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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.

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.

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.

This review focuses upon the use of nitroso Diels−Alder reactions as a structural complexity generating reaction that has been so far a quite scarcely treated topic, despite its potential. In particular, the use of N-acyl-1,2-dihydropyridines as a non-symmetrical diene component in nitroso Diels−Alder reactions encompasses an initial diversification of pathways giving rise to different cycloadducts (direct and inverse). Selective elaborations of these cycloadducts, basically using a reagent-based approach, deliver a discrete number of structurally diverse compounds, including some original heterobicyclic scaffolds and functionalized heterocycles. This forward synthetic planning allowed the individuation of a new biologically active compound based on a novel oxadiaza-bicyclic-[3.3.1]-nonene scaffold which is still under preclinical evaluation.

Clostridium difficile is a Gram-positive bacterium and is the most commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. Despite the emergence of epidemic C. difficile strains having led to an increase in the incidence of the disease, a vaccine against this pathogen is not currently available. C. difficile strains produce two main toxins (TcdA and TcdB) and express three highly complex cell-surface polysaccharides (PSI, PSII and PSIII). PSII is the more abundantly expressed by most C. difficile ribotypes offering the opportunity of the development of a carbohydrate-based vaccine. In this paper, we evaluate the efficacy, in naive mice model, of PSII glycoconjugates where recombinant toxins A and B fragments (TcdA_B2 and TcdB_GT respectively) have been used as carriers. Both glycoconjugates elicited IgG titers anti-PSII although only the TcdB_GT conjugate induced a response comparable to that obtained with CRM197. Moreover, TcdA_B2 and TcdB_GT conjugated to PSII retained the ability to elicit IgG with neutralizing activity against the respective toxins. These results are a crucial proof of concept for the development of glycoconjugate vaccines against C. difficile infection (CDI) that combine different C. difficile antigens to potentially prevent bacterial colonization of the gut and neutralize toxin activity.

The incidence of infectious diseases caused by several bacterial pathogens such as Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis, has been dramatically reduced over the last 25 years through the use of glycoconjugate vaccines. The structures of the bacterial capsular polysaccharide (CPS) antigens, extracted and purified from microbial cultures and obtained with very high purity, show that many of them are decorated by O-acetyl groups. While these groups are often considered important for the structural identity of the polysaccharides, they play a major role in the functional immune response to some vaccines such as meningococcal serogroup A and Salmonella typhi Vi, but do not seem to be important for many others, such as meningococcal serogroups C, W, Y, and type III Group B Streptococcus. This review discusses the O-acetylation status of CPSs and its role in the immunological responses of these antigens.

This paper presents an assessment of machine-learned turbulence closures, trained for improving wake-mixing prediction, in the context of LPT flows. To this end, a three-dimensional cascade of industrial relevance, representative of modern LPT bladings, was analyzed, using a state-of-the-art RANS approach, over a wide range of Reynolds numbers. To ensure that the wake originates from correctly reproduced blade boundary-layers, preliminary analyses were carried out to check for the impact of transition closures, and the best-performing numerical setup was identified. Two different machine-learned closures were considered. They were applied in a prescribed region downstream of the blade trailing edge, excluding the endwall boundary layers. A sensitivity analysis to the distance from the trailing edge at which they are activated is presented in order to assess their applicability to the whole wake affected portion of the computational domain and outside the training region. It is shown how the best-performing closure can provide results in very good agreement with the experimental data in terms of wake loss profiles, with substantial improvements relative to traditional turbulence models. The discussed analysis also provides guidelines for defining an automated zonal application of turbulence closures trained for wake-mixing predictions.

This paper presents an innovative design for reducing the impact of secondary flows on the aerodynamics of low-pressure turbine (LPT) stages. Starting from a state-of-the-art LPT stage, a local reshaping of the stator blade was introduced in the end-wall region in order to oppose the flow turning deviation. This resulted in an optimal stator shape, able to provide a more uniform exit flow angle. The detailed comparison between the baseline stator and the redesigned one allowed for pointing out that the rotor row performance increased thanks to the more uniform inlet flow, while the stator losses were not significantly affected. Moreover, it was possible to derive some design rules and to devise a general blade shape, named ‘snaked’, able to ensure such results. This generalization translated in an effective parametric description of the ‘snaked’ shape, in which few parameters are sufficient to describe the optimal shape modification starting from a conventional design. The “snaked” blade concept and its design have been patented by Avio Aero. The stator redesign was then applied to a whole LPT module in order to evaluate the potential benefit of the ‘snaked’ design on the overall turbine performance. Finally, the design was validated by means of an experimental campaign concerning the stator blade. The spanwise distributions of the flow angle and pressure loss coefficient at the stator exit proved the effectiveness of the redesign in providing a more uniform flow to the successive row, while preserving the original stator losses.

A time-resolved particle image velocimetry (TR-PIV) system has been employed to investigate the unsteady propagation of upstream wakes in a low-pressure turbine cascade. Data are obtained in the steady state condition and for two passing wake reduced frequencies. The study is focused on the identification and split of the different dynamics responsible for deterministic and random oscillations, thus loss generation by means of a new procedure based on proper orthogonal decomposition (POD). The paper takes advantage of the properties of POD that reduce the data set to a low number of modes that represent the most energetic dynamics of the system. It is clearly shown that the phase averaged flow field can be represented by a few number of POD modes related to the wake passing event for the unsteady cases. Proper orthogonal decomposition is also able to capture flow features affecting the instantaneous flow field not directly related to the wake passage (i.e., the vortex shedding phenomenon induced by the intermittent separation developing between adjacent wakes), which are smeared out in the phase averaged results. A procedure exploiting the biorthogonality condition of the POD modes, and the related temporal coefficients, has been developed for the quantification of the contribution due to the different POD modes to the overall turbulence kinetic energy production, or, equivalently, the mean flow energy dissipation rate. Results into the paper clearly show that losses due to wake migration, boundary layer and vortex shedding related phenomena can be distinguished and separately quantified for the different tested conditions.