal killing. Eight of eleven carriage isolates presenting capsule locus mutations expressed no or reduced amounts of capsule. One isolate with a stop codon in the O-acetyltransferase gene expressed non-O-acetylated polysaccharide, and was not recognized by anti-capsule antibodies. Capsule and O-acetylation deficient mutants were resistant to complement deposition and killing mediated by anti-capsular antibodies, but not by anti-lipopolysaccharide antibodies. Two capsule polymerase mutants, one carriage and one case isolate, showed capsule over-expression and increased resistance against bactericidal activity of both capsule- and lipopolysaccharide-specific antibodies. Meningococci have developed multiple strategies for changing capsule expression and structure, which is relevant both for colonization and virulence. Here we show that point mutations in the capsule synthesis genes substantially contribute to the repertoire of genetic mechanisms in natural populations leading to variability in capsule expression.

Conformations of Neisseria meningitidis serogroup A and X polysaccharides: The effects of chain length and O-acetylation.

Neisseria meningitidis is a major cause of bacterial meningitis worldwide especially in Africa. The capsular polysaccharide (CPS) is the main virulence factor and the target antigen for polysaccharide and conjugate vaccines. The high burden of serogroup A disease in the Meningitis Belt of sub-Saharan Africa led to the introduction of MenAfriVac®, which has successfully reduced the number of cases of group A disease. However, several outbreaks caused by other serogroups have been reported, including those due to serogroup X. The capsular polysaccharides of serogroups A and X are both homopolymers of amino sugars (α-D-ManNAc and α-D-GlcNAc) containing phosphodiester linkages at C-6 and C-4, respectively. The similarity of the primary structures of the two polysaccharides suggests that serogroup A vaccination may provide cross-protection against serogroup X disease. Molecular dynamics simulations of a series of serogroup A and X oligosaccharides reveal that the MenA CPS behaves as a flexible random coil which becomes less conformationally defined as the length increases, whereas serogroup X forms a more stable regular helical structure. The presence of the MenX helix is supported by NMR analysis; it has four residues per turn and becomes more stable as the chain length increases. Licensed MenA vaccines are largely O-acetylated at C-3: simulations show that these O-acetyl groups are highly solvent exposed and their presence favors more extended conformations compared to the more compact conformations of MenA without O-acetylation. These findings may have implications for the design of optimal conjugate vaccines.

Evaluation of candidate international standards for meningococcal serogroups A and X polysaccharide.

Polysaccharide (PS) based meningococcal vaccines are primarily evaluated by physicochemical methods to ensure batches are consistently manufactured. As PS content is determined by different methods across numerous laboratories, there is a need for International Standards (IS) to calibrate the assays. Following the successful introduction of the WHO Meningococcal group C (MenC) IS in 2011, NIBSC initiated projects to prepare similar standards for groups A, W, Y and X (MenA/W/Y/X) to standardise all meningococcal- PS based vaccines. On the basis of results from a collaborative study to evaluate preparations of MenA and MenX PS, both were established by the WHO Expert Committee on Biological Standardization in Oct 2015 as; the First WHO International Standard for the Meningococcal Group A polysaccharide with a content of 0.845 ± 0.043 mg MenA PS per ampoule (expanded uncertainty with coverage factor of k=2.45 corresponding to a 95% level of confidence); the First WHO International Standard for the Meningococcal Group X polysaccharide with a content of 0.776 ± 0.089 mg MenX PS per ampoule (expanded uncertainty with coverage factor of k=2.45), as determined by quantitative NMR. The standards are available from NIBSC, who act as guardians and distributors of the material under the auspices of WHO.

Quantification of each serogroup polysaccharide of Neisseria meningitidis in A/C/Y/W-135-DT conjugate vaccine by high-performance anion-exchange chromatography-pulsed amperometric detection analysis.

Invasive bacterial meningitis caused by Neisseria meningitidis can be prevented by active immunization with meningococcal polysaccharide or polysaccharide-protein conjugate vaccines. In a tetravalent A/C/Y/W-135-DT meningococcal conjugate vaccine vial, or in a final formulated bulk, accurate identification and quantification of each polysaccharide are critical in product release. Determination of sialic acid serogroups (C, W-135, and Y) unambiguously is complex since all these serogroups contribute to the sialic acid monosaccharide peaks that overlap in the high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD). We report a quantification method that involves generation of monosaccharide standard plots for respective sugars mannosamine-6-phosphate, sialic acid, galactose- and glucose-derived from hydrolysis of mixtures of the four serogroups A, C, W, and Y reference polysaccharides. These plots were then used to obtain the unknown polysaccharide concentrations of A/C/Y/W-135 in vialed vaccine or from formulated final bulks. We also present our results of the HPAEC-PAD profiles on groups C, W-135, and Y polysaccharides when hydrolyzed individually and/or in mixtures to discuss the individual sialic acid peak contributions.

Four monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, C, Y and W135: its application in identity tests.

Murine hybridoma monoclonal antibodies (MAbs) were produced against the capsular polysaccharide (CPs) of serogroups A, C, W135 and Y meningococci (MenA, MenC, MenW, MenY) in order to develop immunological reagents for the identification of meningococcal polysaccharides. Each serogroup-specific MAb reacted with the CPs from its homologous serogroup only and did not react with CPs from the other three serogroups. The affinity constant (Ka) of the four MAbs measured by non-competitive ELISA was 6.62 × 10(9), 2.76 × 10(9), 1.48 × 10(9) and 3.8 × 10(9) M(-1) for MenA, MenC, MenW and MenY MAbs respectively. The application of these MAbs for identity tests was demonstrated by their abilities to correctly identify the CPs from serogroups A, C, W135 and Y in meningococcal CPs-based vaccines through ELISA. The MAbs obtained in this work are a very valuable set of tools for study meningococcal polysaccharides vaccines.

Synthesis and preliminary biological evaluation of carba analogues from Neisseria meningitidis A capsular polysaccharide.

The Gram-negative encapsulated bacterium Neisseria meningitidis type A (MenA) is a major cause of meningitis in developing countries, especially in the sub-Saharan region of Africa. The development and manufacture of an efficient glycoconjugate vaccine against MenA is greatly hampered by the poor hydrolytic stability of its capsular polysaccharide, consisting of (1→6)-linked 2-acetamido-2-deoxy-α-d-mannopyranosyl phosphate repeating units. The replacement of the ring oxygen with a methylene group to get a carbocyclic analogue leads to the loss of the acetalic character of the phosphodiester and consequently to the enhancement of its chemical stability. Here we report the synthesis of oligomers (mono-, di- and trisaccharide) of carba-N-acetylmannosamine-1-O-phosphate as candidates for stabilized analogues of the corresponding fragments of MenA capsular polysaccharide. Each of the synthesized compounds contains a phosphodiester-linked aminopropyl spacer at its reducing end to allow for protein conjugation. The inhibition abilities of the synthetic molecules were investigated by a competitive ELISA assay, showing that only the carba-disaccharide is recognized by a polyclonal anti-MenA serum with an affinity similar to a native MenA oligosaccharide with average polymerization degree of 3.

Relative stability of meningococcal serogroup A and X polysaccharides.

Prior to the introduction of the MenAfriVac™ serogroup A glycoconjugate vaccine in September 2010, serogroup A was the major epidemic disease-causing meningococcal serogroup in the African meningitis belt. However, recently serogroup X meningococcal (MenX) disease has received increased attention because of outbreaks recorded in this region, with increased endemic levels of MenX disease over the past 2 years. Whereas polysaccharide-protein conjugate vaccines against meningococcal serogroups A, C, W and Y (MenA, MenC, MenW, MenY) are on the market, a vaccine able to protect against MenX has never been achieved. The structure of serogroup A, C, W and Y meningococcal polysaccharides has been already fully elucidated by NMR. MenX capsular polysaccharide (MenX CPS) structure is also documented but fewer characterization data have been published. We have applied here (1)H NMR, (31)P NMR and HPLC to evaluate the stability of MenX CPS in aqueous solution as compared to MenA capsular polysaccharide (MenA CPS). The stability study demonstrated that MenA CPS is more susceptible to hydrolytic degradation than MenX CPS. The different stereochemistry of the N-acetyl group at position C(2) of mannosamine (MenA CPS) and glucosamine (MenX CPS) respectively might play a fundamental role in this susceptibility to polysaccharide chain degradation. The satisfactory stability of MenX CPS predicts the possibility that a stable fully-liquid MenX polysaccharide or glycoconjugate vaccine could be developed.

Purified capsular polysaccharide of Neisseria meningitidis serogroup A as immune potentiator for antibody production.

The development of new immune potentiators for human vaccines is an important and expanding field of research. In the present study, the ability of the capsular polysaccharide from Neisseria meningitidis serogroup A (CPS-A), a mannose-containing carbohydrate, to enhance the antibody production against a co-administered model vaccine antigen, is examined. A protein-meningococcal serogroup C capsular polysaccharide (CPS-C) conjugate was selected as the model antigen for this study. After subcutaneous immunization of Balb/C mice, the conjugate mixed with CPS-A induced higher anti-CPS-C IgG and IgG(2a) antibody levels and higher anti-meningococcal serogroup C bactericidal titers than the conjugate alone or mixed with CPS-C. The immuno-stimulatory properties exhibited by CPS-A and the fact that vaccines based on purified CPS-A has been safely used during decades to fight the serogroup A meningococcal disease, support the proposal to use CPS-A as immune potentiator for human vaccination studies.

Conformational studies of the capsular polysaccharide produced by Neisseria meningitidis group A.

The effect of different cations on the conformational and morphological properties of the capsular polysaccharide produced by Neisseria meningitidis group A was investigated. Circular dichroism studies showed that the presence of Na(+), NH4+ or Ca(2+) ions induced different local conformations of the polysaccharide chain through interactions with the phosphodiester group bridging the saccharide residues in the polymer chain. Atomic force microscopy experiments confirmed that the morphology of the polysaccharide chains was different depending on the nature of the counterion. Ammonium ions were associated with the presence of single polymer chains in an elongated conformation, whereas sodium ions favored the folding of the chains into a globular conformation. The addition of calcium ions produced the aggregation of a limited number of globular polysaccharide chains to form a 'toroidal-like' structure.

Synthesis and biological evaluation of phosphono analogues of capsular polysaccharide fragments from Neisseria meningitidis A.

Neisseria meningitidis type A (MenA) is a Gram-negative encapsulated bacterium that may cause explosive epidemics of meningitis, especially in the sub-Saharan region of Africa. The development and manufacture of an efficient glycoconjugate vaccine against Neisseria meningitidis A is greatly hampered by the poor hydrolytic stability of its capsular polysaccharide, which is made up of (1-->6)-linked 2-acetamido-2-deoxy-alpha-D-mannopyranosyl phosphate repeating units. Since this chemical lability is a product of the inherent instability of the phosphodiester bridges, here we report the synthesis of phosphonoester-linked oligomers of N-acetyl mannosamine as candidates for stabilised analogues of the corresponding phosphate-bridged saccharides. The installation of each interglycosidic phosphonoester linkage was achieved by Mitsunobu coupling of a glycosyl C-phosphonate building block with the 6-OH moiety of a mannosaminyl residue. Each of the synthesised compounds contains an O-linked aminopropyl spacer at its reducing end (alpha- or beta-oriented) to allow for protein conjugation. The relative affinities of the synthetic molecules were investigated by a competitive ELISA assay and showed that a human polyclonal anti-MenA serum can recognise both the phosphonoester-bridged fragments 1-3 and their monomeric subunits, glycosides 20 and 21. Moreover, the biological results suggest that the abilities of these compounds to inhibit the binding of a specific antibody to MenA polysaccharide are dependent on the chain lengths of the molecules, but independent on the orientations of the anomeric linkers.

In vivo determination of Neisseria meningitidis serogroup A capsular polysaccharide by whole cell high-resolution magic angle spinning NMR spectroscopy.

High resolution-magic angle spinning (HRMAS) NMR spectroscopy was applied to serogroup A Neisseria meningitidis (NMA) to determine precise structures of capsular polysaccharide (CPS) expressed on the meningococcal surface. Both the O-acetylated (OAc) NMA parent and a mynC::aphA3 OAc- mutant demonstrated characteristic CPS-derived NMR signals indicating cell-surface expression of CPS, but only the parent expressed O-3 and O-4 acetylation signals. A capsule-defective strain showed no NMR signals for CPS. The (1)H NMR HRMAS spectral patterns correlated with the purified CPS (1)H NMR profiles. HRMAS NMR can distinguish detailed complex carbohydrate structures expressed on bacteria. NMA express both O-3 and O-4 acetylated polymers but not in equimolar ratio amounts in vivo.

Synthesis of structures corresponding to the capsular polysaccharide of Neisseria meningitidis group A.

Four differently substituted trimers of the CPS repeating unit have been synthesised in order to investigate the dependence on oligosaccharide size, acetylation and mode of phosphorylation of glycoconjugate vaccines against Neisseria meningitidis group A. A spacer-containing starting monomer, a H-phosphonate elongating monomer and a 6-O-phosphorylated H-phosphonate cap monomer have been synthesised and coupled together to afford, after deprotection, the target trimer structures differing in their acetylation and phosphorylation substitution pattern.

Development and characterisation of outer membrane vesicle vaccines against serogroup A Neisseria meningitidis.

Neisseria meningitidis bacteria of serogroup A are causing recurring meningitis epidemics on the African continent. An outer membrane vesicle (OMV) vaccine against serogroup A meningococci made from a subgroup III serogroup A meningococcal strain was previously shown to induce antibodies with serum bactericidal activity (SBA) in mice. We have here further investigated the properties of OMV vaccines made from five different subgroup III serogroup A meningococcal strains grown in a synthetic medium with low iron content. In addition to the major outer membrane proteins (PorA, PorB, RmpM, Opa and OpcA), small amounts of the NadA, TdfH, Omp85, FetA, FbpA and NspA outer membrane proteins, as well as lipooligosaccharides, were detected in the vaccines. The OMV vaccines were used to immunise mice. Anti-meningococcal IgG antibodies in the mouse sera were analysed by immunoblotting and by enzyme-linked immunosorbent assay against OMVs, and against live meningococcal cells in SBA and a flow-cytometric assay. The vaccines induced antibodies with high SBA and opsonophagocytic activity. The strongest IgG responses were directed against PorA. Significant SBA responses were also observed against a subgroup III strain, which did not express PorA, whereas no SBA was observed against a clone IV-1 serogroup A strain. An OMV vaccine from serogroup A meningococci may be an alternative to polysaccharide and conjugate polysaccharide vaccines for Africa.