The development and characterization of an E. coli O25B bioconjugate vaccine.

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

O-acetylation of typhoid capsular polysaccharide confers polysaccharide rigidity and immunodominance by masking additional epitopes.

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.

Characterization of the Salmonella Typhimurium core oligosaccharide and its reducing end 3-deoxy-d-manno-oct-2-ulosonic acid used for conjugate vaccine production.

One of the strategies adopted for the development of a bivalent conjugate vaccine against invasive nontyphoidal Salmonella consists of linking the O-antigen component of S. Typhimurium and S. Entertidis lipopolysaccharides to the carrier protein CRM197, a non-toxic variant of diphtheria toxin. The conjugation reaction uses the reducing end residue 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core to which the O-antigen chain is bound (OAg-core). OAg-core chains are cleaved from the lipid A directly in the fermentation broth by mild acid treatment. Kdo has been reported to undergo structural changes under these conditions and therefore the Kdo at the reducing end was thoroughly analysed to verify its structural integrity. For this purpose, low molecular mass OAg-core chains extracted from S. Typhimurium wild type bacteria and core oligosaccharides extracted from S. Typhimurium bacteria mutated not to produce O-antigen repeats were characterized by GLC-MS, MALDI-TOF-MS and NMR spectroscopy. Moreover, a combination of 1H-1H and 1H-13C experiments confirmed the linkage positions, sequence and structure of the octasaccharide core with 5-linked Kdo present at the reducing end in its native structure: α-GlcpNAc-(1→2)-α-Glcp-(1→2)-α-Galp-(1→3)-[α-Galp-(1→6)]-α-Glcp-(1→3)-[α-Hepp-(1→7)]-α-Hepp-(1→3)-α-Hepp-(1→5)-Kdo.

Investigation on Sugar-Protein Connectivity in Salmonella O-Antigen Glycoconjugate Vaccines.

Invasive nontyphoidal Salmonella disease, for which licensed vaccines are not available, is a leading cause of bloodstream infections in Africa. The O-antigen portion of lipopolysaccharide is a good target for protective immunity. Covalent conjugation of the O-antigen to a carrier protein increases its immunogenicity and O-antigen based glycoconjugate vaccines are currently under investigation at the preclinical stage. We developed a conjugation chemistry for linking O-antigen to CRM197 carrier protein, through sequential insertion of adipic acid dihydrazide (ADH) and adipic acid bis( N-hydroxysuccinimide) ester (SIDEA) as linkers, without impacting O-antigen chain epitopes. Here the resulting sugar-protein connectivity has been investigated in detail. The core portion of the lipopolysaccharide was used as a model molecule to prepare CRM197 conjugates, making structural investigations easier. The first step of reductive amination with ADH involves the terminal 3-deoxy-d- manno-oct-2-ulosonic acid (KDO) residue of the core region. The second reaction step resulted not to be selective, as SIDEA reacted with both ADH and pyrophosphorylethanolamine (PPEtN) of the core region, independently from the pH at which the reaction was performed. Peptide mapping analysis of the deglycosylated core-CRM197 conjugates confirmed that lysine residues of CRM197 were linked to SIDEA not only through KDO-ADH but also through PPEtN. This analysis also confirmed that the conjugation chemistry is random on the protein, involving a large number of lysine residues, particularly the surface exposed ones. The method for core-CRM197 characterization was successfully extended to O-antigen-CRM197 conjugate, confirming the results obtained with the core. This study not only allowed full characterization of OAg-CRM197 conjugates, but can be applied to optimize synthesis and characterization of other OAg-based glycoconjugate vaccines. Analytical methods to investigate saccharide-protein connectivity are also of fundamental importance to study the relationship between glycoconjugate structure and immune response induced.

Multiple Techniques for Size Determination of Generalized Modules for Membrane Antigens from Salmonella typhimurium and Salmonella enteritidis.

In the last years, outer membrane vesicles have attracted a lot of attention for the development of vaccines against bacterial pathogens. Extracellular vesicles can be obtained in high yields by genetic mutations, resulting in generalized modules for membrane antigens (GMMA). Methods to check the quality, consistency of production, and stability of GMMA vaccines are of fundamental importance. In this context, analytical methods for size distribution determination and verifying the integrity and possible aggregation of GMMA particles are strongly needed. Herein, GMMA particle size distribution has been evaluated by means of three different techniques. Dynamic light scattering (DLS), multiangle light scattering (MALS) coupled with high-performance liquid chromatography-size exclusion chromatography (SEC), and nanoparticle tracking analysis (NTA) have been compared to characterize GMMA from different mutants of Salmonella typhimurium and Salmonella enteritidis strains. We found that the presence of O-antigen chains on GMMA determined higher Z-average diameters by DLS compared to size estimation by MALS and that the hydrodynamic diameter increased with the number of O-antigen chains per GMMA particle. In the case of SEC-MALS, the size of the whole population better reflects the size of the most abundant particles, whereas DLS diameter is more influenced by the presence of larger particles in the sample. SEC-MALS and NTA are preferable to DLS for the analysis of bimodal samples, as they better distinguish populations of different size. MALS coupled to a size exclusion chromatography module also allows checking the purity of GMMA preparations, allowing determination of generally occurring contaminants such as soluble proteins and DNA. NTA permits real-time visualization with simultaneous tracking and counting of individual particles, but it is deeply dependent on the choice of data analysis parameters. All of the three techniques have provided complementary information leading to a more complete characterization of GMMA particles.

Structural determination of the polysaccharide isolated from biofilms produced by a clinical strain of Klebsiella pneumoniae.

Klebsiella pneumoniae are Gram negative opportunistic pathogens producing capsular (K) polysaccharides. Seventy-seven different K antigens have been described and they are the basis for K serotyping. Capsular polysaccharides are important virulence factors and have a relevant role for the structure of biofilm communities. Nevertheless, little information is available on the polysaccharides produced in biofilm matrices by Klebsiella spp. In the present study, a clinical isolate of Klebsiella pneumoniae was grown both on cellulose membranes deposited on agar plates, where it formed an adherent biofilm, and in liquid medium, where it formed floating biofilms (flocs). Extraction and purification of the polysaccharide fraction showed that only one main carbohydrate polymer was present in both adherent biofilms and flocs. Composition and linkage analysis, Smith degradation followed by ESI-MS, 1D and 2D NMR spectroscopy revealed that the polysaccharide belong to the type K24 and has the following structure.