Towards glycoconjugate vaccines against carbapenem-resistance Klebsiella pneumoniae

Antimicrobial resistant bacteria account for high mortality rates due to the lack of effective methods to combat the pathogens. Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a bacterial group of huge concern that is resistant to nearly all available antibiotics. CRKP has been classified as an “urgent threat” to public health with a dire need to develop new drugs alternative treatments. Antibody therapy or even better, a vaccine, targeting this group would be a viable alternative to resolve this threat. Current strategies to target CRKP rely mostly on the bacterial capsular polysaccharides as antigens. However, the high variability in the CPS composition and low epidemiological correlation of clinical isolates would lead to vaccines with limited target spectrum and numerous antibodies for each CPS variant would be needed for acute treatment, making it a very costly approach. Contrary, O-antigens that are part of the bacterial LPS, account with just four serotypes for the majority of clinically relevant strains, with the O2afg serotype as the most frequent in the CRKP group. The use of native LPS-based antigens for vaccine development is hampered by the low immunogenicity, the lack of a T-cell dependent immune answer, and endotoxin contaminations in LPS batches, which can lead to lethal side effects. Thus, no LPS based vaccines are currently in development. In my thesis I address this bottleneck by exploring a semi-synthetic glycoconjugate vaccine using a highly pure and well-characterized synthetic oligosaccharide that mimics the native O2afg antigen of CRKP. The antigen was designed based on a hexasaccharide repeating unit of O2afg and synthesized by Dr. Dacheng Shen. The glycan was subsequently conjugated to CRM197, a carrier protein, and adsorbed into alum as adjuvant, creating a semi-synthetic glycoconjugate vaccine against CRKP based on approved and validated technology. I provide conclusive evidence that the novel glycoconjugate vaccine induces a strong T-cell dependent supported by an immune response with a long-term memory effect at very low antigen concentrations. The resulting antibodies activate complement deposition, thereby activating the opsophagocytic pathway. The generated antibodies are well suited for passive immunization and result in a significant improvement of several physiological parameters in a mouse model for acute pneumonia. This included lower levels of cytotoxic enzymes, neutrophil infiltration and the reduction of permeability and lung edema. Hospital-acquired pneumonia is the major problem caused by K. pneumoniae, especially among individuals with critical illness, for example HIV and cancer in intensive care units. Recently, the pathogen has also been associated with SARS-CoV-2 pulmonary co-infections contributing to chronic obstructive pulmonary diseases, a severe COVID-19 condition with high mortality rates. Thus, the antibodies would significantly reduce the burden as a result of co-infection and increase the chances of survival. I show that a highly effective and low cost glycoconjugate vaccine targeting conserved LPS structures can be developed based on proven technology and opens up for a safer alternative to fight antimicrobial resistant pathogens without the effect of evolutionary selection of resistant strain caused by the currently treatment with antibiotics

Persistence of plasmids that encodes New-Delhi-Metalo-β- lactamase

As metalo-β-lactamases (MBL) são capazes de hidrolisar os carbapenêmicos, a classe de antimicrobianos com maior potência para o tratamento de infecções graves e de maior uso clinico. Dentre as MBL, o grupo mais recentemente descrito e que apresentou rápida disseminação em todo o mundo é o da New-Delhi-Metalo- β-lactamases (NDM). Nas enterobactérias, os genes que codificam essas enzimas estão mais frequentemente localizados em plasmídeos. O estudo da estabilidade de plasmídeos que albergam o gene blaNDM-1 é importante para entender a predominância de espécies que carregam esses plasmídeos, desvendar mecanismos moleculares envolvidos na sua persistência e para desenvolver novas drogas que possam diminuir a sua persistência. Estudos recentes sobre estabilidade plasmidial evidenciaram que a maprotilina é capaz de induzir perda plasmidial de até 90% em E. coli K12. Neste trabalho, foi estudado o efeito da maprotilina na indução de cura de plasmídeos, que albergam o gene blaNDM-1, em diferentes espécies da família Enterobacteriaceae. Nove isolados pertencentes a diferentes espécies foram incluídas no estudo. Os plasmídeos foram caracterizados quanto ao seu tamanho por eletroforese e por sequenciamento de DNA no sistema Illumina. A persistência plasmidial foi determinada pelo método de contagem em placa em LB ágar com e sem tratamento com maprotilina em concentrações sub-inibitórias (50mg/L). O experimento foi conduzido por 10 dias, representando aproximadamente 100 gerações. Neste estudo evidenciou-se que o grupo das enterobactérias estão envolvidas na disseminação de plasmídeos com blaNDM-1, sendo que plasmídeos do grupo IncF estão mais relacionados a essa dispersão. A maprotilina teve efeito de cura plasmidial em todos os isolados exceto em E. hormaechei \"subsp. oharae\" e C. freundii. O isolado P. rettgeri apresentou maior taxa de perda plasmidial e a análise comparativa da sequência nucleotídica do plasmídeo indicou que a presença da IS5 pode estar relacionada com a diminuição da persistência plasmidial. Diferenças na persistência plasmidial, quando tratados com maprotilina, entre E. hormaechei \"subsp. steigerwaltii\" e E. hormaechei \"subsp. oharae\" sugerem que E. hormaechei \"subsp. oharae\" pode ser um possível disseminador de plasmídeos albergando blaNDM-1, devido a processos de adaptação co-evolutivos.Metallo- β-lactamases (MBL) are able to hydrolase carbapenems, an antimicrobial class in clinical use with high potency in the treatment of severe infections. The most recently decribed group of MBL is the New-Delhi-Metallo-β-lactamases (NDM). This group is mostly correlated to the spread of resistance mediated by plasmid in Enterobacteriaceae. Understanding the plasmid persistence pattern is important in order to understand the predominance of a given species related to antimicrobial resistance plasmids spread, to unveil molecular mechanisms involved in the increase of plasmid persistence and to develop new drugs which could decrease its persistence. Recent studies have associated maprotiline to a decrease in 90% of plasmid persistence in E. coli K12. In this work, we evaluated the effect of maprotiline in curing plasmids carrying blaNDM-1 in different species of Enterobacteriaceae. Nine isolates belonging to different species were evaluated. Plasmids were characterized by agarose gel electrophoresis and by DNA sequencing with Illumina platform. The plate counting method was used to determine plasmid persistence, with and without sub-inhibitory (50 mg/L) concentration of maprotiline during 10 days, representing approximately 100 generations. We found that Enterobacteriaceae are involved in the spread of NDM-1 plasmid-mediate resistance and the IncF group is the plasmid incompatibility group more frequently involved in this dissemination. Maprotiline showed a plasmid-curing effect in all isolates, except against plasmids of E. hormaechei \"subsp. oharae\" and C. freundii. The P. rettgeri isolate had the highest plasmid-curing rate. Sequencing analysis revealed an IS5 in the plasmid, which could be associated to a decrease in plasmid persistence. The difference between plasmid persistence pattern of plasmids isolated from E. hormaechei \"subsp. steigerwaltii\" and E. hormaechei \"subsp. oharae\", when treated with maprotiline, suggest that E. hormaechei \"subsp. oharae\", could be associated to the spread of plasmids carrying blaNDM-1 due to co-evolution adaptation