A multivalent capsule vaccine protects against Klebsiella pneumoniae bloodstream infections in healthy and immunocompromised mice

Abstract

Klebsiella pneumoniae is a leading cause of nosocomial infections, bacteremia, and worldwide mortality. Further, a drastic rise in antibiotic-resistant isolates poses an urgent threat to humanity. Unfortunately, despite its clinical importance, a licensed K. pneumoniae vaccine is not yet available. Here, we report on the production and characterization of the broadest K. pneumoniae capsule bioconjugate vaccine to date. We tested this vaccine for its immunogenicity, functionality, efficacy, and antibody durability against a variety of K. pneumoniae isolates in a murine bacteremia model. We also established an immunocompromised murine model of bacteremia to better recapitulate human infection and tested our vaccine's efficacy in this background. The tetravalent capsule vaccine is highly immunogenic in mice, generating a robust immune response against all capsule types included (K1, K2, KL102, and KL107). Further, the generated antibodies persist for at least 6 months. The vaccine-induced antibodies are highly functional against a variety of clinical isolates of K. pneumoniae, including both classical and hypervirulent strains. Finally, the vaccine led to increased survival after bacteremia challenge compared to placebo-immunized mice. Our findings confirm that a capsule-based bioconjugate vaccine has clinical potential in preventing K. pneumoniae infections. These experiments signify much-needed progress towards a multivalent vaccine to combat this increasingly troublesome pathogen.

Fig. 1. Detection of IgG generated by K4V-EPA immunization in mice.

Sera from carrier protein alone- (Control) or K4V-immunized mice were used to measure specific IgG concentrations over the course of the immunization series as measured by ELISA against glycoengineered E. coli expressing A K1 polysaccharide, B K2 polysaccharide, C KL102 polysaccharide, or D KL107 polysaccharide. Graphs display immunoglobulin concentrations of 1:100 serum dilutions as calculated from a standard curve. Error bars represent standard deviation.

Fig. 2. Klebsiella pneumoniae isolate-specific IgG titers over the course of immunization.

Control or K4V-EPA mouse sera were used to measure specific IgG kinetics over the course of the immunization by ELISA against whole bacterial strains: A NTUH-K2044, B cKp120, C ATCC 43816, D KR174, E BEI 669448, or F BEI 702325. Graphs display immunoglobulin concentrations of 1:100 serum dilutions as calculated from a standard curve. Error bars represent standard deviation.

Fig. 3. Serum bactericidal assays with vaccinated mouse serum.

Serum bactericidal assays of day 42 sera from mice immunized with either EPA carrier protein or K4V-EPA bioconjugate vaccines as measured against A NTUH-K2044, B cKp120, C ATCC 43816, D KR174, E BEI 669448, or F BEI 702325. Each data point represents a single mouse. Statistical analyses were performed via Mann-Whitney U test in comparison to EPA survival. Exact p values: ****p < 0.0001. Error bars represent standard deviation.

Fig. 4. Opsonophagocytic killing assay with vaccinated mouse serum.

OPKA using day 42 immune serum from mice immunized with EPA carrier protein alone or K4V-EPA bioconjugate vaccines as measured against strains A NTUH-K2044, B cKp120, C ATCC 43816, D KR174, E BEI 669448, or F BEI 702325. As indicated by legend below each graph, bacteria were incubated with combinations of heat-inactivated diluted mouse serum +/− HL-60 cells with baby rabbit complement (BRC). Each data point represents a single mouse. Statistical analyses were performed using Mann-Whitney U tests in comparison to EPA survival or between K4V-EPA groups +/− HL-60 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. Error bars represent standard deviation. Exact p values: A **** p < 0.0001, ** p = 0.0029, ***p = 0.0002. B**** p < 0.0001, ***p = 0.0003. C **** p < 0.0001, ***p = 0.0003, ns p = 0.0603. D ****p < 0.0001, ***p = 0.0003. E ***p = 0.0003, *p = 0.0115, ns p = 0.0753. F ****p < 0.0001, ns p = 0.4813.

Fig. 5. Survival of bioconjugate-vaccinated mice after lethal bacteremia challenge.

Mice were vaccinated with either the carrier protein EPA alone or K4V-EPA multi-valent capsule vaccine on days 0,14, and 28 followed by intraperitoneal injection with K. pneumoniae isolates A NTUH-K2044, B cKp120, C ATCC 43816, D KR174, E BEI 669448, or F BEI 702325. Mice were infected with ~2000 CFU for NTUH and 43816, ~10⁷ CFU for BEI 702325, ~10⁸ CFU for cKp120 and KR174, and ~10⁹ CFU for BEI 669448. Each group contains n = 10 mice combined over two independent experiments. Statistical analyses were performed via Log-rank (Mantel-Cox) tests comparing against EPA control group. *p < 0.05, ****p < 0.0001, ns not significant. Exact p values: A ****<0.0001. B ns p = 0.2626. C ****<0.0001. D *p = 0.0145. E *p = 0.0293. F ns p = 0.1990.

Fig. 6. Survival of immunocompromised bioconjugate-vaccinated mice after lethal bacteremia challenge.

Mice were vaccinated with either the carrier protein EPA alone or the K4V-EPA multi-valent capsule vaccine on days 0,14, and 28. An immunocompromising state was then induced by three injections of cyclophosphamide at 4 days prior to infection (150 mg/kg), one day prior to infection (100 mg/kg), and two days post-infection (100 mg/kg). Mice were then given an intraperitoneal injection with classical K. pneumoniae isolates A cKp120, B KR174, C BEI 669448, or D BEI 702325. Mice were infected with ~10⁶ CFU for cKp120 and BEI 702325, ~10⁷ CFU for KR174, ~10⁸ CFU for BEI 669448. Each group contains n = 10 mice combined over two independent experiments. Statistical analyses were performed via Log-rank (Mantel-Cox) tests comparing against EPA control group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Exact p values: A ***p = 0.0001. B ****p < 0.0001. C *p = 0.0245. D **p = 0.0061.

Fig. 7. Antibody longevity of each bioconjugate vaccine in K4V.

Mice were immunized with monovalent formulations of EPA, K1-EPA, K2-EPA, KL102-EPA, or KL107-EPA. Mice were immunized on days 0, 14, and 28 and were bled every month for 6 months. Six-month serums were tested in a serum bactericidal assay against strains A NTUH-K2044, B cKp120, C, ATCC 43816, D KR174, E BEI 669448, or F BEI 702325. After six months, the EPA and K1-EPA immunized cages were challenged intraperitoneally with a lethal dose (~2000 CFU) of NTUH-K2044 and monitored for survival (G). Each data point represents a single mouse. Statistical analyses (A−F) were performed via the Mann-Whitney U test or via (G) Log-rank Mantel-Cox test in comparison to EPA survival. *p < 0.05, **p < 0.01. Error bars represent standard deviation. Exact p values: A* p = 0.0159. B **p = 0.0079. C **p = 0.0079. D **p = 0.0079. E *p = 0.0317. F *p = 0.0159. G **p = 0.0031.