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. 2026 Jan 8;16:3570. doi: 10.1038/s41598-025-33619-x

Transfer of anti-Klebsiella pneumoniae immunity following infection in mice is protective against lethal challenge in offspring

Emily Mason 1,2,#, Kaitlin Winter 1,2,#, Jiawei Liang 1, Sina Hakimpour 1,3, Rhienna Patel 1,2, Tony Harn 1, Ken H Chu 1, Hennady Shulha 1, Bing Cai 1, Mandy Lo 1, Manish Sadarangani 1,2,3,✉
PMCID: PMC12848145  PMID: 41507311

Abstract

Klebsiella pneumoniae is a gram-negative, opportunistic pathogen, with high rates of antimicrobial resistance, and is responsible for a wide range of infections of the urinary tract, lungs, and bloodstream, among others. Disease burden is particularly high in neonates, where K. pneumoniae is a leading cause of sepsis. Renewed interest in vaccine development against this critical priority pathogen has focused on this vulnerable population. Vaccination in pregnancy is a promising approach for prevention of neonatal sepsis, however efforts to understand the dynamics, specificity and function of maternally transferred antibodies is ongoing. We report here that K. pneumoniae-specific IgG is readily transferred from dam to pup following wild-type infection in mice, and that maternally-transferred immunity is protective against lethal infection in pups aged 6 weeks. Further work to investigate the mechanisms of protection and explore neonatal challenge models will advance the path to a maternal vaccine to protect against neonatal sepsis.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-33619-x.

Subject terms: Diseases, Immunology, Microbiology

Introduction

Klebsiella pneumoniae is a gram-negative, bacterial species of the Enterobactericeae family, that can be a commensal organism. As a predominantly opportunistic pathogen, K. pneumoniae can cause a range of infections, and frequently causes infections of the urinary tract, respiratory tract, and surgical site incisions1. In severe cases, K. pneumoniae causes invasive infections including meningitis, bacteremia, and sepsis2,3. K. pneumoniae frequently harbours resistance to multiple classes of antibiotics with rates of multidrug resistance (MDR) increasing globally, including resistance to commonly used third generation cephalosporins and carbapenems. Given a concerning increase in the rate of MDR infections, particularly in the pediatric population, the World Health Organization has identified carbapenem-resistant and third generation cephalosporin resistant K. pneumoniae as a critical priority pathogen4,5.

Classical Klebsiella infections are predominantly associated with vulnerable populations and healthcare acquisition, including neonates and individuals with comorbidities. K. pneumoniae is a leading cause of neonatal sepsis and is the leading cause of infectious death in children under the age of five years in Sub-Saharan Africa6–8. This disproportionate impact of K. pneumoniae in neonatal sepsis burden has sparked recent interest in development of novel therapeutics targeted towards protecting neonates9. A recent Bayesian analysis found that the introduction of a vaccine in pregnancy against K. pneumoniae could avert 1–7% of all neonatal sepsis deaths worldwide, with the heaviest benefit in low- and middle-income countries (LMICs) in Africa and South Asia10. Vaccine development against K. pneumoniae has been ongoing for decades with little success. A variety of vaccine strategies have been attempted in preclinical models including whole cell, subunit, and mRNA vaccines targeting a wide array of K. pneumoniae antigens11. Despite many of these vaccines displaying strong immunogenicity and various degrees of protective ability in animal models, none have made it through clinical trials to licensure.

Importantly, none of these vaccines have been assessed in a model of administration during pregnancy, despite this being a key target population for any successful future vaccine. Antibodies are actively transferred to the fetus during pregnancy via the placenta and are thus able to protect the fetus and newborn from infection12. This antibody transfer provides important protection to neonates and young infants during the first few months of life. Young infants are at high risk for severe disease for several reasons, including immunological bias towards tolerance and dampening of pro-inflammatory signalling mechanisms that are important for clearance of pathogens13. Additionally, newborns lack the memory B cell repertoire of older children, which is acquired from exposure to various antigens over time14. Thus, maternally derived antibody provides the key mechanism of antigen-specific support for bacterial clearance and protection from severe disease15. The amount of IgG that is transferred via the placenta is dependent upon a number of factors including the maternal levels of total and specific IgG, gestational age, IgG subclass, and placental health16. There remains a gap in our understanding of Klebsiella-specific immunology kinetics and functionality in the maternal-infant dyad. A robust understanding of immune responses to infection and vaccination, and transfer of protective immune components to the fetus and newborn is a prerequisite for future testing of vaccine candidates for use during pregnancy. The specific presence and protective ability of anti-Klebsiella immunity passed from dam to offspring must be clearly elucidated. A small number of studies have previously demonstrated that placental transfer of K. pneumoniae LPS-specific IgG does occur during human pregnancy, however there have not been comprehensive studies to evaluate this in humans, or in animal models17–19.

We have developed a systemic K. pneumoniae infection model in BALB/c mice and applied it to evaluate the kinetics related to transfer of maternally-derived IgG antibodies following sub-lethal K. pneumoniae infection. The protective ability of immunity transferred from dam to pup were assessed by lethal challenge in the offspring.

Materials and methods

Ethics statement

All procedures were done in accordance with University of British Columbia (UBC) Animal Care Committee, Canadian Council on Animal Care guidelines on animal welfare, and ARRIVE guidelines. The experiments were approved by the UBC Animal Care Committee (Ref: A24-0106). All experiments were performed in accordance with these stated guidelines and the respective regulations.

Bacterial preparations

Since 2019 we have been collecting K. pneumoniae isolates from patients at BC Children’s Hospital, Vancouver, Canada with invasive infections. Of 39 unique infection episodes collected between 2019 and 2024, we have identified 27 distinct K loci with no K locus being attributed to more than 4 infection episodes. Due to the lack of dominant K type at our hospital, we chose a bacteremia causing isolate for use based on its ease and consistency in in vivo and in vitro use. A K. pneumoniae clinical isolate (internal reference 19–0061) from a pediatric bacteremia infection at BC Children’s Hospital was used for all mouse infections, serum bactericidal assays, and enzyme-linked immunosorbent assay (ELISA) coating antigens. The isolate is sequence type 1537 and of capsule K24 and lipooligosaccharide (LOS) O2α serotypes, based on Kleborate v3.2.420 and Kaptive v321. The isolate is encapsulated. To prepare bacteria for infectious challenge, K. pneumoniae was grown overnight in Luria Broth (LB) with added mucin at a concentration of 1 mg/L (LBM). Bacterial preparation was sub-cultured in LBM until it reached mid-exponential phase, with an optical density (OD600) of 0.4–0.6. OD600 used to predict colony forming units (CFU) per mL, based on a standard curve equation relating OD600 with CFU/mL. Doses were prepared by diluting bacterial suspension in 0.9% saline to the desired concentration. Doses were confirmed by plating serial dilutions on LB agar (LBA) plates overnight at 30 °C. In dose titrations, reported doses are within a ± 15% range while all other doses are reported as the exact administered dosage.

To prepare K. pneumoniae cell lysate as the target antigen for IgG ELISAs, a frozen glycerol stock was streaked onto an LB agar plate and incubated overnight at 37 °C. An isolated colony was inoculated into 5mL of LBM and incubated overnight (20 h) with shaking at 37 °C at 200 rpm. A fresh subculture was prepared by adding 2.5mL of overnight culture into 100mL of fresh LBM, which was incubated at 37 °C with 200 rpm shaking until the OD600 reached 0.45–0.55. The culture underwent centrifugation (5000 xg for 20 min, at 4 °C), the supernatant was discarded and pellet was resuspended in 5mL of chilled PBS. A further centrifugation of the solution was performed (5000 xg, 20 min, 4 °C) and the pellet resuspended in 10mL of 70% ethanol. The solution was mixed at room temperature (RT) for two hours. After a final centrifugation, the pellet was resuspended in 5mL PBS, and washed twice. Aliquots were stored at -80 °C. Bacterial protein concentration was quantified using a Pierce BCA Protein Assay Kit (thermo scientific REF 23225, LOT YD365609).

Infectious challenge

BALB/c mice of both sexes were obtained from Charles Rivers Laboratories.

Six-week-old mice of both sexes were used for infectious challenge. Mice receiving an infectious dose of K. pneumoniae were injected with 100µL of bacteria intraperitoneally. Negative control mice for dose titrations and infectious challenge in pups were injected with 0.9% saline via IP injection. Negative control dams received phosphate buffered saline via subcutaneous injection. Mice were monitored regularly following injections and assessed for weight loss, as well as changes in behaviour, appearance, elimination, dehydration, and injection site reactions according to a standardized scoring rubric (Supplemental Table 1). Mice were euthanized if they met humane endpoints of morbidity as indicated by University of British Columbia guidelines including a score of 5/5 in any category or a score of 4/5 in any category for 24 consecutive hours. Lethal doses were defined as the K. pneumoniae CFU/mouse resulting in a lethal response in 70% of mice (LD70 ± 20%). Conversely, a sub-lethal infection was defined as a K. pneumoniae CFU/mouse with a 0% mortality rate (LD0) along with observable clinical symptoms of illness. After IP injection, mice were monitored regularly for at least 48 h post-infection at 6–8 h intervals, after which time monitoring frequency was reduced, dependent upon infection recovery. Mice surviving systemic infection were included in the morbidity assessment. Symptom scores were collected and managed using REDCap electronic data capture tools hosted at BC Children’s Hospital Research Institute22,23. All symptom scores across the first 48 h of monitoring were added together to provide a cumulative symptom score (CSS) for each mouse.

Breeding procedures

Female mice aged 7 weeks were paired with males for 10 days to facilitate breeding. Mice were monitored daily to identify birth dates of litters between 16 and 30 days post pairing.

Sample collection

Non-terminal blood collections were completed by saphenous bleeds. Mice were restrained and blood was collected from the medial saphenous vein in volumes no greater than 15% of the total blood volume using a 23G needle. Terminal blood collections of adult mice were completed via cardiac puncture using a 25G needle. Terminal blood collections of trunk fluid were done for all litters aged less than 42 days. For serum collection from trunk fluid, blood was collected in microtainer serum separator tubes (Grenier Bio-One GmbH). Serum separation was performed according to manufacturer’s instructions and aliquots were stored at -80 °C until use.

In adult mice, animals were first anesthetized using 5% isoflurane; and then exposed to carbon dioxide until breathing ceased followed by a physical euthanasia method. In mice aged less than 10 days, anesthesia was gradually administered until mice were fully anesthetized (5% isoflurane) followed by physical euthanasia methods.

For the evaluation of bacterial burden, mice were euthanized at regular intervals following infection. Blood was collected by cardiac puncture and immediately stored with 10 USP units of heparin. The lungs, liver, heart, kidney and brain were collected in D-PBS (Gibco) and stored on ice. Organs were homogenized. All collected samples were serially diluted and plated on LB agar at 30 °C overnight.

Quantification of anti-K. pneumoniae IgG

An endpoint ELISA was used to quantify K. pneumoniae-specific IgG in mouse sera. Plates were coated with 10 µg/mL of whole-cell K. pneumoniae lysate, which was prepared as described above at 100µL per well. Following washing, plates were blocked for two hours at RT using a 1% bovine serum albumin (BSA) solution. Plates were washed by automated plate washer (BioTek 405DW) using a pre-set wash cycle including 200µL of 1X PBS with 0.1% Tween-20 wash buffer followed by thirty second soaks with five repeats. Remaining liquid was expelled before addition of next solution. Sera were then added in serial two-fold dilutions, starting from a dilution of 1:200, and incubated for two hours at RT. Following a wash step, goat anti-mouse IgG conjugated to horseradish peroxidase was added as the secondary antibody and incubated for 1 h at RT. Plates were washed. Results were visualized using the addition of 3,3′,5,5′-tetramethylbenzidine followed by the addition of 1 M hydrochloric acid to stop the reaction after a 15-minute incubation. The OD450 of all wells were determined immediately following the reaction stop using a VarioSkan Luxe plate reader.

All samples were run in duplicate. Means of plate duplicates were used to compute endpoint titers. The anti-K. pneumoniae IgG endpoint titers were calculated as log2 transformations of the highest dilution yielding an OD450 greater than two times that of blank wells, which contained blocking buffer rather than sera during primary antibody incubations. Samples with titers at or below the lower limit of quantification (LLOQ) of 11.6 were assigned an endpoint titer of 10.6. LLOQ was determined to be the endpoint titer at which sera collected from germ-free mice of both sexes (n = 16) were assessed to be negative on the optimized assay. All plates included blank wells as a negative control and positive control serum, and plates were rejected if negative control ODs exceeded a value of 0.1. Positive control serum was comprised of pooled sera from mice of both sexes that were previously infected with K. pneumoniae by IP injection. Plates were also rejected if the endpoint titer of the positive control was not in the range 14.6–16.6. This range was determined from repeated measurements of positive control sera across users, plates, and days to ensure repeatability. Samples were rejected if the percent coefficient of variance of any duplicate within two dilutions of the endpoint titer exceeded 15%.

Serum bactericidal activity

Serum bactericidal activity was measured using a serum bactericidal assay (SBA). K. pneumoniae (19–0061) was prepared identically to infection (described above). Sera were heat inactivated at 56 °C for 30 min. Sera were diluted serially in two-fold dilutions in PBS, in a total of 20 µL per well, in duplicate on a non-treated 96 well round bottom plate (Falcon). Baby rabbit complement (BRC) (Pel-Freez, lot 30646) diluted in PBS to 60% was added to each well with a final volume of 15%. An estimated 300 CFU of K. pneumoniae diluted in PBS was added to each well. Control columns included a BRC-only control (no serum; complement control) and a heat inactivated BRC with no sera control (viable control). 10 µL of each viable control well (8) were plated on LB agar at time 0 (T0). The 96 well plate was incubated for an hour at 37 °C. 10 µL of each well was then plated on LB agar (T60). LB agar plates were incubated at room temperature overnight, prior to counting colonies. Bacterial survival was calculated as the averaged counts at T60 divided by the averaged viable counts at T0, multiplied by 100%. For a plate to pass, the complement control needed to be > 80%. Bacterial killing was calculated as bacterial survival subtracted from 1, multiplied by 100%. SBA results are reported as the inverse of the highest dilution achieving greater than 50% bacterial killing.

Western blot for identification of immunogenic Klebsiella antigens

A 15 µg of K. pneumoniae lysate was loaded onto a 15% sodium dodecyl sulfate-polyacrylamide gel alongside the Kaleidoscope prestained protein ladder (Biorad, 1610375). Proteins were transferred to a nitrocellulose membrane using the Power Blotter (Invitrogen). After transfer, membranes were washed in PBS and then blocked for an hour with 5% skimmed milk in PBS. Membranes were washed 3 times with 0.1% Tween-20 in PBS and then cut to include one ladder lane and one lysate lane per membrane piece. Sera were pooled from mice at the same timepoint. Pooled mouse antibodies were incubated with the membranes overnight at 4 °C. Membranes were washed again 3 times and then incubated at RT with IRDye conjugated anti-mouse IgG (Licor) diluted 1:2000 in 2.5% skimmed milk and 0.05% Tween in PBS for an hour. Membranes were washed 3 times and then imaged using the iBright1500 (Invitrogen).

Statistical analyses

Analyses were completed and visualized using GraphPad Prism (ver 10.2.2). Kaplan-Meier survival curves were used to visualize survival and mantel-cox log-rank tests were used to assess significance. Cumulative symptom scores, antibody ratios (pup/dam and IgG1/IgG2a), and bacterial burden were assessed using non-parametric tests, either a Mann-Whitney test or a Kruskal-Wallis test with Dunn’s multiple comparisons, as indicated in the figure legend. Log2 of endpoint antibody titers were assessed by parametric methods, either t-tests or a one-way ANOVA with Dunnett’s multiple comparisons, as indicated in the figure legend. For all multiple comparisons only groups with 3 or more samples were included in statistical analyses and Bonferroni correction was used. p-values < 0.05 were considered significant.

Results

Development of sub-lethal and lethal systemic infection models using intraperitoneal injection

Lethal infection doses, that result in less than 30% survival, were determined in mice of both sexes by administration of increasing doses of K. pneumoniae. Female mice were infected with bacterial doses ranging from 4 × 106 to 1.5 × 107 CFU per mouse (Fig. 1A). Mice were grouped as dose groups that received doses that are within 15% of the reported dose. Female mice infected with a dose of 9.5 × 106 CFU had 25% survival, which was significantly lower than negative controls (100% survival) (p = 0.004) (Fig. 1A). Male mice were infected with doses ranging from 2.5 × 106 to 7 × 106 CFU per mouse (Fig. 1B). Male mice infected with a dose of 7 × 106 CFU had a 14% survival rate, which was significantly lower than negative controls (100% survival) (p = 0.010). Thus, doses of 9 × 106 and 7 × 106 CFU per mouse were selected for female and male lethal infectious doses respectively.

Fig. 1.

Fig. 1

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Optimization of sub-lethal and lethal infectious doses of K. pneumoniae in mice of both sexes. Mice aged 6 weeks were injected with varying doses of K. pneumoniae by IP injection and monitored for mortality and symptoms of illness. Differences in survival of female (A) and male mice (B) dosing ranges was assessed using Mantel-Cox log-rank tests (n = 6–16, 2 repeats, DF = 1). Groups shown are pooled between different doses that are within 15% of the reported dose. Groups infected with K. pneumoniae were compared with negative control groups; exact p values are denoted. The cumulative symptom score in female (C) and (D) male mice surviving infection. CSS between mice receiving K. pneumoniae (all groups where n > 2) and negative controls were done by Kruskal-Wallis tests with Dunn’s multiple comparison corrections. Exact p-values are reported for all comparisons (DF = 2 female, DF = 2 male). (E) Mice (n = 4 female, n = 6 male) were infected with sub-lethal infectious doses of K. pneumoniae. Anti-K. pneumoniae IgG endpoint titers as determined by ELISA. Negative control mice of both sexes were pooled as all samples were below the lower limit of quantification. Mean log2 endpoint titers and standard deviation are shown.

A sub-lethal dose was also established in female mice for use in pre-pregnancy infection. A dose of 4.5 × 106 CFU was selected based on survival and further assessed for morbidity and immunological responses. Morbidity scores in mice of both sexes surviving infection followed a dose-dependent response wherein mice infected with higher doses of K. pneumoniae had higher morbidity scores over 48 h of monitoring (Fig. 1C, D). Female mice receiving a sub-lethal infectious dose of K. pneumoniae at 5 × 106 CFU experienced significantly higher morbidity scores compared with negative controls (p = 0.044) (Fig. 1C). In female mice given a sub-lethal infection of 4 × 106 CFU, K. pneumoniae was identified across various organs including the lungs, liver, kidneys, and spleen until at least 96 h post-infection with full clearance by 160 h (Supplemental Fig. 1). Bacteria were identified in the bloodstream and brain at 12- and 24-hours post-infection but were cleared by 36 h post-infection. Antibody responses to infection were characterized in mice of both sexes (Fig. 1E). In mice receiving sub-lethal doses, anti-K. pneumoniae IgG was identified 7 days post-infection and persisted for at least 12 weeks following infection (Fig. 1E).

Dams had a significant immune response to sub-lethal infection of K. pneumoniae

Dams were infected with a sub-lethal dose of 3.8 × 106 CFU K. pneumoniae before breeding (Fig. 2A). Female mice demonstrated observable symptoms including weight loss during the first 48 h post-infection and had higher cumulative symptom scores compared with previous negative control mice. The anti-K. pneumoniae IgG among infected dams at 21 days post-infection, approximately 7 days before birth of litters, was significantly higher than negative control dams (mean log2 endpoint titer 14.5 vs. 10.6, p < 0.001) (Fig. 2B). Following birth of the litters, infected dams had persistently higher anti-K. pneumoniae IgG than controls until weaning (30–57 days post-infection), at which point mean log2 IgG titers were 15.6 (Fig. 2B). Dams experienced a 2–5 fold increase in K. pneumoniae-specific IgG from baseline to 3-weeks post-infection (day − 7), which persisted to day 21 post-birth, by which time mice had a 4–6 fold higher antibody compared with baseline (Fig. 2C). Anti-K. pneumoniae IgG recognized a range of K. pneumoniae proteins by Western blot (Supplemental Fig. 2).

Fig. 2.

Fig. 2

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Anti-K. pneumoniae IgG titers are maintained in dams up to 49 days post infection. (A) Female mice aged 6 weeks were injected with 3.9 × 106 CFU of K. pneumoniae by IP injection. Serum was collected from all dams 21 days after infection. Serum was collected from litters and the corresponding dam 1-, 7- and 14-days post birth. Remaining dam serum was collected at weaning, 21 days post birth and approximately 49 days post infection. Weaned pups were challenged with a lethal dose of K. pneumoniae at 42 days old (created in Biorender. https://BioRender.com/k33md8e). (B) Anti-K. pneumoniae IgG endpoint titers in sera collected from dams were determined by ELISA (n = 1–10). Log2 endpoint titers of 10.64 represents titers below the lower limit of quantification. Significant comparisons are shown with lines and p-values from t-tests between the negative control and sub-lethally infected dams at each timepoint. (C) Fold change from baseline titers (pre-infection) were calculated by subtracting the logged baseline endpoint titer from the endpoint titer on the indicated experimental day (n = 1–10). Mean fold change and standard deviation are shown.

Maternal transfer of Klebsiella-specific IgG persisted to 42 days of age in pups

Anti-K. pneumoniae IgG was assessed in pups at 1, 7, 14, and 42 days of age to determine presence and kinetics of maternally derived antibody (Fig. 3). K. pneumoniae-specific IgG was identified at all timepoints in pups born to dams with prior infection, with the highest titers observed at day 7 of life (mean log2 endpoint titer 13.6–14.6). Offspring from infected dams had significantly higher K. pneumoniae-specific IgG titers than negative control pups at all timepoints (p = 0.013, < 0.001, 0.029 and < 0.001 for days 1, 7, 14 and 42 respectively) (Fig. 3A). There was a decrease in the total quantity of anti-K. pneumoniae IgG from day 7 to day 42, although the titers in the infected group remained significantly higher than negative controls at day 42 (mean log2 endpoint titer 12.6 vs. 10.6, p < 0.001) (Fig. 3A). There was no significant difference between sexes in IgG titers at days 14 and 42 (p > 0.999, p = 0.436, respectively; Welch’s t test). The ratio of pup anti-K. pneumoniae IgG to their dam at the time of sample collection in the infected group increased significantly between day 1 and day 7 (0.82 to 0.93, p < 0.001) and was 0.88 at day 14 (p = 0.049) (Fig. 3B).

Fig. 3.

Fig. 3

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Anti-K. pneumoniae IgG is transferred from dams to pups. (A) Anti-K. pneumoniae IgG endpoint titers in sera collected from pups were determined by ELISA (n = 6–28). 10.64 represents titers below the lower limit of quantification. Titers from pups born of negative control and sub-lethally infected dams at each timepoint were tested by t-test. (B) The ratio of pup/dam anti-K. pneumoniae IgG endpoint titers were calculated by dividing the pup endpoint titer with the endpoint titer of their dam collected at the same time (n = 6–10). (C) The IgG1 over IgG2a ratio was determined by dividing IgG1 endpoint titers by the IgG2a endpoint titer. Dam IgG1/IgG2a ratio 7 days before giving birth are displayed with pup ratios on Day 1, 7, 14 and 42 days after birth (n = 3–8). (D) Dam IgG2a titers 7 days before giving birth are displayed with pup titers on Day 1, 7, 14 and 42 days after birth. Log2 endpoint titers of 8.64 represents titers below the lower limit of quantification (n = 3–8). Significant comparisons are shown with lines and p-values from a one-way ANOVA with Dunn’s multiple comparison test against dam titers 7 days before giving birth. (E) Dam IgG1 titers 7 days before giving birth are displayed with pup titers on Day 1, 7, 14 and 42 days after birth. Log2 endpoint titers of 8.64 represents titers below the lower limit of quantification (n = 3–8). A one-way ANOVA detected no significant differences. (F) Pup/dam IgG2a ratios were calculated by dividing the pup endpoint titer with the endpoint titer of their dam collected at the same time (n = 3–7). (G) Pup/dam IgG1 ratios were calculated by dividing the pup endpoint titer with the endpoint titer of their dam collected at the same time (n = 3–7). Box and whiskers plots display minimum and maximum, all other plots show mean and standard deviation. All pup/dam ratio ratios were tested with a Kruskal-Wallis test with Dunn’s multiple comparison test against all pairings (B,F,G). All significant comparisons are displayed with lines and exact p-values.

Among pups born to infected dams, initial transfer of K. pneumoniae-specific IgG at 1 and 7 days of age was skewed towards IgG2a (IgG1/IgG2a ratio 0.75 and 0.67, respectively) (Fig. 3C). Over time, the remaining IgG profile shifted towards an IgG1 skewed response (IgG1/IgG2a ratio 2.00 and 2.25 at days 14 and 42, respectively). This was primarily due to a change in anti-K. pneumoniae IgG2a levels, with high IgG2a titers in pups at 1 and 7 days after birth that waned over time to 42 days of age (Fig. 3D). In contrast, the IgG1 titers remained stable throughout this period, with minimal waning evident by 42 days of age (Fig. 3E). The pup/dam ratio of anti-K. pneumoniae IgG2a remained consistently above 1 (2.00, 3.33, and 2.00 on days 1, 7 and 14, respectively) (Fig. 3F). The pup/dam ratio of anti-K. pneumoniae were higher at day 7 and 14 when compared with day 1 (0.32, 2, and 2 on days 1, 7 and 14, respectively) (p = 0.002 and p = 0.019) (Fig. 3G).

A limited number of samples were tested for serum bactericidal activity, based on availability of sufficient sample volume for this assay. Both dams 7 days prior to birth (n = 2), and pups at day 7, 14, and 42 (n = 2, 2, & 6; respectively) had bactericidal activity below the limit of detection.

Maternal infection with Klebsiella pneumoniae protected pups against a lethal infection

Pups were infected with a lethal dose of 3.6 × 106 CFU (males) − 6.6 × 106 CFU (females) of K. pneumoniae at 42 days of age. In both male and females, offspring of dams that had been infected before pregnancy had higher survival compared with uninfected controls (female pups: 100% vs. 37.5%, p = 0.030; male pups: 50% vs. 25%, p = 0.538 (Fig. 4). While male pups from infected dams had increased survival compared to controls, the study was underpowered for the protective effect size. In both sexes of pups from infected dams, surviving pups had significantly higher IgG titers at day 42 compared to non-survivors (p = 0.003, Welch’s t test). In a second group of female mice receiving a dose of 5.7 × 106 CFU, bacterial burden in liver, lungs, spleen, and blood were quantified in female mice at 24 h post challenge (Supplemental Fig. 3). While the bacterial concentration was lower in the infected group in blood, lungs, and liver, these differences were found to be statistically insignificant (p = 0.218, p = 0.234 and p = 0.662, respectively). Bacterial burden in the spleen was increased in the pups of sub-lethally infected dams.

Fig. 4.

Fig. 4

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Sublethal infection of dams with K. pneumoniae protects pups from lethal challenge at 42 days old. (A) Female (n = 5–8, individual experiment) pup survival following lethal challenge of 6.6 × 106 CFU K. pneumoniae is shown. (B) Male (n = 8–12, two experiments) pup survival following lethal challenge of 3.6 × 106 CFU K. pneumoniae is shown. Survival curves were assessed using Mantel-Cox log-rank tests, exact p values are denoted.

Discussion

We have demonstrated that following sub-lethal infection with K. pneumoniae, female mice developed a pathogen-specific IgG response which was transferred to their offspring via the placenta and breastmilk, indicated by the presence of IgG from birth, and subsequent increase by day 7. This polyclonal IgG response was reactive to several K. pneumoniae proteins. Further evaluation of anti-K. pneumoniae IgG subtypes revealed that dams infected with K. pneumoniae had a slight skewing towards an IgG1, indicating a Th2-biased response. While this skewing was also observed in pups from day 14 onwards, their initial responses were IgG2a skewed. Importantly, pups from previously infected dams were protected from lethal challenge with K. pneumoniae at 42 days of age when compared with negative controls, and at a time when they had a significantly IgG1-skewed response. Fundamentally, this work provides proof-of-concept in a mouse model that maternally derived IgG against K. pneumoniae is protective against lethal infection and is helpful in further development of vaccines for use in pregnancy.

The presence of anti-K. pneumoniae specific IgG in pups at day 1 of life indicates that placental transfer of pathogen-specific maternal antibody does take place. Further, the increase in ratio between pup-dam K. pneumoniae-specific IgG between day 1 and day 7 suggests that ongoing transfer of maternal anti-K. pneumoniae IgG continued via colostrum and milk after birth. Thus, the total pathogen-specific IgG in breast-fed pups was a combination of waning IgG transferred via the placenta, increasing amounts of IgG transferred by breast milk, and any IgG being produced by the pups themselves. The lack of any IgG in the control group, however, indicates that there was minimal K. pneumoniae-specific IgG being produced by the pups. The interaction between these two maternally derived sources of pathogen-specific IgG likely informed the non-linear IgG kinetics observed in offspring. Due to the less quantitative nature of endpoint titer ELISAs the IgG subtype ratio data is best interpreted by comparing the ratio at different timepoints. Initially, pups had Th1, IgG2a-skewed responses, despite the Th2 IgG1-skewed responses in the dams evident at 7 days prior to birth. Over time, the IgG1/IgG2a ratio increased in pups, and this appeared to be from increased waning of anti-K. pneumoniae specific IgG2a over time, whereas the IgG1 titers held steady across all timepoints with a slight decrease by 42 days of age. Interestingly, while the pup/dam ratio of anti-K. pneumoniae IgG2a remained consistently around 2 over the first 14 days of life, the IgG1 pup/dam ratio increased from 0.33 at Day 1 to 2.00 for days 7 and 14. This suggests that IgG1 was transferred with increased efficacy through milk.

Higher survival in pups of infected dams coupled with significantly higher anti-K. pneumoniae IgG at day 42 compared with negative controls strongly suggests that maternal IgG against K. pneumoniae is protective against lethal infection. Protection is likely mediated by a variety of factors including quantity of antibodies, antigen specificity, functionality or binding affinity, and possibly aspects of adaptive cellular immunity11,24–26. We screened a very limited number of sera samples for serum bactericidal activity. All samples had activity below our level of detection. This suggests protection is not a result of serum bactericidal activity, which aligns with the recent work of Roscioli et al.27. Roscioli et al. demonstrated that bactericidal activity of monoclonal antibodies was not directly correlated with their protective efficacy in mice. While not performed in this study, the use of alternative models such as knockout mice and passive transfer of IgG from infected dams could be used in future to confirm the protective role of IgG. Additional in vitro assays such as the opsonophagocytic killing assay, a repeat of the SBA and examining the ability of antibodies to elicit enchained growth may reveal a mechanism of protection by IgG. It has been previously established that specific antibody responses to classical and hypervirulent K. pneumoniae is protective against K. pneumoniae re-infection in adult mice, though this protective effect appears to be strain dependent28. CPS and LPS are well-established antigens that may be required for protection against infection and are often serotype specific. In our study, pups in the sub-lethal infection group were challenged with the same strain as was used to infect the dams prior to pregnancy. It is possible that the antibodies stimulated by sub-lethal infection were protective because they targeted serotype-specific polysaccharide epitopes. The potential role of antigen specificity in protection from challenge is further supported by differences in proteinaceous antigen specificity of dam sera by western blot (supplemental Fig. 2). Antigens may be targeted by different IgG subtypes, as Rosen et al. found that a capsule-based vaccine elicited more IgG2b and IgG2c antibodies in outbred CD-1 mice compared with an O1-based vaccine29. Beyond antigen targets, antibody subtype and functionality can also contribute to protection. Antibody-mediated phagocytic activity has been demonstrated to be a significant clearance pathway for Klebsiella and other encapsulated bacteria, which requires a robust Th2 response9,30. Further investigations not only into protective mechanisms in adult mice, but also in pups via maternal transfer are vital for better K. pneumoniae vaccine design.

To better understand the maternal transfer of Klebsiella-specific antibody, we sought to establish an infection model in mice. Initially, we established a dosing range of K. pneumoniae that delivered intraperitoneally resulted in 30%-100% survival in 6-week-old female mice and 10%-100% survival in male mice. Administration of live K. pneumoniae to mice via intraperitoneal injection has been previously used to establish systemic and abdominal-focus infections in mice of various strains31–33. While administered doses vary significantly between groups and experiments, largely owing to pathogenic differences in specific bacterial isolates used for infection, 106-107 CFU has been used for ‘classical’ infections previously28. Our data suggest that systemic infection was established with bacteria identifiable in various organ systems until at least 4 days post-infection; interestingly, bacteria were found in the peritoneal cavity, thoracic cavity, and central nervous system at various points in the infection course, though bacteria were cleared from the blood within 24 h. This suggests that while the clinical isolate in this study likely uses the bloodstream to disseminate effectively to distal infection sites, it lacks the ability to persist in the circulatory system itself at sub-lethal infectious doses.

The use of mice to study maternal transfer of immunity does have inherent limitations. Following birth, dams provide additional immunity through breast-feeding until pups are weaned at about 21 days of age. We and others have demonstrated that pathogen-specific IgG concentrations in pups are maintained at the same concentration or higher than observed at birth for several weeks34,35. In humans, however, the evidence for the quantity and function of IgG or IgA transferred via colostrum or breast milk is less established36.

The intention of K. pneumoniae vaccination in pregnancy is to protect young infants in the first few months of life9. Lethal challenge of pups was administered at 42 days of age, and sexual maturity of mice is reached between 42 and 56 days of age, while antibody production and waning kinetics follow comparable patterns in timing to humans37. Administration of lethal challenge to pups at adulthood presents some limitation in applicability of these results to human infection biology. In order to more accurately model the impact of maternal immunity in protecting neonates, lethal challenge of neonatal pups may be required. However, it is highly encouraging that pathogen-specific IgG was maintained until at least 42 days of age in mice, which indicates that longer-lasting immunity against K. pneumoniae in human infants may be achievable.

Conclusion

We demonstrated, for the first time, that anti-K. pneumoniae IgG is transferred via placenta and colostrum in mice from dam to pup. We further showed protection from lethal challenge following sub-lethal infection in dams, which was accompanied by significantly higher anti-K. pneumoniae IgG at time of infection compared with negative controls, indicating antibody-mediated protective mechanisms, which is extremely encouraging for K. pneumoniae vaccine development. Neonates continue to be an important target population for vaccine development against K. pneumoniae; the use of maternal transfer models such as the one described are imperative to specifically develop vaccines to prevent neonatal sepsis and reduce infant mortality.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (12.2KB, xlsx)
Supplementary Material 2 (653.5KB, docx)

Acknowledgements

We would like to thank the Veterinarian team at University of British Columbia’s Animal Care Services for their support in developing our animal models. We would like to thank Wei Ling Kuan, Selina (Jiayun) Cai, and Lianne Presley for their help with the optimization of and performing the SBAs.

Author contributions

E.M., K.W., J.L., and M.S. contributed to the study and experimental design. E.M., K.W., J.L., S.H., R.P., T.H, and B.C. performed animal experiments and sample processing. E.M., K.W., J.L., S.H., R.P., T.H., K.C., and M.L. performed ELISAs and SBAs. E.M., K.W., H.S., and M.S. performed the analysis of the data and prepared the manuscript.

Funding

This work was supported by funding from the BC Children’s Hospital Research Institute and the Canadian Lung Association awarded to M.S. K.W. received fellowships from BC Children’s Hospital Research Institute and Michael Smith Health Research BC. This work was supported by an Early Career Award Program Grant awarded by the Thrasher Research Fund and an ESCMID Research Grant awarded by the European Society of Clinical Microbiology and Infectious Diseases awarded to K.W. M.S. is supported via a salary award from the BC Children’s Hospital Foundation.

Data availability

Data is provided within the manuscript or supplementary information files. Additional data requests can be sent to the corresponding author.

Declarations

Competing interests

In the last 3 years, M.S. has been an investigator on unrelated projects funded by GlaxoSmithKline, Merck, Moderna, Pfizer, and Sanofi-Pasteur. All funds have been paid to his institute, and he has not received any personal payments. Other authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Emily Mason and Kaitlin Winter contributed equally to this work.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (12.2KB, xlsx)
Supplementary Material 2 (653.5KB, docx)

Data Availability Statement

Data is provided within the manuscript or supplementary information files. Additional data requests can be sent to the corresponding author.

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