Innovative Reversed-Phase Chromatography Platform Approach for the Fast and Accurate Characterization of Membrane Vesicles' Protein Patterns.

Outer membrane vesicles (OMVs) have been widely explored to develop vaccine candidates for bacterial pathogens due to their ability to combine adjuvant properties with immunogenic activity. OMV expresses a variety of proteins and carbohydrate antigens on their surfaces. For this reason, there is an analytical need to thoroughly characterize the species expressed at their surface: we here present a simple and accurate reversed-phase ultrahigh-performance liquid chromatography (RP-UPLC) method developed according to quality by design principles. This work provides an analytical alternative to the classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization. The higher selectivity and sensitivity of the RP-UHPLC assay allow for the identification of additional protein species with respect to SDS-PAGE and facilitate its precise relative abundance quantification. According to validation results, the assay showed high accuracy, linearity, precision, repeatability, and a limit of quantification of 1% for less abundant proteins. This performance paves the way for improved production campaign consistency while also being analytically simple (no sample pretreatment required), making it suitable for routine quality control testing. In addition, the applicability of the assay to a wider range of vesicle classes (GMMA) was demonstrated.

Analytical Insights into Protein-Alum Interactions and Their Impact on Conformational Epitope.

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.

Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine candidates.

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases. The covalent conjugation of relevant polysaccharide antigens to immunogenic carrier proteins enables the induction of a long-lasting and robust IgG antibody response, which is not observed for pure polysaccharide vaccines. Although there has been remarkable progress in the development of glycoconjugate vaccines, many crucial parameters remain poorly understood. In particular, the influence of the conjugation site and strategy on the immunogenic properties of the final glycoconjugate vaccine is the focus of intense research. Here, we present a comparison of two cysteine selective conjugation strategies, elucidating the impact of both modifications on the structural integrity of the carrier protein, as well as on the immunogenic properties of the resulting glycoconjugate vaccine candidates. Our work suggests that conjugation chemistries impairing structurally relevant elements of the protein carrier, such as disulfide bonds, can have a dramatic effect on protein immunogenicity.

Epigenetic-based therapy in allogenic hematopoietic stem cell transplantation: Novel opportunities for personalized treatment.

Current management of patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) lacks immunosuppressant drugs able to block the host immune response toward the graft antigens. Novel treatments may include epigenetic compounds (epidrugs) some of which have been yet approved by the Food and Drugs Administration for the treatment of specific blood malignancies. The most investigated in clinical trials for allo-HSCT are DNA demethylating agents (DNMTi), such as azacitidine (Vidaza) and decitabine (Dacogen) as well as histone deacetylases inhibitors (HDACi), such as vorinostat (Zolinza) and panobinostat (Farydak). Indeed, azacitidine monotherapy before allo-HSCT may reduce the conventional chemotherapy-related complications, whereas it may reduce relapse risk and death after allo-HSCT. Besides, a decitabine-containing conditioning regimen could protect against graft versus host disease (GVHD) and respiratory infections after allo-HSCT. Regarding HDACi, the addition of vorinostat and panobinostat to the conditioning regimen after allo-HSCT seems to reduce the incidence of acute GVHD. Furthermore, panobinostat alone or in combination with low-dose decitabine may reduce the relapse rate in high-risk patients with acute myeloid leukemia patients after allo-HSCT. We discuss the phase 1 and 2 clinical trials evaluating the possible beneficial effects of repurposing specific epidrugs which may guide personalized therapy in the setting of allo-HSCT.

Evaluation of Immune Responses to Group B Streptococcus Type III Oligosaccharides Containing a Minimal Protective Epitope.

Recent structural studies demonstrated that the epitope recognized by a monoclonal antibody representative of the protective response against the type III group B Streptococcus polysaccharide was comprised within 2 of the repeating units that constitute the full-length native structure. In the current study, we took advantage of this discovery to design a novel vaccine based on multivalent presentation of the identified minimal epitope on a carrier protein. We show that highly glycosylated short oligosaccharide conjugates elicit functional immune responses comparable to those of the full-length native polysaccharide. The obtained results pave the way to the design of well-defined glycoconjugate vaccines based on short synthetic oligosaccharides.

The Conformation of the Mannopyranosyl Phosphate Repeating Unit of the Capsular Polysaccharide of Neisseria meningitidis Serogroup A and Its Carba-Mimetic.

Neisseria meningitidis serogroup A (MenA) is an aerobic diplococcal Gram-negative bacterium responsible for epidemic meningitis disease. Its capsular polysaccharide (CPS) has been identified as the primary virulence factor of MenA. This polysaccharide suffers from chemical lability in water. Thus, the design and synthesis of novel and hydrolytically stable structural analogues of MenA CPS may provide additional tools for the development of therapies against this disease. In this context, the structural features of the natural phosphorylated monomer have been analyzed and compared to those of its carba-analogue, where the endocyclic oxygen has been replaced by a methylene moiety. The lowest energy geometries of the different molecules have been calculated using a combination of quantum mechanical techniques and molecular dynamics simulations. The predicted results have been compared and validated using NMR experiments. The results indicate that the more stable designed glycomimetics may adopt the conformation adopted by the natural monomer, although they display a wider flexibility around the torsional degrees of freedom.