Bacterial infections are one of the leading causes of death, which have become a major threat to human health. Many infections are attributable to Gram-negative bacteria like Neisseria meningitidis or Acinetobacter baumannii. The number of infections has drastically increased due to treatment limitations and the rise of antimicrobial resistance. Unfortunately, as traditional antibiotics are no longer effective, alternative strategies are needed to fight bacteria and the associated diseases. Monoclonal antibodies (mAbs) and vaccination are two approaches which confer protection and prevention with a much lower probability of resistance emergence. Some commercial glycoconjugate vaccines using native capsular polysaccharides (CPSs) are available, but the isolation of CPS is complex and may lead to impurities. To address this, synthetic and pure oligosaccharides that mimic native CPS and are conjugated to a carrier protein can be used to ultimately promote specific immune responses. In the first part of my thesis, I demonstrated that using a fluorinated synthetic glycan conjugated to carrier proteins induced a strong immune response. The fluorinated glycan mimicked the epitope of N. meningitidis serotype C, which was conjugated to two carrier proteins, CRM197 or Porin A (PorA), respectively. After immunization, I showed that the raised antibodies recognized the fluorinated glycan as well as the native epitope of serotype C. Moreover, as PorA is expressed by serotype B, employing this carrier protein led to the creation of a bivalent glycoconjugate targeting both serotypes. I also proved the protective effect of the glycoconjugates since the pathogens were successfully cleared in vitro by the action of the complement and phagocytic cells, induced by the generated antibodies. Consequently, this strategy presents as a novel platform for developing glycoconjugate vaccine leads. In the second part of my thesis, I worked on the development of mAb candidates and vaccine leads against A. baumannii. On one hand, I produced three mAbs against the K1 capsule of A. baumannii. The mAbs recognized specifically both the synthetic glycan and the native CPS K1. The protective effects of the mAbs were studied in vitro, demonstrating their activity in reducing the bacterial load of A. baumannii. On the other hand, I also developed glycoconjugate vaccine leads containing synthetic oligosaccharides mimicking CPS types K1 and K3 of A. baumannii. The immunized mice produced a robust and long-lasting immune response, generating different subclasses of IgGs that are linked to a higher protective potential. The semi-synthetic glycoconjugates induced the production of antibodies with higher or similar binding to native CPS compared to those generated by the traditional CPS-based glycoconjugates, which were also evaluated in mice. Moreover, the protective activity in vitro induced by the semi-synthetic glycoconjugates was superior. An in vivo study with a mouse model of A. baumannii infection revealed that immunization with the semi-synthetic glycoconjugate candidates led to the reduction of bacterial burden in organs and alleviated disease symptoms, especially against the strain with K1 capsule. However, the results obtained with the K3 CPS strain showed a moderate efficacy, likely due to its higher virulence or the higher inoculum dose. In summary, my work during this thesis demonstrates that the utilization of synthetic oligosaccharides is a great alternative for the generation of effective glycoconjugate vaccine leads and mAbs against N. meningitidis and A. baumannii. Therefore, making them promising approaches for the treatment and/or prevention of infections, especially in high-risk or immunocompromised patients.
