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Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

Controlling the global COVID-19 pandemic depends, among other measures, on developing preventive vaccines at an unprecedented pace. Vaccines approved for use and those in development intend to use neutralizing antibodies to block viral sites binding to the host’s cellular receptors. Virus infection is mediated by the spike glycoprotein trimer on the virion surface via its receptor binding domain (RBD). Antibody response to this domain is an important outcome of the immunization and correlates well with viral neutralization. Here we show that macromolecular constructs with recombinant RBD conjugated to tetanus toxoid induce a potent immune response in laboratory animals. Some advantages of the immunization with the viral antigen coupled to tetanus toxoid have become evident such as predominant IgG immune response due to affinity maturation and long-term specific B-memory cells. This paper demonstrates that subunit conjugate vaccines can be an alternative for COVID-19, paving the way for other viral conjugate vaccines based on the use of small viral proteins involved in the infection process.