Hexasaccharide epitope2was significantly better recognized by human antibodies than trisaccharide1, when compared to the negative sera control (Figure2). awaiting preclinical development. == Introduction == Klebsiella pneumoniaeis a Gram-negative bacterium naturally occurring in the gastrointestinal tract of humans. Pathogenic strains, when invading other tissues can cause diseases such as pneumonia, urinary tract infection, sepsis, liver abscesses, and meningitis.1Hospital-acquired infections (HAIs) are a critical global health crisis affecting millions annually.2Carbapenem-resistantK. pneumoniae(CR-Kp) has emerged as a common cause of these infections, with studies reporting mortality rates as high as 3060%.3 The combination of high mortality rate, global dissemination, and their resistance to nearly all currently used antibiotics, resulted in CR-Kpto be classified as an urgent public health threat.4A specific CR-Kplineage belonging to the sequencing type 258 (ST258) is the major cause of infections in the USA and Europe,58a group that includes the serotype O2afg.9The O2afg serotype is found in over 80% of the CR-Kpisolates globally.10TheO-andK-carbohydrate antigens are well-described virulence factors that contribute toK. pneumoniaesurvival by avoiding complement-mediated killing.11,12Both antigens have been explored as targets for glycoconjugate vaccine development due to their prevalent exposure on the bacterial surface.13In contrast to more than 79K-serotypes,14just 12O-serotypes compose the relevant CR-Kpclinical strains and among those, four serotypes (O1, O2, O3, and O5) are responsible for more than 90% of allK. pneumoniaeinfections worldwide.15Serotypes O1 and O2 are particularly important as they are associated with high antimicrobial resistance and high prevalence.1618AlthoughK. pneumoniaeO2afg strains are markedly more TSPAN31 serum sensitive than the O1 strains, humans produce very low numbers of specific B cells against the O2afg antigens. The poor immunogenicity of O2afg antigens may explain the high frequency of CR-Kpglobal propagation of this serotype.18 Licensed glycoconjugate vaccines againstHaemophilus influenzaetype B, meningococcus,Streptococcus pneumoniae, andSalmonella typhi(19,20) are very effectively used to Amyloid b-peptide (42-1) (human) protect humans.K. pneumoniaevaccines based on whole cell preparations or subunits21have been studied extensively. A multivalentO-antigen glycoconjugate vaccine lead containing O1, O2, O3, and O5 serotypes conjugated toPseudomonas aeruginosaflagellin A or B proteins resulted in high IgG-titers in rabbits against all antigens including the carrier proteins and protected mice againstK. pneumoniaeinfection. The vaccine candidate did not enter clinical trials, likely due to the adverse effects caused by the endotoxin lipid A from bacterial cultures.22The Phase I/II study of a tetravalent bioconjugate vaccine candidate (kleb4 V) targeting fourO-serotypes was completed, but results remain unpublished.23 Here, we report the chemical synthesis of two definedO-antigen oligosaccharides containing one or two repeating units resembling the O2afg serotype and the immune response of CRM197glycan conjugates in rabbits. Passive immunization with antibodies that target the O2afg serotype glycans was also evaluated in a murine model of acute pneumonia. Amyloid b-peptide (42-1) (human) == Results == == Synthetic Strategy to Obtain Epitopes Mimicking Natural Occurring O2afg Antigen == K. pneumoniaeO2afg serotype bacteria are covered by polysaccharides composed ofd-galactan-III (Gal-III) (3)–d-Galf-(1 3)-[-d-Galp-(1 Amyloid b-peptide (42-1) (human) 4)]–d-Galp-(1) repeating units (RUs)16(Figure1A). We synthesized two oligosaccharide antigens mimicking the natural occurring trisaccharide containing one RU (1) and an hexasaccharide containing two RUs (Figure1B). A fluorinated C3aminoalkyl linker24at the reducing end of both antigens allowed for efficient conjugation to the carrier protein and immobilization on microarrays. == Figure 1. == Glycoconjugate vaccine design and retro-synthetic analysis to obtain the epitopes. (A) Trisaccharide repeating unit ofGal-IIIfromKPST258. (B) Synthetic trisaccharide repeating unit1and hexasaccharide2containing two repeating units, each connected to an amine linker. (C) Retrosynthetic analysis of trisaccharide1and hexasaccharide2. The dense antigen structure with branching at the C3 and C4 positions of the central Galps to the bridging Galfresidue, and the presence of fragile Galfglycosidic bonds renders the synthesis challenging. No syntheses of theK. pneumoniaeO2afg antigens have been reported so far. The core galactopyranose of our target trisaccharide1connects to the other two sugar residues via C3 and C4 while its anomeric position is connected to a linker. The key strategic consideration is the design of core Galpbuilding block4. It needs to be glycosylated with building blocks5and6and connected to linker7on correct positions with proper stereo selectivity. Protected hexasaccharide8is assembled by combining two building blocks with trisaccharide-repeating-unit structures together, donor9and acceptor10(Figure1C). == Synthesis of Trisaccharide1and Hexasaccharide2 == The acetyl esters on C4 and C6 of galactopyranose building block5participate to ensure -glycoside formation,25galactofuranose6, and 2,2-difluoro-5-aminopropanol linker7favoring -glycosylation26were prepared in anticipation of the assembly of target trisaccharide1. Core building block4was synthesized from commercially available -d-galactose pentaacetate (see theSupporting Informationfor details.). With all of the building blocks in hand, thioglycoside4was reacted with linker7. Following glycosylation with building block5and Fmoc cleavage yielded disaccharide acceptor11. The HC coupling constants (170 Hz) of both glycosidic linkages confirmed the formation of two.