RESUMEN
Klebsiella pneumoniae (Kp) is a Gram-negative bacterium, and a leading cause of neonatal sepsis in low- and middle-income countries, often associated with anti-microbial resistance. Two types of polysaccharides are expressed on the Kp cell surface and have been proposed as key antigens for vaccine design: capsular polysaccharides (known as K-antigens, K-Ags) and O-antigens (O-Ags). Historically, Kp has been classified using capsule serotyping and although 186 distinct genotypes have been predicted so far based on sequence analysis, many structures are still unknown. In contrast, only 11 distinct OAg serotypes have been described. The characterization of emerging strains requires the development of a high-throughput purification method to obtain sufficient K- and O-Ag material to characterize the large collection of serotypes and gain insight on structural features and potential cross-reactivity that could allow vaccine simplification. Here, this was achieved by adapting our established method for the simple purification of O-Ags, using mild acetic acid hydrolysis performed directly on bacterial cells, followed by filtration and precipitation steps. The method was successfully applied to purify the surface carbohydrates from different Kp strains, thereby demonstrating the robustness and general applicability of the purification method developed. Further, antigen characterization showed that the purification method had no impact on the structural integrity of the polysaccharides and preserved labile substituents such as O-acetyl and pyruvyl groups. This method can be further optimized for scaling up and manufacturing to support the development of high-valency saccharide-based vaccines against Kp.
RESUMEN
Several human diseases are caused by enteroviruses and are currently clinically untreatable, pushing the research to identify new antivirals. A notable number of benzo[d][1,2,3]triazol-1(2)-yl derivatives were designed, synthesized, and in vitro evaluated for cytotoxicity and antiviral activity against a wide spectrum of RNA positive- and negative-sense viruses. Five of them (11b, 18e, 41a, 43a, 99b) emerged for their selective antiviral activity against Coxsackievirus B5, a human enteroviruses member among the Picornaviridae family. The EC50 values ranged between 6 and 18.5 µM. Among all derivatives, compounds 18e and 43a were interestingly active against CVB5 and were selected to better define the safety profile on cell monolayers by transepithelial resistance test (TEER). Results indicated compound 18e as the hit compound to investigate the potential mechanism of action by apoptosis assay, virucidal activity test, and the time of addition assay. CVB5 is known to be cytotoxic by inducing apoptosis in infected cells; in this study, compound 18e was proved to protect cells from viral infection. Notably, cells were mostly protected when pre-treated with derivative 18e, which had, however, no virucidal activity. From the performed biological assays, compound 18e turned out to be non-cytotoxic as well as cell protective against CVB5 infection, with a mechanism of action ascribable to an interaction on the early phase of infection, by hijacking the viral attachment process.
RESUMEN
Antimicrobial resistance (AMR) is emerging as the next potential pandemic. Different microorganisms, including the bacteria Acinetobacter baumannii, Clostridioides difficile, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, Neisseria gonorrhoeae, Pseudomonas aeruginosa, non-typhoidal Salmonella, and Staphylococcus aureus, and the fungus Candida auris, have been identified by the WHO and CDC as urgent or serious AMR threats. Others, such as group A and B Streptococci, are classified as concerning threats. Glycoconjugate vaccines have been demonstrated to be an efficacious and cost-effective measure to combat infections against Haemophilus influenzae, Neisseria meningitis, Streptococcus pneumoniae, and, more recently, Salmonella typhi. Recent times have seen enormous progress in methodologies for the assembly of complex glycans and glycoconjugates, with developments in synthetic, chemoenzymatic, and glycoengineering methodologies. This review analyzes the advancement of glycoconjugate vaccines based on synthetic carbohydrates to improve existing vaccines and identify novel candidates to combat AMR. Through this literature survey we built an overview of structure-immunogenicity relationships from available data and identify gaps and areas for further research to better exploit the peculiar role of carbohydrates as vaccine targets and create the next generation of synthetic carbohydrate-based vaccines.
