Article pubs.acs.org/bc
Cite This: Bioconjugate Chem. 2018, 29, 1736−1747
Investigation on Sugar−Protein Connectivity in Salmonella O‑Antigen Glycoconjugate Vaccines Gianluigi De Benedetto,†,‡ Laura Salvini,§ Stefano Gotta,∥,# Paola Cescutti,‡ and Francesca Micoli*,† †
GSK Vaccines Institute for Global Health (GVGH) S.r.l., via Fiorentina 1, 53100 Siena, Italy Dipartimento di Scienze della Vita, Università degli Studi di Trieste, Ed. C11, via L. Giorgieri 1, 34127 Trieste, Italy § Fondazione Toscana Life Sciences, via Fiorentina 1, 53100 Siena, Italy ∥ GSK Vaccines S.r.l., via Fiorentina 1, 53100 Siena, Italy ‡
ABSTRACT: 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 Oantigen 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 Oantigen 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 3deoxy-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.
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INTRODUCTION Salmonella enterica serovars Typhimurium and Enteritidis are the leading cause of invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa.1 iNTS disease, for which licensed vaccines are not available, is a severe, extra-intestinal invasive bacteraemia, mostly contracted via food-borne transmission. iNTS septicaemia has a profound impact on the African population with malaria, anemia, HIV, and malnutrition and in children.2 3.4 million cases of iNTS disease occur globally each year. In Africa, iNTS disease incidence is high (227 cases per 100,000) and it is associated with case fatality rates of 20%.3 Antibiotics are not always available in African rural settings, and increasing levels of multidrug resistance are limiting their effectiveness.4,5 Difficulties of a proper and specific clinical diagnosis of the bloodstream infection make iNTS disease a high priority for vaccine development.6,7 Efforts are ongoing to identify promising targets of protective immunity and conjugation of the O-antigen portion of nontyphoidal Salmonella lipopolysaccharide (LPS) to an © 2018 American Chemical Society
appropriate carrier protein has been proposed as a strategy for vaccine development.8 LPS consists of a lipid A molecule linked to the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) terminus of a conserved core region, which is attached to the O-antigen chain. The serovar specific O-antigen is a repetitive glycan polymer and represents the immunodominant portion of the LPS molecule.9−11 We have proposed a bivalent vaccine against Salmonella Typhimurium and Enteritidis, by independent chemical conjugation of their OAg (the O-antigen chain repeating units and the core region are here referred to as OAg) to the carrier protein CRM197, a nontoxic recombinant form of diphtheria toxin.12,13 OAg-CRM197 conjugates induced functional OAg-specific serum antibodies in mice13−16 and were protective in a mouse challenge study.12 The conjugation Received: March 9, 2018 Revised: April 20, 2018 Published: April 26, 2018 1736
DOI: 10.1021/acs.bioconjchem.8b00178 Bioconjugate Chem. 2018, 29, 1736−1747
Article
Bioconjugate Chemistry
resolution electrospray ionization mass spectrometry (HR-ESIMS) analysis (negative ion mode) of core-ADH confirmed the effective involvement of KDO ketone group in reductive amination with ADH. The doubly charged ion at 1052.3 u (z = 2) was observed and the accurate mass, measured at resolution 35,000, corresponded to the elemental composition of C69H123O61N6P3 (0.33 ppm), as expected for the product of the reaction (Figure 3A). The above-mentioned ion was analyzed by high resolution tandem mass spectrometry (MS/MS or MS2), to elucidate the fragmentation pathway. Among many species yielded by loss of PPEtN, PEtN, ethanolamine (EtN), phosphate (P), and water (H2O), ions at 790.2 and 667.2 u (z = 1) were observed, attributed to the species Hep(PPEtN)-KDO-ADH and Hep(P)KDO-ADH, respectively. High-performance liquid chromatography−size exclusion chromatography (HPLC-SEC) analysis of core-ADH showed a single peak (refractive index detection), confirming no dimer formation (data not shown). Core and Core-ADH Derivatization with SIDEA Linker. SIDEA linker was first reacted with underivatized core, to confirm PPEtN reactivity (Scheme 1a). Reaction conditions were adjusted to result in high degree of derivatization with no dimer formation, and with reduced hydrolysis of introduced active ester groups essential for conjugation to CRM197. The reaction was performed in the presence of HCl and with a large excess of SIDEA equivalents respect to amino (−NH2) groups of the core. MALDI-MS analysis confirmed introduction of SIDEA (ion at 2075.4 u in Figure 2B), with 64.4% of PPEtN groups derivatized, as estimated by TNBS colorimetric assay, and 75.6% of active ester groups available for subsequent conjugation, as determined by RP-HPLC analysis. Core-SIDEA intermediate was also analyzed by HPLC-SEC, which showed no dimer formation (data not shown). Reaction of core-ADH with SIDEA was then investigated (Scheme 1B). The presence of ADH could direct the reaction toward the linker introduced and make the reaction more selective, with no PPEtN involvement. Also in this case, MALDI-MS analysis revealed simultaneous reaction of SIDEA with both ADH-NH-NH2 and PPEtN-NH2 groups present on the oligosaccharide chains (ion at 2343.5 u in Figure 2C). No species with only one SIDEA linker were identified, including possible cyclic product deriving from linkage of SIDEA to ADH and PPEtN on the same molecule. 93% of total −NH2 groups were derivatized, with 47% active ester groups. In this case dimer formation was not completely abolished as revealed by the presence of a shoulder at higher molecular weight in a HPLC-SEC chromatogram (refractive index detection) (data not shown). Conjugation of Core Structures to CRM197 Carrier Protein. Once verified that reaction with SIDEA occurred on two different positions on core-ADH, we wanted to verify if both active ester groups were effectively involved in the conjugation to CRM197. Core-ADH-(SIDEA)2 and core-SIDEA were conjugated to CRM 197 . Analysis by HPLC-SEC (fluorescence detection) and sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the formation of conjugates with both oligosaccharides, showing a clear shift of the protein at higher molecular mass. MALDI-MS analysis revealed an average of 5 oligosaccharide chains linked per protein for both conjugates (Figure 4). Saccharide-Protein Connectivity Investigated by Mass Spectrometry in Both Core and OAg Conjugates.
chemistry adopted involves the KDO sugar at the end of the core region in the linkage to CRM197, without impact on Oantigen epitopes.17,18 Reductive amination of KDO with adipic acid dihydrazide (ADH) and activation of free ADH hydrazide group with adipic acid bis(N-hydroxysuccinimide) ester (SIDEA) are then followed by conjugation to the lysine (Lys) residues of CRM197.17 In a previous study we had verified that the pyrophosphorylethanolamine (PPEtN) substituent of the core region can also react with SIDEA and could be involved in the conjugation to CRM197.17 Here we report an indepth investigation of the linkers reaction selectivity on the OAg component and of the Lys residues involved in the glycosylation on CRM197. Analytical methods used in this study can be applied to other glycoconjugate vaccines to verify consistency of batch-to-batch production and to investigate the relationship between saccharide−protein connectivity and immune response.19−21 In fact, recent studies have suggested the possible role of linkage position, both on the saccharide chain and on the carrier protein, in modulating the immune response elicited by the corresponding glycoconjugate vaccines.22−25
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RESULTS With the aim of facilitating the investigation on OAg-CRM197 conjugate structure, the core region of Salmonella LPS molecules was used as model molecule (Figure 1).
Figure 1. Salmonella LPS core region structure, as reported in the literature for Salmonella strains.26 After acid hydrolysis for lipid A removal, only one terminal KDO remains attached to the core.
Core oligosaccharide was extracted and purified from Salmonella Typhimurium O-antigen-negative generalized modules for membrane antigens (GMMA) not producing Oantigen chain repeats, as previously reported.27,28 Extracted sugar was separated from residual proteins through P-10 chromatography, as confirmed by micro BCA analysis of purified core showing undetectable level of residual proteins (
