In this work, we explore the effects of O-acetylation on the physical and immunological characteristics of the WHO International Standards of Vi polysaccharide (Vi) from both Citrobacter freundii and Salmonella enterica serovar Typhi. We find that, although structurally identical according to NMR, the two Vi standards have differences with respect to susceptibility to de-O-acetylation and viscosity in water. Vi standards from both species have equivalent mass and O-acetylation-dependent binding to a mouse monoclonal antibody and to anti-Vi polyclonal antisera, including the WHO International Standard for human anti-typhoid capsular Vi PS IgG. This study also confirms that human anti-Vi sera binds to completely de-O-acetylated Vi. Molecular dynamics simulations provide conformational rationales for the known effect of de-O-acetylation both on the viscosity and antigenicity of the Vi, demonstrating that de-O-acetylation has a very marked effect on the conformation and dynamic behavior of the Vi, changing the capsular polysaccharide from a rigid helix into a more flexible coil, as well as enhancing the strong interaction of the polysaccharide with sodium ions. Partial de-O-acetylation of Vi revealed hidden epitopes that were recognized by human and sheep anti-Vi PS immune sera. These findings have significance for the manufacture and evaluation of Vi vaccines.

An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8×10(6) g/mol to larger than 20×10(6) g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines.

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.

Physico-chemical analysis of pneumococcal polysaccharide (PS)-protein conjugate vaccine components used for two commercially licensed vaccines was performed to compare the serotype- and carrier pro...

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 andO-acetyl content of polysaccharide. We investigated a key stability-indicating method which measures the percent free saccharide ofSalmonella entericasubspeciesentericaserovar 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 againstSalmonellaTyphi andShigellaspecies is described in this paper. This study should aid the development and licensing of new glycoconjugate vaccines aimed at the prevention of enteric diseases.