ABSTRACT
A new generation multi-component vaccine, principally directed against serogroup B Neisseria meningitidis (4CMenB), has recently been developed. One of its components, identified through reverse vaccinology, is the neisserial heparin-binding antigen (NHBA) which is included in the formulation as a novel NHBA-GNA1030 fusion protein (NHBA-FP). We describe here the biophysical characteristics of this vaccine antigen to understand better its structural properties in solution and concurrent immunogenicity prior to formulation. By deliberately stressing the protein to lose its immune responses, we were able to study the protein's structural changes at the molecular level. The unmodified NHBA-FP was found to be mainly monomeric with mass of 67kDa and secondary structure dominated by ß-sheets and turns (57% average). The antigen was very stable in storage buffer. It could be forced to unfold in a low-salt buffer resulting in the exposure of one of its two tryptophan residues at 50°C. Long-term stress studies (10-15 days at 37°C) showed modification in the chromatographic and electrophoretic profiles with progressive degradation and aggregation. Since there was little change in secondary structure (as monitored by circular dichroism and tryptophan fluorescence spectroscopy), the loss of functional immunogenicity of the thermal stressed protein could be mainly attributed to the observed fragmentation and aggregation. We therefore conclude that the maintenance of the intact, non-fragmented state of the NHBA-FP is important to preserve its functional immunogenicity. This may thus be utilised as an assay for the control testing of the protein.
Subject(s)
Adhesins, Bacterial/immunology , Antigens, Bacterial/immunology , Meningococcal Vaccines/immunology , Neisseria meningitidis, Serogroup B/immunology , Recombinant Fusion Proteins/immunology , Adhesins, Bacterial/genetics , Adhesins, Bacterial/metabolism , Animals , Antibodies, Bacterial/biosynthesis , Antibodies, Bacterial/immunology , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Circular Dichroism , Heparin/immunology , Heparin/metabolism , Hot Temperature , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Meningococcal Vaccines/genetics , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Folding , Protein Stability , Protein Structure, Secondary , Proteolysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray IonizationABSTRACT
The physico-chemical characterization of NadADelta(351-405), a recombinant protein discovered by reverse vaccinology, component of a candidate vaccine against Neisseria meningitidis serotype B is presented. Analytical methods like mass spectrometry, electrophoresis, optical spectroscopy and SEC-MALLS have been applied to unveil the structure of NadADelta(351-405), and to evaluate Product-Related Substances. Moreover, analysis of the protein after intentional denaturation has been applied in order to challenge the chosen methods and to determine their appropriateness and specificity. All the obtained results were inserted in a model allowing in-depth understanding of the antigen NadADelta(351-405): it is present in solution as a homo-trimer, retaining a high percentage of alpha-helix secondary structure, and able to reassemble from monomeric subunits after thermal denaturation; this structural organization is consistent with that foreseen for MenB NadA (Neisseria Adhesin A). The analytical data sets produced during process development for clinical phases I-III material confirm product quality and manufacturing consistency.
Subject(s)
Adhesins, Bacterial/chemistry , Meningococcal Vaccines/chemistry , Neisseria meningitidis, Serogroup B/chemistry , Recombinant Proteins/chemistry , Adhesins, Bacterial/immunology , Amino Acid Sequence , Animals , Circular Dichroism , Female , Meningococcal Vaccines/immunology , Mice , Molecular Sequence Data , Neisseria meningitidis, Serogroup B/immunology , Peptide Mapping , Protein Conformation , Recombinant Proteins/immunology , Spectrometry, Fluorescence , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunologyABSTRACT
Bacterial capsular polysaccharides covalently linked to an appropriate carrier protein represent the best tool to induce a protective immune response against a wide range of bacterial diseases, such as meningococcal infections. We describe here the physico-chemical characterisation of glycoconjugate molecules designed to prepare a vaccine against Neisseria meningitidis serogroups A, C, W135 and Y. The use of a selective conjugation chemistry resulted in well characterised, reproducible and traceable glycoconjugate that can be consistently manufactured at large scale. A pool of physical and spectroscopic methods was used to establish glycosylation ratio, identity, molecular weight profiles, integrity of carrier protein and sites of glycosylation, assuring effective and consistent lots of vaccines.
Subject(s)
Meningitis, Meningococcal/prevention & control , Meningococcal Vaccines/chemistry , Meningococcal Vaccines/standards , Circular Dichroism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Vaccines, Conjugate/chemistry , Vaccines, Conjugate/standardsABSTRACT
A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.
