Abstract Capsular polysaccharides are the principal constituents of S. pneumoniae capsules and major determinants of bacterial virulence. As key antigenic targets, they represent the foundation for pneumococcal vaccine development. Cell wall polysaccharides (CWPSs) commonly persist as a process-related impurity during fermentation and purification and therefore require reliable quantification throughout vaccine manufacturing. In this work, a multinuclear quantitative NMR strategy based on 1H and 31P detection was developed for the determination of CWPSs in purified pneumococcal polysaccharide preparations. The analytical workflow was evaluated using a representative panel of ten pneumococcal serotypes selected to encompass different polysaccharide structural classes, charge characteristics, and CWPS variants. Both NMR methods were demonstrated to be fit for the purpose, exhibiting specificity, linearity, and accuracy across the investigated concentration range. The complementary use of 1H NMR and 31P NMR enabled reliable CWPS quantification while providing additional structural information and discrimination between CWPS variants. The proposed multinuclear NMR approach represents a robust, selective, and quantitative analytical platform for monitoring CWPS impurities during pneumococcal polysaccharide vaccine development and quality control.

Glycosylation is the most critical post-translational modification occurring on proteins and its specific pattern depends both on the cells used for their production, such as mammalian cells, insect cells and yeast, and the expression conditions (e.g., mannosidase inhibitors, gene deletion). The aim of this study is to characterize the glycans of recombinant glycoprotein B (gB) from Human Cytomegalovirus (HCMV), expressed in CHO cells, by using different hyphenated mass spectrometry techniques in order to enrich the knowledge about glycosylation pattern changes in the protein production process and to develop versatile analytical platforms for the rapid analysis of protein glycosylation. The settled workflows focus on two main classes of analytes originating from the glycoprotein studied: glycopeptides and glycans. The analysis of the first category of molecules relies on a bottom-up proteomic approach in which the glycosites and the correspondent glycan heterogeneity and occupancy have been investigated by using high resolution tandem mass spectrometry; the analysis of the second class of molecules occurs by glycan shaving followed by fluorescence-liquid chromatography and mass-spectrometry characterization. These analyzes have been performed on the same glycoprotein expressed in a different culture media to demonstrate the capability of these analytical methods to monitor in detail oligosaccharide pattern changes. Finally, the impact of changes in glycosylation pattern on antigen immunogenicity has been assessed, showing that the presence of only high mannose oligosaccharides elicits higher titer of neutralizing antibodies.

Group B Streptococcus (GBS) is a bacterial pathogen associated with significant morbidity and mortality in pregnant women and infants, particularly in resource-limited settings. A hexavalent vaccine candidate in development incorporates the capsular polysaccharides (CPSs) from the most prevalent serotypes: Ia, Ib, II, III, IV, and V. Vaccine production is facilitated by a standardized CPS purification process. In the final purification step, a 30 kDa membrane filter gives high-yield recovery for five of the six CPSs, but <50 % for type II (GBSII), despite similar CPS structure and size. However, a smaller 10 kDa membrane improves recovery to about 90 %, suggesting that CPS conformation affects retention. Here comparative molecular modeling - corroborated by through-space NMR correlations - reveals that GBSII forms compact, globular conformations, while type III (GBSIII) forms an elongated zig-zag. This explains GBSII's poor retention during filtration: GBSII's compact globules pass through the 30 kDa membrane more easily than GBSIII's elongated forms. Additionally, we identify distinct epitopes and compare their interactions with a GBSIII-specific fragment antibody to clarify the lack of cross-reactivity between GBSII and GBSIII. This work provides valuable mechanistic insight into physically observed behavior to inform development of multivalent GBS vaccines to reduce maternal and infant mortality.

Carbohydrate-based therapeutic vaccines are actively pursued as targeted immunotherapy to treat cancer. Aberrant glycosylation is indeed of paramount importance in tumors, leading to the formation of “neo-epitopes”, known as tumor-associated carbohydrate antigens (TACAs), crucial in cancer onset, development and spread. Accordingly, the over-simplified mucin-type O-glycans Tn and STn have been confirmed among the most promising candidates for the development of cancer vaccines. In this work, we first propose genetically manipulated bacteria outer membrane vesicles (OMVs), namely GMMA, as a vaccine formulation platform to display glycan antigens. GMMA were glycosylated with multiple copies of structurally locked Tn mimetic or STn mimetic as cancer vaccine prototypes. These constructs, in non-adjuvanted formulations, showed sounding immunogenic properties in vivo and impressive efficacy in a mouse model of aggressive triple-negative breast cancer. This example of tailor-made therapeutic vaccine might revolutionize the approach to cancer therapy.

Glycosylation is the most prominent modification important for vaccines and its specific pattern depends on several factors that need to be considered when developing a new biopharmaceutical. Tailor-made glycosylation can be exploited to develop more effective and safer vaccines; for this reason, a deep understanding of both glycoengineering strategies and glycans structures and functions is required. In this review we discuss the recent advances concerning glycoprotein expression systems and the explanation of glycans immunomodulation mechanisms. Furthermore, we highlight how glycans tune the immunological properties among different vaccines platforms (whole virus, recombinant protein, nucleic acid), also comparing commercially available formulations and describing the state-of-the-art analytical technologies for glycosylation analysis. The whole review stresses the aspect of glycoprotein glycans as a potential tool to overcome nowadays medical needs in vaccine field.

Streptococcus pneumoniae is one of the globally important encapsulated human pathogens and more than 100 different serotypes have been identified. Despite very extensive genetic and immune-serological studies, the capsular polysaccharide repeating unit structure of several serotypes has not been determined yet, including the type 38 (type 38 in Danish nomenclature; type 71 in US nomenclature). Physicochemical data revealed that type 38 polysaccharide is composed of a pentasaccharide repeat unit →3)-[β-D-Galf(1→2)]-β-D-GalpA6(L-Ser)-(1→3)-α-D-GlcpNAc-(1→3)-α-D-Sugp-(1→4)-α-D-Galp(2OAc)-(1→. The polysaccharide is O-acetylated at position C2 of the α-Gal residue at approximately (68-87%) of the repeat units.

Several alum-adjuvanted vaccines have been licensed in the past 40 years. Despite its extensive and continuous use, the immune mechanism of action of alum adjuvants is not yet completely understood. Many different variables during the formulation process have been assessed as critical for alum-adjuvanted vaccines, although most of them are still not yet fully understood. The absence of a clear understanding of all the possible variables regulating the mechanism of action and the behavior that alum adjuvant imposes on the protein antigen may also be related to analytical challenges. For this reason, there is an urgent need for a fast and simple tool that is possible without a preliminary sample manipulation and is able to control the amount and the degree of antigen adsorption levels and their consistency across different production processes. This work attempts to develop new analytical tools with the aim of directly quantifying and assessing both the content and/or the purity of formulated alum-adsorbed antigens, without any preliminary sample manipulation (e.g., antigen desorption) being reported. In addition, the different confirmation/behavior in terms of the response to specific monoclonal antibodies in the presence of different ratios of alum-OH adsorbent antigens have been investigated. As a proxy to develop new analytical tools, three recombinant protein adsorbed models were used as follows: Neisseria adhesin A (NadA), Neisserial Heparin Binding Antigen (NHBA), and factor H binding protein (fHbp) as antigens, as well as aluminum hydroxide (AH) as an adjuvant system. The selection of the adjuvanted system model was dictated due to the substantial quantity of the literature regarding the protein structure and immunological activities, meaning that they are well characterized, including their adhesion rate to alum. In conclusion, three different analytical tools were explored to quantify, detect, and study the behavior of antigens in the presence of the alum adjuvant.

Glycoconjugate vaccines play a major role in the prevention of infectious diseases worldwide, with significant impact on global health, enabling the polysaccharides to induce immunogenicity in infants and immunological memory. Tetanus toxoid (TT), a chemically detoxified bacterial toxin, is among the few carrier proteins used in licensed glycoconjugate vaccines. The recombinant full-length 8MTT was engineered in E. coli with eight individual amino acid mutations to inactivate three toxin functions. Previous studies in mice showed that 8MTT elicits a strong IgG response, confers protection, and can be used as a carrier protein. Here, we compared 8MTT to traditional carrier proteins TT and cross-reactive material 197 (CRM197), using different polysaccharides as models: Group A Streptococcus cell-wall carbohydrate (GAC), Salmonella Typhi Vi, and Neisseria meningitidis serogroups A, C, W, and Y. The persistency of the antibodies induced, the ability of the glycoconjugates to elicit booster response after re-injection at a later time point, the eventual carrier-induced epitopic suppression, and immune interference in multicomponent formulations were also evaluated. Overall, immunogenicity responses obtained with 8MTT glycoconjugates were compared to those obtained with corresponding TT and, in some cases, were higher than those induced by CRM197 glycoconjugates. Our results support the use of 8MTT as a good alternative carrier protein for glycoconjugate vaccines, with advantages in terms of manufacturability compared to TT.

Several glycoconjugate-based vaccines against bacterial infections have been developed and licensed for human use. Polysaccharide (PS) analysis and characterization is therefore critical to profile the composition of polysaccharide-based vaccines. For PS content quantification, the majority of Ultra High Performance Liquid Chromatography (UHPLC) methods rely on the detection of selective monosaccharides constituting the PS repeating unit, therefore requiring chemical cleavage and tailored development: only a few methods directly quantify the intact PS. The introduction of charged aerosol detector (CAD) technology has improved the response of polysaccharide analytes, offering greater sensitivity than other detector sources (e.g., ELSD). Herein, we report the development of a universal UHPLC-CAD method (UniQS) for the quantification and quality evaluation of polysaccharide antigens (e.g., Streptococcus Pneumoniae, Neisseria meningitidis and Staphylococcus aureus). This work laid the foundation for a universal UHPLC-CAD format that could play an important role in future vaccine research and development helping to reduce time, efforts, and costs.

Multivalent vaccines addressing an increasing number of Streptococcus pneumoniae types (7-, 10-, 13-, 15-, 20-valent) have been licensed over the last 22 years. The use of polysaccharide-protein conjugate vaccines has been pivotal in reducing the incidence of invasive pneumococcal disease despite the emergence of non-vaccine serotypes. Notwithstanding its undoubtable success, some weaknesses have called for continuous improvement of pneumococcal vaccination. For instance, despite their inclusion in pneumococcal conjugate vaccines, there are challenges associated with some serotypes. In particular, Streptococcus pneumoniae type 3 remains a major cause of invasive pneumococcal disease in several countries.Here a deep revision of the strengths and weaknesses of the licensed pneumococcal conjugate vaccines and other vaccine candidates currently in clinical development is reported.

The increasing diffusion of antimicrobial resistance (AMR) across more and more bacterial species emphasizes the urgency of identifying innovative treatment strategies to counter its diffusion. Pathogen infection prevention is among the most effective strategies to prevent the spread of both disease and AMR. Since their discovery, vaccines have been the strongest prophylactic weapon against infectious diseases, with a multitude of different antigen types and formulative strategies developed over more than a century to protect populations from different pathogens. In this review, we review the main characteristics of vaccine formulations in use and under development against AMR pathogens, focusing on the importance of administering multiple antigens where possible, and the challenges associated with their development and production. The most relevant antigen classes and adjuvant systems are described, highlighting their mechanisms of action and presenting examples of their use in clinical trials against AMR. We also present an overview of the analytical and formulative strategies for multivalent vaccines, in which we discuss the complexities associated with mixing multiple components in a single formulation. This review emphasizes the importance of combining existing knowledge with advanced technologies within a Quality by Design development framework to efficiently develop vaccines against AMR pathogens.

For the preparation of glycoconjugate vaccines, polysaccharide antigens can usually be chemically modified to generate reactive functional groups (e.g., the formation of aldehyde groups by periodate oxidation of adjacent diols) for covalent coupling with proteins. In a recent issue of JBC, Duke et al. showed that an alternative agent, galactose oxidase (GOase) isolated from the fungus Fusarium sp. can generate aldehyde groups in a unique chemoenzymatic approach to prepare a conjugate vaccine against Streptococcus pneumoniae. These findings introduce a new strategy for the design and development of glycoconjugate vaccines.

Glycoconjugate vaccines have been effectively used in humans for about 40 years. The glycoconjugates have substituted plain polysaccharide vaccines that have many limitations, especially in infants. The covalent linking of protein to carbohydrates has allowed to overcome T-cell-dependent type-2 response of sugars. Glycoconjugates can show improved responses (over plain saccharides) also in elderly and immunocompromised (and depending on the endpoint also in immunocompetent adults), but infants represent the main target of these vaccines because of their unique immune system. Differently from the plain polysaccharide vaccines, the glycoconjugates are also able to induce Immunoglobulin G (IgG) response in infants.

GlaxoSmithKline (GSK) is currently developing a fully liquid presentation to ease the administration of the licensed quadrivalent conjugate vaccine (Menveo) against meningococcal serogroup A, C, W, and Y (MenACWY) infections. Herein, we report a new method for determining the free saccharide (FS) content of CRM197-MenACWY conjugated antigens, with the aim of improving accuracy and reproducibility. Mathematical models have been used to support technical knowledge in reducing the need for experimental development. This results in an improved, faster, and platform-based technique for FS separation with one single pretreatment applicable to all antigens of the multivalent meningococcal vaccine.

GSK is currently working to improve the commercial presentation of the licensed quadrivalent conjugate vaccine (Menveo) for use against meningococcal serogroup A, C, W, Y (MenACWY) infections. Menveo consists of a primary, lyophilized vial, containing the serogroup A antigen that is reconstituted with the content of a second, liquid, vial that contains the serogroup C, W, Y antigens, to give the final liquid MenACWY product.Since the MenA structure is prone to hydrolytic degradation in liquid formulations, we used mathematical models to rationally design a clinical Phase 2 development plan and provide end of shelf-life (EoSL) and release specification setting for the MenACWY liquid product.By using development and clinical stability data, statistical models were built and used to predict both the MenA free saccharide (FS) and O-Acetyl (OAc) content during long-term storage conditions at 5 degrees C and stressed (accelerated) stability studies at 15 degrees C, 22.5 degrees C, 25 degrees C, 37 degrees C and 50 degrees C. This approach allowed us to define an aging plan for the clinical material to reach at least the required levels of MenA FS and OAc levels at product EoSL. The clinical material was then exposed to a temperature of 22.5 +/- 2.5 degrees C for 59 days to generate FS OAc content of about 35% and 40%, respectively, which was then delivered to the patients in the clinical trial.To the best of our knowledge, this work represents the first example in the field of vaccine research where statistical models have been used to rationally design tailored lots, with the goal of setting EoSL and release specification limits based on data collected on artificially aged clinical material, in which the FS and OAc levels tested were intended to support a product shelf-life of at least 24 months.0, 2022 Elsevier Ltd. All rights reserved.

Several glycoconjugate vaccines have been licensed or are currently in clinical development to prevent bacterial infections. Here we report the development of a single analytical assay to quantify the conjugated saccharide content, as alternative to two separated total and free (unconjugated) saccharide assays used so far, for a quadrivalent conjugate vaccine containing meningococcal serogroup A polysaccharide (α-1,6-linked N-acetylmannosamine phosphate repeating unit partly O-acetylated at position C3 or C4) coupled with CRM197 protein. The results confirm a high linear correlation among the two approaches (conjugated saccharide content vs. difference of total saccharide and free saccharide). Conjugated saccharide content estimation is therefore demonstrated to be a suitable method to monitor the product quality of vaccines containing meningococcal serogroup A conjugate antigen, in the final filled presentation as demonstrated here and potentially on the bulk conjugate before formulation.

Burkholderia cenocepacia is an opportunistic pathogen isolated from cystic fibrosis patients where it causes infections that are extremely difficult to treat with antibiotics, and sometimes have a fatal outcome. Biofilm is a virulence trait of B. cenocepacia, and is associated with infection persistence and increased tolerance to antibiotics. In biofilms exopolysaccharides have an important role, conferring mechanical stability and antibiotic tolerance. Two different exopolysaccharides were isolated from B. cenocepacia H111 biofilms: a water-soluble polysaccharide rich in rhamnose and containing an L-Man residue, and a water-insoluble polymer made of glucose, galactose and mannose. In the present work, the product encoded by B. cenocepacia H111 bepA-L gene cluster was identified as the water-insoluble exopolysaccharide, using mutant strains and NMR spectroscopy of the purified polysaccharides. It was also demonstrated that the B. cenocepacia H111 wild type strain produces the water-insoluble exopolysaccharide in pellicles, thus underlining its potential importance in in vivo infections.

The higher-order structure (HOS) of protein therapeutics has been confirmed as a critical quality parameter. In this study, we compared 2D 1H-13C ALSOFAST-HMQC NMR spectra with immunochemical ELISA-based analysis to evaluate their sensitivity in assessing the HOS of a potent human monoclonal antibody (mAb) for the treatment of coronavirus disease 2019 (COVID-19). The study confirmed that the methyl region of the 2D 1H-13C NMR spectrum is sensitive to changes in the secondary and tertiary structure of the mAb, more than ELISA immunoassay. Because of its highly detailed level of characterization (i.e., many 1H-13C cross-peaks are used for statistical comparability), the NMR technique also provided a more informative outcome for the product characterization of biopharmaceuticals. This NMR approach represents a powerful tool in assessing the overall higher-order structural integrity of mAb as an alternative to conventional immunoassays.

Neisseria meningitidis is a major cause of bacterial meningitidis worldwide. Children less than five years and adolescents are particularly affected. Nearly all invasive strains are surrounded by a polysaccharide capsule, based on which, 12 N. meningitidis serogroups are differentiated. Six of them, A, B, C, W, X, and Y, cause the vast majority of infections in humans. Mono- and multi-valent carbohydrate-based vaccines against meningococcal infections have been licensed or are currently in clinical development. In this mini-review, an overview of the past and present approaches for producing meningococcal glycoconjugate vaccines is provided.