Abstract
The ‘silent pandemic’ caused by antimicrobial resistance requires innovative therapeutic approaches. Human monoclonal antibodies (mAbs), which are among the most transformative and safe drugs in oncology1 and autoimmunity2, are rarely used for infectious diseases and not yet used for antimicrobial resistance3. Here we applied an antigen-agnostic strategy to isolate extremely potent human mAbs against Klebsiella pneumoniae sequence type 147 (ST147), a hypervirulent and pandrug-resistant lineage that is spreading globally. Isolated mAbs target the KL64 capsule and the O-antigen. However, although mAbs displayed bactericidal activity in the picomolar range in vitro, only the capsule-specific mAbs were protective against fulminant bloodstream infection by ST147 and two geographically and genetically distant carbapenem-resistant KL64-bearing K. pneumoniae. Protection observed in vivo correlated with in vitro bacterial uptake by macrophages and enchained bacterial growth. Our study thus describes a mAb that protects against pandrug-resistant K. pneumoniae and provides a strategy to isolate mAbs and identify mAbs that confer protection against bacteria with antimicrobial resistance.
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Data availability
Source data are available under BioProject accession PRJNA1067287. Data that were previously available and were used in this study to support our conclusions are accessible from RefSeq (GCF_904865815.1), BioSample ID SAMEA3357277 and BioSample ID SAMN46792556. All data supporting the findings in this study are available within the article.
Code availability
All code used for data analysis and image generation is available at https://github.com/dasch-lab/K_pneumoniae-anti_capsule_mabs.
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Acknowledgements
The authors thank all patients who participated in this study; A. Paolini, A. Frosini and C. Tinti for funding acquisition; F. Romano, G. Fantoni, G. Pierleoni and the Clinical Studies team at Fondazione Toscana Life Sciences for technical support and help with clinical study documentation; V. Galfo for the contribution to patient sampling and enrolment; S. Tavarini and C. Sammicheli for help with cell sorting and flow cytometry; M. G. Pizza for insightful discussions; F. Micoli, C. Giannelli, O. Rossi, G. Gasperini for technical advice on Klebsiella antigen purification; C. Whitfield and S. Kelly for the gift of Kp LPS; and M. G. Cusi, D. Hung, J. L. Chao. L. C. Wong and G. Frankel for providing Kp strains and insightful discussion. This project was supported by funding from Fondazione Toscana Life Sciences, the Tuscan Regional Center for Precision Medicine (Centro Regionale per la Medicina di Precisione, CREMEP) and by PROREACT project of the Italian Ministry of Health (CUP: D65F21001420001). This work was also supported within the IHU SEPSIS project sustained by the French National Research Agency-France 2030 program (grant ANR-23-IAHU-0004).
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Contributions
Conceptualization and design of the study: C.S., A.K., R.R. Supervision of experimental work: C.S. and A.K. Investigation: E.R., V.Z.G.F., S.S.B., I.P., G.C., G.B., S.S., S.Y., L. Capoccia, M.R., M.T., N.M., C.M., C.D.S., A.C., L. Cerofolini and M. Fragai. Bioinformatic and data analysis: G.M., D.C. and D.L. Resources: G.T., C.G., K.W., L.D., S.B., F.M. and M. Falcone. Mouse experiments: E.R., S.S., S.Y., N.M., C.D.S., D.P.N. and K.A. Writing: E.R., V.Z.G.F., S.S.B., G.M., G.B., D.P.N., K.A., C.S., R.R. and A.K. Data visualization: E.R., V.Z.G.F., S.S.B., G.M. and A.K.
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Competing interests
Patent application 102023000000924 describing isolated mAbs has been filed by Fondazione Toscana Life Sciences. E.R., V.Z.G.F., S.S.B., I.P., G.B., D.C., G.M., A.K. and C.S. are listed as inventors and declare no competing financial interests. R.R. holds shares in the GSK group of companies and declares no other financial or non-financial relationships and activities. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Analysis of ST147 genomes reported on the Pathogenwatch database, related to Fig. 1.
Top panel: annual frequency of the ten most frequent STs among reported Kp isolates. Percentage was computed towards all STs. Middle pannel: percentage of reported isolates with virulence score equal or greater than 3, for the ten most frequent ST. Bottom pannel: percentage of reported isolates with resistance score equal or greater than 2, for the ten most frequent ST. Scores were derived from Pathogenwatch.
Extended Data Fig. 2 Agnostic-based approach for antibody isolation and selection, related to Fig. 2.
(A) Schematic workflow depicting the strategy used to isolate and select anti-Kp antibodies from human donors. Silhouette from https://publicdomainvectors.org/ (Creative Commons: CC0 1.0). (B) Gating strategy for single-cell sorting of total memory B cells. Flow cytometry plots show the sequential gating used to identify live cells, lymphocytes, singlets, CD19+B cells, CD19+CD27+IgD− memory B cells, and CD19+CD27+IgD−IgM− B cells (representing memory B cells expressing IgG, IgA, or IgE). Plots were analyzed using FlowJo v10 (BD Biosciences). (C) ELISA screening of secreted monoclonal antibodies for binding to Kp. Heatmap shows reactivity against two Kp ST147NDM-1 clinical isolates (c.i.). Antibodies were considered positive when the OD405 signal was at least 3-fold higher than the blank. (D) Gating strategy for the detection of mAb binding to live bacteria by flow cytometry. Plots illustrate sequential gating to identify bacterial cells, singlets, and Alexa Fluor 488+ stained bacteria. Plots were generated using NovoExpress v1.6.2 (Agilent Technologies).
Extended Data Fig. 3 Phylogenetic analysis of the Kp strains used for flow cytometry, ELISA and immunoblot experiments and flow cytometry analysis of the mAb binding properties against other Kp species and commensals, related to Fig. 2.
(A) Maximum likelihood SNP tree calculated with IQTree, based on the 4189 core genes identified with Roary. K locus, O locus and O type are annotated on the side. Evolutionary model: GTR + F + ASC. (B) Heatmap showing the summary of high-throughput flow cytometry screening of 20 functional anti-Kp mAbs against Klebsiella oxytoca and Klebsiella variicola isolates, as well as against commensals. MFI values were normalized on no mAb controls and transformed in logarithmic scale.
Extended Data Fig. 4 Characterization of ST147NDM-1 c.i.1 and ST147NDM-9, related to Fig. 2.
(A) Annotation of K and O locus on selected strains. Sequence fragment resulting from the alignment of wbbO gene in selected strains, illustrating the single-point deletion at position 474 causing a premature stop codon in ST147NDM-9. (B) For each sequence, the presence of K- (blue) and O-locus (red) genes is presented; genes with premature stop codons are visualized as shades. (C) Heatmap of ELISA values obtained with 08O09 (cluster 1 mAb) and cluster 2 mAbs against ST147NDM-1 c.i.1, ST147NDM-9 (wt), ST147NDM-9/pSEVA23a1_wbbO (wbbO+), ST147NDM-9/pSEVA23a1 (wbbO−) strains. (D-F) Immunoblot (IB) of 05N02 (panel D), 05D08 (panel E) and 08O09 (panel F) against ST147NDM-1 c.i.1, ST147NDM-9 (wt), ST147NDM-9/pSEVA23a1_wbbO (wbbO+), ST147NDM-9/pSEVA23a1 (wbbO−) strains. IBs were repeated two times for each mAb obtaining similar results.
Extended Data Fig. 5 Polysaccharide characterization of O-antigen-deficient strain ST147NDM-9, purification of capsule type KL64 from ST147NDM-1 Kp and purification of different O-Antigen subtypes, related to Fig. 2.
(A) SEC-HPLC analysis of total sugar content. (B) Silver staining analysis of total sugar extract from ST147NDM-1 c.i. 1 and ST147NDM-9 Kp strains. ST147NDM-9 lacks LPS ladder-like signal in the 25–50 kDa range, which is present in the ST147NDM-1 loaded sample. Silver staining was performed two times obtaining similar results. (C) SEC-HPLC emission (λ = 280 nm) and RIU normalized profiles of purified KL64 capsule from ST147NDM-1 c.i. 1. ST147NDM-1 capsular polysaccharide was purified following the protocol reported in Materials and Methods. (D) Theoretical structure of capsule type 64, as described in literature27. (E) SEC-HPLC profiles of O2a purified from ST147NDM-1 c.i.1 and (F) O1v2 purified from ST13OXA-48. (G) Molecular structures of O1, O2a, and O2afg O-antigen types as reported in literature28,43.
Extended Data Fig. 6 NMR analysis of capsule KL64 and O2a-antigen, related to Fig. 2.
(A) 1D 1H NMR spectrum of capsule KL64 acquired on a spectrometer operating at 400 MHz and 298 K. The spectrum perfectly nicely matches the data reported in the literature for KL64 capsule55. The signals of the anomeric protons of the five sugars of the repetitive unit have been pointed out with a star. (B) 1D 1H NMR spectrum of O2a-antigen acquired on a spectrometer operating at 950 MHz and 298 K.
Extended Data Fig. 7 Representative immunoblots (IB) of selected anti-Kp mAbs, related to Fig. 2, probed against total sugar extracts of indicated strains.
(A) mAbs against the high molecular weight KL64 capsule type, including 08O09. (B) 05N02, 05B17 and 08F04 recognize O-antigens with broad molecular weight distribution belonging to O2- and O1 types. (C) 05D08, 05D14, 05C11 and 05M13 recognize medium molecular weight O-antigen structures from O2-carrying Kp strains. IBs were repeated two times for each mAb obtaining similar results.
Extended Data Fig. 8 Binding on additional anti-Kp mAbs and ROI definition and spot detection for mAb binding characterization, related to Fig. 3.
(A) Scatter plot displaying the Mean Fluorescence Intensity (MFI) at the single bacterium level of the mAb binding against ST147NDM-1 c.i. 1 (black lines show median with interquartile range). Median values ± SD are the following: 1419 ± 976 a.u., n = 28503 bacteria over 2 independent experiments for 08D18; 1246 ± 643 a.u., n = 29643 bacteria over 2 independent experiments for 08K19; 731 ± 450 a.u., n = 27873 bacteria over 2 independent experiments for 05M13; 1189 ± 561 a.u., n = 29066 over 2 independent experiments bacteria for 08F04; 1034 ± 524 a.u., n = 25337 bacteria over 2 independent experiments for 05C11; 118.2 ± 1.46 a.u., n = 29118 bacteria over 2 independent experiments for unrelated. One-way ANOVA with Tukey’s multiple comparisons with 95% C.I. to compare each anti-Kp mAb to the unrelated mAb resulted in the following P values, **** p < 0.0001. (B) Images show ST147NDM-1 c.i. 1 stained with 08D18, 08K19, 05M13, 08F04, 05C11 mAbs labelled with anti-human A488 conjugated secondary antibody (green). Bacterial DNA is stained with DAPI (blue). Experiments were repeated two times obtaining similar results. Scale bar 2 µm. (C) Images on the left show the binding pattern of some A488-labelled anti-Kp mAbs on ST147NDM-1 c.i. 1 bacteria. Images in the middle show the ROI where A488 signal was detected whereas on the right it is shown the morphology of A488 spot. Experiments were repeated four times obtaining similar results. Scale bar 2 µm.
Extended Data Fig. 9 Sequence analysis of 20 selected mAbs, related to Fig. 2.
(A) IGHV and IGHJ genes pairing heatmap (left), IGKV and IGKJ genes pairing heatmap (right) targeting either the bacterial capsule (functional cluster 1, violet circles) or the O-antigen (functional cluster 2, purple circles). (B) Violin plots depicting somatic hypermutation levels in the VDJ genes of both heavy (left) and light (right) chains across the two functional clusters. (C-D) Sequence similarity heatmap of heavy and light chains. Antibodies similarity was analyzed by calculating a distance matrix using CLUSTAL Omega for the heavy (C) and light (D) chains separately. Dendrogram tips were colored according to the associated mAb cluster.
Extended Data Fig. 10 F-SBA profiling and killing efficacy of 20 functional mAbs against complement-sensitive pathogenic Kp strains and comparision of 08O09 binding levels and activity in LB vs. M9 media, related to Fig. 4.
F-SBA curves of 20 bactericidal anti-Kp mAbs against ST147NDM-1 c.i.1 (A), ST147NDM-1 c.i.2 (B), ST147NDM-9 (C) and ST307NDM-5 (D). Single experiments were normalized to no mAb controls. Green, orange and dark red curves correspond to 08O09, 05N02 and 05D08 mAbs, respectively. For each mAb, the bactericidal curve and the associated IC50 value were obtained using the [Inhibitor] vs. normalized response, variable slope analysis on GraphPad Prism 10.1.0. IC50 values are reported in Fig. 4A. (E) The heatmap displays the percentage of reduction in resazurin fluorescence as a readout of bacterial viability, normalized to controls. Results were extrapolated from F-SBA experiments with single mAbs. (F) Scatter plot displaying the Mean Fluorescence Intensity (MFI) at the single bacterium level of the mAb binding against ST147NDM-1 c.i. 1(black lines show median with interquartile range). Median values ± sd are the following: 1480 ± 1057 a.u., n = 29842 bacteria examined over 2 independent experiments for LB; 2359 ± 1638 a.u., n = 11089 bacteria examined over 2 independent experiments for M9. Unpaired two-tailed t-test with 95% C.I., **** p < 0.0001. (G) Normalized luminescence values showing bactericidal activity of 08O09 against ST147NDM-1 grown in LB vs M9. In each experiment technical replicates (in brackets) were analysed. (H) Fold uptake by THP-1 macrophages of ST147NDM-1 c.i. 1-sfmCherry bacteria grown in LB vs M9 relative to the condition without mAb. In each experiment technical replicates (in brackets) were analysed.
Extended Data Fig. 11 Evaluation of the in vivo therapeutic and combined efficacy of 08O09, 05D08 and 05N02 mAbs in immunocompetent ST147NDM-1 bacteremia model, related to Fig. 5.
(A) Spleen load of ST147NDM1 c.i.1 one hour after the injection of 1.6 × 106 CFU formulated in hog gastric mucin (n = 18 animals examined in 3 independent experiments). (B) Survival analysis of 08O09 treatment (TX) regimens versus sham controls (n = 10 mice per group in 1 experiment; i.p. challenge with 1.6 × 106 CFU ST147NDM-1 c.i.1 formulated in hog gastric mucin). Kaplan-Meier survival analysis and the log rank (Mantel-Cox) test to compare each 08O09-treated group versus the respective control group resulted in the following P values: 0.0002 for 1 mg/kg; 0.0005 for 5 mg/kg, and <0.0001 for 10 and 20 mg/kg. (C) Spleen load of ST147NDM-1 in 08O09 TX study. Log10 CFU/spleen at endpoint (n = 10 animals per group in 1 experiment) was normalized to non-infected controls (n = 6 animals in 1 experiment). Two-tailed Mann-Whitney test, * p = 0.0241, **** p < 0.0001. (D) Survival analysis of 08O09 PPX, TX and PPX + TX regimens at 1 mg/kg doses (n = 10 mice per group in 1 experiment). Indicated P values were calculated by log-rank (Mantel-Cox) test and refer to a comparison of PPX + TX group versus either PPX (* p = 0.0378) or TX group (*** p = 0.0008). (E) Survival analysis of 08O09 PPX regimen at 5 mg/kg versus sham controls (n = 12 mice per group, 4 mice were monitored for 96 h and 8 mice were monitored for 192 h over 2 independent experiments; i.p. challenge with 107 CFU ST147NDM-1 c.i.1 formulated PBS). Kaplan-Meier survival analysis and the log rank (Mantel-Cox) test to compare the groups resulted in a P value of 0.0018. (F) Spleen load of ST147NDM1 c.i.1 in 08O09 PPX study (i.p. challenge with 107 CFU ST147NDM-1 c.i.1 formulated PBS). Log10 CFU/spleen at endpoint for PPX group (n = 8 animals examined over 2 independent experiments) and non-infected controls (n = 7 animals examined over 2 independent experiments). Two-tailed Mann-Whitney test, * p = 0.036.
Extended Data Fig. 12 Evaluation of antibody binding, bactericidal activity, opsonophagocytosis and enchained growth of ST231 and ST2096, related to Fig. 5.
(A) Philogenetic tree of Kp strains of different KL and ST types considered for the inclusion in the in vivo study. Strains used in the in vivo study, related to Fig. 5, are highlighted with red rectangles. Maximum likelihood tree calculated with FastTree GTR+Gamma model, based on the 2,358 core genes. K locus, O locus and O type are annotated on the side. (B) Flow cytometry analysis of binding of the anti-capsular 08O09 to ST147NDM-1 c.i.1, ST231 and ST2096. (C) Complement sensitivity assay using increasing concentrations of active baby rabbit complement (BRC) supplemented with 150 ng/mL of the anti-KL64 bactericidal mAb 08O09. N = 1 including technical replicates (in brackets). (D) Fold uptake by THP-1 macrophages of ST147NDM-1 c.i.1, ST231 and ST2096-sfmCherry bacteria relative to the condition without mAb. For ST147NDM-1 c.i.1, Two-way Anova test with Šídák’s multiple comparisons to compare 08O09 to the unrelated mAb resulted in the following P values with 95% C.I., at the minimum effective dose of 0.037 mg/mL, * p = 0.0003. N = 3 independent experiments including technical replicates (mean values ± SD are shown). For ST231 and ST2096 were performed N = 2 and N = 1 independent experiments, respectively, including technical replicates (in brackets). (E-F) Live imaging of ST231 and ST2096 incubated for 120 min in RPMI medium supplemented with 10% fetal bovine serum (FBS) without mAb and with 100 µg/mL or 10 µg/mL of 08O09. Experiments were repeated two times for each strain obtaining similar results. Scale bar 20 µm.
Supplementary information
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Supplementary Figs. 1 and 2
Supplementary Tables
Supplementary Tables 1–6
Supplementary Video 1
Time-lapse imaging of ST147NDM-1-sfmCherry treated with 100 μg ml−1 of anti-Kp mAb 08O09.
Supplementary Video 2
Time-lapse imaging of ST147NDM-1-sfmCherry treated with 100 μg ml−1 of anti-Kp mAb 05N02.
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Roscioli, E., Zucconi Galli Fonseca, V., Bosch, S.S. et al. Monoclonal antibodies protect against pandrug-resistant Klebsiella pneumoniae. Nature (2025). https://doi.org/10.1038/s41586-025-09391-3
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DOI: https://doi.org/10.1038/s41586-025-09391-3
