Patient-Derived Antibody Data Yields Development of Broadly Cross-Protective Monoclonal Antibody against ST258 Carbapenem-Resistant Klebsiella pneumoniae

Generation of monoclonal antibody against wzi50 capsular polysaccharide.

With the knowledge that anti-wzi50 patient antibodies cross-react and protect against other capsular types (24), we utilized wzi50 as an immunogen to develop novel MAbs. We vaccinated BALB/c mice with unconjugated purified wzi50 CPS, or with wzi50 CPS conjugated to Bacillus anthracis Protective Antigen (wzi50-BaPA) (Fig. S1A in the supplemental material). Titer responses were poor for both groups of mice, requiring additional booster doses of CPS with adjuvant (incomplete Freud Adjuvant) at 6 and 8 weeks to achieve sufficient titers (>1:10,000). This regiment was still insufficient to elicit strong anti-capsular titers in the BaPA-CPS vaccinated mice (≤1:6,400), whereas mice vaccinated with unconjugated wzi50 reached and maintained high titers (>1:10,000) by 6 weeks post-initial vaccination against both wzi50 CPS and wzi29 CR-Kp bacteria (Fig. S1B).

Splenic fusions with two hybridoma cell lines were screened for activity against wzi50, and 3 positive clones with NSO^bcl2 backgrounds were identified for downstream subcloning and soft agar selection. Of these, we selected and subcloned IgG2b-producing hybridoma 24D11 for further study (Fig. S1A), as it had superior reactivity relative to the other 2 clones. Enzyme-linked immunosorbent assay (ELISA) binding estimated the 50% effective concentration (EC₅₀) of 24D11 against wzi50 as 4.72 nM (Fig. S1C) and that against wzi154 as 5.8 nM, respectively (Fig. S1D).

Anti-wzi50 antibody 24D11 mediates whole-blood killing of multiple CR-Kp strains.

To explore the anti-infective potential of MAb 24D11, we examined the antibody-mediated killing of multiple strains of CR-Kp in whole blood. As a control, we utilized our previously characterized murine IgG3 (mIgG3) 17H12, developed against CPS of a wzi154-carrying CR-Kp strain (18). The CFU of all tested CR-Kp strains dropped to undetectable levels regardless of condition at 2h. At 1 h, both 24D11 and 17H12 reduced the survival of wzi50-carrying SBU116 (~83% reduction in bacterial survival) significantly more than their corresponding control antibodies did (P < 0.0001) (Fig. 1A). Interestingly, 24D11 cross-reacted and significantly reduced wzi29 MMC36 CR-Kp bacterial levels in whole blood by 90% (P < 0.0001), whereas 17H12 did not significantly affect bacterial levels relative to its control antibody (Fig. 1B). Additionally, both 24D11 and 17H12 promoted the significant killing of wzi154-carrying MMC34 (~90% reduction in bacterial survival) compared to their isotype controls (P < 0.0001) (Fig. 1C). In addition, we also utilized CR-Kp SBU255, a strain belonging to the emerging clonal group CG307 Kp, which is genetically distinct from the ST258 clonal group and expresses an unrelated wzi173 CPS (25), to test the specificity of 24D11. As expected, 24D11 did not promote the killing of both CG307 CR-Kp strains (SBU100 and SBU255) in the whole blood assay at 1 h (Fig. S2). Furthermore, we also observed increased cross-reactive killing of MMC34 by 24D11 and of SBU116 by 17H12 relative to the controls (Fig. 1A and C) (P < 0.0001). This observation led us to investigate whether both MAbs bind to any conserved epitopes on wzi50 and wzi154 CPS by performing modified competitive ELISAs. Binding curves indicated that 17H12 began to compete with 24D11 binding to wzi50 CPS and wzi154 CPS at 5 μg/mL, whereas 24D11 began to compete with 17H12 binding to wzi154 at 10 μg/mL (Fig. S3). These results demonstrate the ability of 24D11 to promote the killing of many different ST258 strains in human blood.

MAb 24D11 promoted cross-protective opsonophagocytosis of multiple CR-Kp strains.

Macrophages and monocytes are important players in CR-Kp clearance, and the antibody-mediated opsonophagocytic uptake by macrophages is important for cell-mediated protection against CR-Kp (26–28). Therefore, we investigated the potential of 24D11 to promote opsonophagocytic uptake of different CR-Kp strains by the murine macrophage-like cell line J774A.1. Our data showed that 24D11 promoted opsonophagocytosis of 5/6 CR-Kp strains, including two wzi29 strains and one wzi154 strain (Fig. 2). Three of these five strains required serum for 24D11 to significantly improve opsonophagocytosis relative to the controls (SBU116, MMC34, MMC36), while two strains (MMC38, SBU207) showed serum-independent promotion of opsonophagocytosis. For one strain (MMC38), opsonophagocytosis of CR-Kp by 24D11 did not differ with the controls in the presence of serum, with opsonophagocytosis levels equivalent to those in the presence of 24D11 without serum (Fig. 2).

To ensure that the cross-reactivity of 24D11 was dependent on the presence of CPS and not due to nonspecific binding to the bacteria, we tested its ability to promote opsonophagocytosis of CR-Kp 33576 (wzi154) and its acapsular mutant 33576Δwzy (19) (Fig. S4). 24D11 mediated phagocytosis of the capsular 33576 bacteria but did not promote phagocytic uptake of the acapsular 33576Δwzy. Additionally, when tested against CG307 strain SBU255, 24D11 did not promote phagocytosis of SBU255, as expected (Fig. S4). Taken together, these data confirm that 24D11 enhances phagocytosis of ST258 strains with different polysaccharide capsules.

Intraperitoneal delivery of MAb 24D11 provides cross-protection in intratracheal lung infection model.

We followed our in vitro observation by exploring the cross-protective efficacy of 24D11 in vivo by testing its ability to reduce organ burden in mice intratracheally infected with wzi50-, wzi29-, or wzi154-carrying CR-Kp strains. For this investigation, we used our previously utilized pre-opsonized model and a prophylactic model where 24D11 was given intraperitoneally (IP) 4 h prior to intratracheal (i.t.) infection to explore whether 24D11 was able to decrease bacterial lung burden and dissemination in C57BL/6 mice (Fig. 3). Mice injected with 10⁶ CFU/inoculum pre-opsonized wzi50 SBU116 exhibited a moderate 1-log₁₀ reduction in the lung with no effect on the dissemination of the bacteria to the liver and spleen (Fig. 3A). Furthermore, mice infected with 10⁷ CFU/inoculum pre-opsonized SBU116 also showed a 1-log₁₀ decrease in bacterial lung burden with no significant reduction observed in bacterial dissemination to the liver or spleen (Fig. 3B). On the contrary, prophylactic administration of 24D11 prior to CR-Kp intratracheal infection significantly reduced the lung bacterial burden by 2-log₁₀ and the bacterial spread by 3-log₁₀ for both inoculums of SBU116 (P < 0.001) (Fig. 3A and B). Furthermore, at a high-inoculum dose of SBU116, we observed better lung CR-Kp clearance as well as inhibition of bacterial dissemination in 3 out of 6 mice within the pre-treated group compared to that in the pre-opsonized group (Fig. 3A and B). In mice infected with 24D11-opsonized wzi29 MMC36 strain (Fig. 3C) and wzi154 MMC34 strain (Fig. 3D), we observed more significant CFU reductions of 3 and 2 log₁₀-fold in lung tissue, respectively. Furthermore, a moderate drop in the bacterial load in the liver and spleen in antibody-opsonized MMC36- and MMC34-infected mice was observed (Fig. 3C and D). Pre-treatment with 24D11 had similar effect on the reduction of lung bacterial burden, but for both MMC36 (Fig. 3C) and MMC34 (Fig. 3D), almost all animals showed significant inhibition in bacterial dissemination compared to their pre-opsonized counterparts (Fig. 3C and D).

Next, we investigated whether 24D11 also protected CR-Kp infected mice if administered intraperitoneally 4 h post-surgery (Fig. 4). To better mimic clinical therapy, we treated CR-Kp infected mice 4 h after i.t. infection. These data demonstrated a significant drop of 3 log₁₀-fold in the lung burden of SBU116 CR-Kp infected mice compared that in the control groups (Fig. 4A). In addition, SBU116-infected mice treated intraperitoneally with MAb 24D11 showed a moderate 1-log₁₀ reduction in the bacterial CFU disseminated to other organs (Fig. 4A). For MMC29 (wzi29)- and MMC34 (wzi154)-infected mice, IP treatment with 24D11 post-i.t. surgery also demonstrated a significant decrease in the bacterial organ load in lungs (Fig. 4B and C). Furthermore, IP treatment with MAb 24D11 prevented dissemination to the limit of detection to liver and spleen in 3 out of 5 MMC36-infected mice (Fig. 4B) and reduced bacterial spread to other affected organs in MMC34-infected mice (Fig. 4C). In summary, these murine experiments demonstrate that 24D11 also exhibits cross-reactive efficacy in vivo. In addition, 24D11 can be used as a prophylactic a well as a therapeutic reagent.

CR-Kp lung clearance is improved by MAb 24D11 in neutropenic mice.

Previously published data showed that 17H12 could promote clearance of MMC34 from the lung in neutropenic mice (29). Therefore, we tested whether MAb 24D11 was also protective in neutropenic mice infected with SBU116 CR-Kp strain. Ly6G-mediated neutrophil depletion and generation of neutropenic mice was confirmed by flow cytometry (Fig. 5A). Our data showed that mice depleted of neutrophils with Ly6G and infected with SBU116 (10⁶ CFU/inoculum) exhibited a higher bacterial lung burden compared to immunocompetent untreated and antibody-treated mice (Fig. 5B). Intraperitoneal treatment with MAb 24D11 decreased the bacterial burden in the lung tissue in both immunocompetent and neutropenic mice by more than 100-fold relative to the untreated controls (Fig. 5B). 24D11 treatment also prevented dissemination in all immunocompetent mice, but not in neutropenic mice (Fig. 5B). MAb 24D11 had no effect in neutropenic mice when a higher inoculum was used (Fig. S5 in Text S1).

To better understand the immune cells involved in mediating the anti-infective efforts of 24D11, we studied and compared innate immune cells in both immunocompetent and neutropenic mice (Fig. 5C and Fig. S6 in Text S1). First, we observed and confirmed >80% depletion of neutrophils (black box) in Ly6G-treated mice (Fig. 5C). CD45⁺ cells were further subtyped. Immunophenotyping of the innate cells showed no difference in the population of M1 macrophages, but a significant change in M2 macrophage population was observed (Fig. 5C). Interestingly, neutropenic mice exhibited higher numbers of M2 macrophages in lung tissue, which was decreased by 24D11 treatment of neutropenic mice to values similar to those observed in the immunocompetent mice. Furthermore, in the lungs of neutropenic and wild-type mice, MAb treatment increased the presence of inflammatory monocytes (Fig. 5C). Non-classical resident monocytes were increased by 47% in neutropenic mice relative to the immunocompetent mice but were not affected by MAb treatment (Fig. 5C). We observed no change in CD45⁺CD3⁺ T cell populations across all experimental groups (data not shown). Additionally, we investigated the cytokine levels of interleukin (IL)-17 and tumor necrosis factor alpha (TNF-α) and observed a decrease between immunocompetent versus neutropenic mice, but no difference was reported within both groups when they were treated with phosphate-buffered saline (PBS) or 24D11 (Fig. 5D and E). In summary, protective efficacy of MAb 24D11 was still observed in neutropenic mice infected with SBU116 and was associated with lower recruitment of M2 macrophages to infected lung tissue.

24D11 monotherapy has the same efficacy compared to 17H12 and 24D11 combination therapy against wzi154 CR-Kp infection.

Because we had observed binding competition between 24D11 and 17H12, we sought to determine how this interaction would affect opsonophagocytosis and in vivo efficacy if they were given together. We tested whether a combination of the two MAbs promoted effectiveness in clearing wzi154 CR-Kp intratracheal infection. First, we explored whether phagocytic uptake by macrophages was enhanced when 24D11 and 17H12 were combined. We found that the combination therapy in the absence of serum increased opsonophagocytis relative to PBS treatment, but not relative to 17H12 treatment alone (Fig. 6A, left panel). We also found that although both 17H12 and 24D11 promoted opsonophagocytosis in the presence of serum, the combination of the two MAbs did not further enhance phagocytosis (Fig. 6A, right panel).

In an intratracheal lung infection model, we observed that MMC34-infected mice treated intraperitoneally with 17H12 and 24D11 (10 mg/kg/antibody) combined therapy had a significant drop in the organ burden load and a drop in dissemination by >3 log₁₀-fold (Fig. 6B) which was slightly higher than the 2.5 log₁₀ achieved. We found that 17H12 alone had a limited effect on reducing bacterial spread to liver and spleen (Fig. 6B) whereas 24D11 monotherapy prevented dissemination to other organs, which was not further enhanced by the combination of 24D11 with 17H12 (Fig. 6B). We repeated the opsonophagocytosis study with SBU116 CR-Kp strain, and only bacteria pre-opsonized with 24D11, whether alone or in combination, were phagocytosed by macrophages under both conditions. No difference in phagocytosed CFU was observed between the 24D11 monotherapy and combination therapy groups (Fig. S7 in Text S1). These data indicate that the protective efficacy of monotherapy with 24D11 is not further enhanced by combining it with 17H12 therapy. It also confirms that 24D11 has potent efficacy against a wzi154-carrying clade 2 CR-Kp strain.

Ethics statement.

Animal study protocols were approved by the Animal Committee (IACUC) at SBU (approval no. 628253). Healthy donors consented with Institutional Review Board (IRB)- and SBU Human Subjects Committee-approved protocols and gave blood donations under IRB no. 718744. No potentially identifiable human images or data are presented in this study.

Capsular polysaccharide purification and conjugation.

Capsular polysaccharide from carbapenem-resistant K. pneumoniae strain MMC38 (wzi50) was purified according to the methods of Banerjee et al. (24) and detailed in the Methods section in Text S1 in the supplemental material. Conjugation to the protective antigen of Bacillus anthracis (wzi50-BaPA) using the I-cyano-4dimethylaminopyridinium tetra-fluoroborate (CDAP) method was performed as previously described (55).

Vaccinations and titer calculations.

BALB/c mice (Taconic) were administered either 10 μg of BaPA-conjugated wzi50 CPS, unconjugated wzi50 CPS, or no polysaccharide in 100 μL of a 1:1 mixture of PBS with Complete Freund’s Adjuvant by intraperitoneal injection (13, 56). Booster injections were given using a 1:1 mixture of Incomplete Freund’s Adjuvant until sufficient titers (1:10,000) were reached. Serum titers in mice were measured from clotted serum extracted by facial bleed using ELISA with plates coated with unconjugated wzi50 CPS and conjugated wzi50 CPS, and also against methanol-fixed whole CR-Kp bacteria, as per protocol (24, 52). Serum titer was defined as the lowest serum dilution at an optical density (O.D.) of vaccinated mouse serum of ≥2.5 times the O.D. of naive mouse serum.

Monoclonal antibody generation and purification.

Fusion and cloning were performed as described previously (56) using polyethylene glycol-mediated fusion of splenocytes collected 2 weeks after the final booster with unconjugated wzi50 CPS to myeloma lines Ag.8 or NSO^bcl2, followed by selection in hypoxanthine-aminopterin-thymidine (HAT) medium. Details on cloning and selection of positive hybridomas and antibody production are described in the Methods S2 section of Text S1. Antibodies were produced weekly over 6 months and were purified using Pierce Protein G Affinity Chromatography per the manufacturer’s protocol and then concentrated by centrifugal filtration (AMICON 30K), filter-sterilized, snap-frozen in liquid nitrogen, and stored at −80°C until use. Concentration was determined by Bradford assay and absorbance at 280 nM (extinction coefficient = 1.4).

Binding affinity analysis.

The EC₅₀ of the MAbs was calculated using ELISA, as described previously (55, 57). Briefly, polystyrene plates (Corning 3690) were coated overnight with 0.5 mg/mL of wzi50 CPS in PBS, then blocked with 1% PBS-BSA (bovine serum albumin). Antibody was detected using an AP-conjugated goat anti-mouse IgG2b secondary antibody (Southern Biotech, 1091-04, 1:1,000). Control antibodies were run in parallel as negative controls. Binding curves were calculated in GraphPad Prism 6 using a four-parameter variable slope log agonist response curve.

Competitive ELISA.

For competitive ELISA, we modified the assay previously described by Banerjee et al. (24). Briefly, 96-well plates were coated overnight at 4°C with 0.5 μg/well of detergent-free purified CPS34 and CPS38. Next, plates were blocked with 2% PBS-BSA for 1 h. After blocking, an antibody cocktail containing one constant anti-CPS MAb at 40 μg/mL and a second competing anti-CPS antibody at increasing concentrations of 0 to 80 μg/mL was added to the coated plates. The decrease in binding affinity to the target CPS was detected by AP-labeled secondary antibodies (1:1,000) against the constant antibody. The competitive binding curve was plotted using GraphPad Prism 6.

In vitro whole blood resistance assay.

The in vitro whole blood resistance assay was modified from a previously described assay (58) and is described in Methods S3 in Text S1. Briefly, blood samples collected from healthy donors were diluted 1:1 with sterile pre-warmed (37°C) RPMI 1640. Next, 200 μL of the mixture was added to each well of 96-well round-bottomed plates (Becton Dickinson). After this, 1 × 10⁵ CFU per well of bacterial suspensions (MMC34, MMC36, SBU116) was added to blood samples and they were simultaneously treated with either 200 μg of anti-capsular MAbs or isotype controls. At 1 and 2 h, 100 μL of culture was collected and serially plated for CFU/mL quantitation. For all experiments, conditions were performed in triplicate wells, and experiments were all repeated three times.

Macrophage phagocytosis.

Macrophage cell line J774A.1 (ATCC) was used for macrophage phagocytosis assays with anti-capsular antibodies using published protocols (24, 29) and as described in the Methods S4 in Text S1. Briefly, 10⁶ CFU/well of bacteria was opsonized for 60 min in Dulbecco’s Modified Eagle Medium (DMEM) only containing 40 μg/mL of MAb 24D11 or 17H12 and was added at a multiplicity of infection (MOI) = 1 to each well of a 96-well containing 10⁶ macrophages per well. For complement-dependent phagocytosis assay, macrophages were treated with 10% normal human serum (NHS) in DMEM. For all experiments, conditions were performed in triplicate wells, and experiments were all repeated three times.

Intratracheal infection of mice.

We used C57BL/6 mice (Taconic) aged 6 to 14 weeks for all mouse experiments. Pulmonary infections and analyses of CFU/mL present in collected organs were performed as performed previously (24) and described in the Methods S5 in Text S1.

For pre-opsonized mice, bacterial strains were opsonized with 5 mg/mL of MAb 24D11 for 1 h prior to instilling the opsonized bacteria into the surgically exposed trachea. For the prophylactically treated group, mice were pretreated intraperitoneally with either 100 μL of PBS or 10 mg/kg of MAb 24D11 4 h prior to infecting mice intratracheally with 50 μL of PBS containing 6 × 10⁸ CFU of bacterial inoculum. For the therapeutic group, mice were infected with 50 μL of PBS containing 6 × 10⁸ CFU of bacterial inoculum. Four h post-surgery, they received 10 mg/kg of MAb 24D11 or MAb 17H12 through intraperitoneal injection.

Intratracheal infection of neutropenic mice.

To elucidate the efficacy of MAb 24D11 in neutropenic mice, mice were pretreated with 225 μg of rat anti-mouse Ly6G (1A8) or a control rat anti-mouse IgG2a (2A3) (BioXcell) as described previously (29). After 4 h, neutropenic mice received anti-wzi50 capsular MAbs intraperitoneally, and after 24 h, mice were euthanized, and lung tissues were dissected into two portions. The first halves of the lungs were collected in PBS containing 1× Pierce Proteinase Inhibitor, serially diluted, and plated on LB agar plates to enumerate CFU. Livers and spleens were processed in 1× PBS and serially plated for CFU. IL-17 and TNF-α cytokine analyses in lung homogenates were tested using a BioLegend ELISA Max Deluxe set (cat no. 436204 and 430904) according to the manufacturer’s protocol.

The remaining portions of lung tissues were processed and labeled for flow cytometry analysis to confirm neutrophil depletion and immunophenotyping, as described in Methods S6 in Text S1. Immune cells were first gated as live/dead cells, followed by CD45⁺ gating, and then the immune cells were gated as follows: neutrophils (CD45⁺CD11b⁺Ly6G⁺), M1 macrophages (CD45⁺CD11b⁺CD11c⁺F4/80⁺), M2 macrophages (CD45⁺CD11b⁺F4/80⁺), inflammatory monocytes (CD45⁺CD11b⁺Ly6C⁺Ly6G^–), resident monocytes (CD45⁺CD11b⁺Ly6C^–Ly6G), and T cells (CD45⁺CD3⁺) as shown in Fig. S6 in Text S1. All flow cytometry data were processed in BD FACSDiva and Flowing Software (Turku Bioscience, Finland).

Statistical analysis.

Statistical tests were performed with GraphPad Prism 6 for Windows. For multigroup comparisons of parametric data (e.g., phagocytosis, serum-resistance assays, and animal experiments), analysis of variance (ANOVA) with post hoc analysis using Tukey’s, Sidak’s, or Dunnett’s comparison tests was used. For two-group comparisons of parametric data, we performed paired t tests corrected for multiple comparisons using the Holm-Sidak method.