Capsular polysaccharide inhibits vaccine-induced O-antigen antibody binding and function across both classical and hypervirulent K2:O1 strains ofKlebsiella pneumoniae

Abstract

Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen.

A heptavalent O-antigen bioconjugate vaccine exhibits differential functional antibody responses against diverseKlebsiella pneumoniaeisolates

“…Strains that demonstrated high levels of antibody reactivity also demonstrated the most bactericidal killing and vice versa; except for strain 28893, which displayed high antibody binding but no bacterial killing, and strain 4759, which displayed modest levels of antibody binding but high bacterial killing. We hypothesized that the level of capsule produced by some strains may explain the poor O-antibody binding and function against these strains, as it has been demonstrated that capsule masking of O-antigen occurs in some K. pneumoniae isolates (21, 28, 29). Using a glucuronic acid detection assay, we quantified the relative amount of capsule produced by each of the 18 strains.…”

“…Six of the K. pneumoniae O-antigen clusters were produced in E. coli (O1v1, O2v1, O2v2, O3, O3b, and O5); nuclear magnetic resonance spectroscopy analysis of extracted polysaccharides from E. coli confirmed structural matches to the native K. pneumoniae O-antigen polysaccharides, including terminal caps on the O3, O3b, and O5 polysaccharides (Supplemental Figures 1-5). The NMR structure of the O1v1 bioconjugate was previously reported (21). The O1v2 bioconjugate was produced directly in a genetically modified K. pneumoniae NTUH-K2044 strain (hereafter referred to as NTUH), which naturally produces the O1v2 O-antigen polysaccharide.…”

“…The resulting strain was then transformed with a plasmid encoding the carrier protein and oligosaccharyltransferase (OTase). The carrier protein used for all K. pneumoniae bioconjugates was a genetically inactivated Pseudomonas aeruginosa exotoxin A (EPA) engineered to contain two internal 23-amino acid sequons that are glycosylated by the Acinetobacter baylyi ADP1 PglS OTase (21,25).To produce the bioconjugates, EPA and PglS were co-expressed from plasmid pVNM245 (21) alongside the K. pneumoniae O-antigen expression plasmids in E. coli strain CLM24 (26) or K. pneumoniae NTUH∆wcaJ∆waaL. Bacteria were cultured in Terrific Broth and induced with 0.1 mM IPTG at mid-log followed by overnight growth.…”

“…Serum bactericidal assays for K. pneumoniae were adapted from methods previously described (21). K. pneumoniae isolates and derived mutants were grown under static conditions in LB broth for 16 hours at 37 o C. Cultures were centrifuged at 8000 x g for 10 min and resulting pellets were resuspended in sterile PBS to an OD600 ~1.0.…”

“…Conjugate vaccines have demonstrated great success in targeting other bacterial pathogens including Streptococcus pneumoniae , Haemophilus influenzae , and Neisseria meningitidis , and therefore have been an area of focus for K. pneumoniae vaccine development. A unique method of polysaccharide protein conjugation, termed bioconjugation, has previously been applied to produce K. pneumoniae polysaccharide-protein conjugates that feature an unaltered polysaccharide antigen (21, 22). Our bioconjugation technology synthesizes the polysaccharide-protein conjugate entirely within a glycoengineered bacterial expression system, allowing for rapid production of diverse polysaccharide protein conjugates targeting either capsule or O-antigen (23).…”