Vaccination with Carbapenemase KPC-2 and Virulence Factor Pal Provided Robust Protection Against Klebsiella pneumoniae Lung Infection

TL;DR

A vaccine combining KPC-2 and Pal proteins effectively protects against a dangerous type of Klebsiella pneumoniae lung infection.

Contribution

A novel dual-target vaccine, KPC-Pal, is developed to combat carbapenem-resistant and hypervirulent Klebsiella pneumoniae.

Findings

KPC-Pal vaccination significantly improved survival and reduced bacterial load in the lungs of mice.

The vaccine induced a strong immune response with Th2-biased humoral and mixed Th1/Th2/Th17 cellular profiles.

KPC-Pal antibodies enhanced meropenem efficacy and showed cross-reactivity against KPC-3 and KPC-33 variants.

Abstract

Objectives: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) merges multidrug resistance with hypervirulence, posing unprecedented therapeutic challenges. This study aimed to evaluate the efficacy of a recombinant fusion protein vaccine, KPC-Pal, designed to target both the carbapenemase KPC-2 and the virulence-associated peptidoglycan-associated lipoprotein Pal. Methods: The KPC-Pal fusion protein was constructed, expressed, and purified. Its protective efficacy was systematically assessed in a murine pneumonia model by measuring antigen-specific antibodies, cytokine profiles, and memory cell populations. The synergistic effect with the antibiotic meropenem was evaluated both in vitro and in vivo. Furthermore, the interaction with innate immune signaling via TLR2 was investigated. Results: Immunization with KPC-Pal conferred superior protection, resulting in significantly higher survival rates and reduced bacterial burdens in the lungs compared to immunization with either KPC-2 or Pal alone. It induced a robust Th2-biased humoral response and a mixed Th1/Th2/Th17 cellular immune profile, along with enhanced formation of tissue-resident memory T cells. Antibodies generated against KPC-Pal enhanced the efficacy of meropenem in vitro and in animal models, demonstrating a synergistic effect. While Pal alone strongly activated TLR2-driven inflammatory pathways, the KPC-Pal fusion selectively modulated MAPK signaling, mitigating excessive cytokine production. Additionally, KPC-Pal vaccination elicited cross-reactive antibodies against KPC-3 and KPC-33 variants. Conclusions: KPC-Pal functions as both an antigen and a self-adjuvant, offering a promising dual-target strategy for combating K. pneumoniae infections.

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1. Introduction

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative opportunistic pathogen and a leading cause of nosocomial infections [1], responsible for approximately 1.8% of hospital-acquired pneumonia and 8–12% of ventilator-associated infections worldwide [2]. Bloodstream infections caused by K. pneumoniae carry a mortality rate of 50–100% in patients with alcoholism or sepsis [3]. The rise of carbapenem-resistant (CRKP) and carbapenem-resistant hypervirulent (CR-hvKP) strains [4,5] has compromised the effectiveness of carbapenems as the “last line of defense” against multidrug-resistant Gram-negative bacteria [6]. Traditional antibiotic development is unable to keep pace with CRKP evolution, leading to a shift in focus towards rapid diagnostics, optimized combination therapies, and new antibacterial targets [7,8]. Proactive prevention, particularly through prophylactic vaccines that induce antigen-specific immunity, offers a potential strategy to overcome resistance mechanisms [9]. Consequently, vaccine development against CRKP is considered as a highly promising strategic solution [10,11].

The antigen is the “core” component of any vaccine, determining the strength and potency of the immune response. Current K. pneumoniae vaccines focus on three main approaches: (1) traditional whole-cell or inactivated vaccines; (2) conjugate vaccines using O-specific polysaccharides or capsular polysaccharides (CPS); and (3) recombinant protein subunit vaccines. Recombinant subunit vaccines contain only pathogen-derived antigenic components, avoiding the virulence-reversion risk of live-attenuated strains and the adverse reactions caused by irrelevant components in inactivated preparations [12]. By eliminating non-essential antigens, they enable induction of humoral and cellular immunity against key protective targets [13], offering superior safety, targeted efficacy, and mature manufacturing processes [14], making them highly translatable [15]. Several conserved surface-exposed proteins, such as outer-membrane proteins (e.g., OmpA, OmpK36), iron-acquisition proteins (IutA, Cfu), virulence/adhesion factors (MrkD, PilQ, Pal) [16,17], and pilus components (FimA) [18], have been identified as promising antigens for K. pneumoniae vaccines. Animal studies have confirmed their immunogenicity and protective potential. However, vaccines targeting a single antigen often have limited coverage and may not address both resistance and virulence simultaneously [13,19]. Future research should focus on expanding the breadth of antigens, increasing potency [20], and elucidating immune mechanisms to develop a vaccine that effectively targets both resistance and virulence, essential for controlling CR-hvKP infection and spread [21,22].

Previously, we identified five immune-dominant antigens with strong protective efficacy and immunoreactivity by screening the entire genome of K. pneumoniae. We have already reported the protective effects of three of these antigens, including GlnH, FimA, and KPN_00466 [23]. The remaining two candidates are KPC-2 and Pal. Among them, KPC-2 is encoded by the plasmid-borne blaKPC-2 gene on mobile genetic elements and represents the most prevalent carbapenemase in CRKP, with over 240 known variants [24]. Although this diversity hampers the development of specific inhibitors, KPC-2 is an attractive vaccine target because it can elicit neutralizing antibodies that block β-lactamase activity and thereby restore the efficacy of β-lactam antibiotics [25]. Pal is comparatively understudied in K. pneumoniae. In other Gram-negative bacteria such as Escherichia coli, Pal functions as a peptidoglycan–outer-membrane linker, contributing to cell-envelope integrity, antibiotic resistance, and virulence [26]. Notably, Pal derived from Burkholderia mallei has been shown to improve host survival and demonstrate promise as a vaccine antigen [27]. Furthermore, studies indicate that Pal can act as a Toll-like receptor 2 (TLR2) agonist, and exert an adjuvant effect by activating the TLR2/MyD88 signaling pathway [28]. This promotes dendritic cell maturation and antigen presentation capacity, enhances CD4^+^ T cell activation and antibody class switching [29].

In this study, we genetically fused KPC-2 and Pal to generate the KPC-Pal fusion antigen. We systematically assessed its immunoprotective efficacy in a murine model, investigated the underlying protective immune mechanisms, examined synergistic interactions with antibiotics, and explored its role in regulating innate immune signaling. This research offers strong theoretical and experimental support for the development of a dual-target vaccine against CR-hvKP.

2. Materials and Methods

2.1. Ethical Statement

All animal experiments conducted in this study were approved by the Animal Ethics and Experiment Committee of Army Medical University (Approval No.: AMUWEC20250087). All procedures complied with national and institutional guidelines for the care and use of experimental animals. To minimize suffering and ensure humane euthanasia, mice were anesthetized with sodium pentobarbital during procedures and euthanized by CO_2_.

2.2. Animals

Female BALB/c mice (18–20 g, 6–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were housed under specific pathogen-free (SPF) conditions with a 12 h automatic light-dark cycle. Female New Zealand rabbits (SPF-grade) weighing approximately 2.5 kg were purchased from Chongqing Tengxin Biotechnology Co., Ltd. (Chongqing, China) and housed in the Experimental Animal Center of the SouthWest Hospital.

2.3. Bacterial Strains

The standard K. pneumoniae strain (ATCC700721) was purchased from the American Type Culture Collection. Clinical K. pneumoniae strains KP-YBQ and KP45147 (KPC-2 positive) were isolated from the Clinical Laboratory Department of SouthWest Hospital. The capsular polysaccharide (CPS) serotypes of these strains were K52, K20, and K47, respectively. Bacteria were cultured in tryptic soy broth, washed and diluted with sterile PBS to an appropriate cell concentration determined spectrophotometrically at 600 nm (OD_600_).

2.4. Cloning, Expression, and Purification of Recombinant Proteins

The sequences encoding Pal (GenBank: CDO13556.1), KPC-2 (GenBank: EU784136.1), Pal, KPC-3 (GenBank: ARW72954.1), and KPC-33 (GenBank: XUT16033.1) were retrieved from PubMed. The fragments Pal_70–120_, KPC-2_30–293_, KPC-3_30–293_ and KPC-33_30–293_ were cloned into the BamHI–XhoI sites of the prokaryotic expression vector pGEX-6p-1. A fusion construct comprising KPC-2_30–293_ linked to Pal_70–120_ via a GGGGS linker (named KPC-Pal) was synthesized by Gene Create (Wuhan, China), and inserted into the same sites of pGEX-6p-1. Protein expression and purification followed the previously described protocol. Endotoxin levels in the final preparations were measured with the Limulus Amebocyte Lysate (LAL) assay, and protein concentrations were determined by the bicinchoninic acid (BCA) assay.

2.5. Immunization, Infection, and Drug Treatment

To evaluate the protective efficacy of the recombinant antigens, mice (n = 10) were immunized intramuscularly on days 0, 7 and 14 with 30 µg of each antigen (KPC-Pal, KPC-2 or Pal) formulated with 0.3 mg of aluminum hydroxide (Al(OH)3) adjuvant in a total volume of 200 µL, and PBS was used as a control. On day 21, mice were anesthetized with sodium pentobarbital and challenged via intratracheal inoculation with 20 µL of a bacterial suspension containing K. pneumoniae strains ATCC 700721 (3 × 10^8^ CFU), KP-YBQ (6 × 10^6^ CFU) or KP45147 (3 × 10^7^ CFU), respectively. In the drug-treated group, meropenem (5 mg/kg) was administered intravenously on days 22 and 23. Survival was recorded daily for 7 days post-infection or post-treatment.

2.6. Bacterial Burden and Histopathological Analysis

Seven days after the final immunization, mice (n = 5) were challenged intratracheally with 20 µL of K. pneumoniae strains ATCC 700721 (3 × 10^7^ CFU), KP-YBQ (9 × 10^5^ CFU) or KP45147 (2 × 10^6^ CFU), respectively. The drug-treated group received meropenem (5 mg/kg) via tail-vein injection on days 22 and 23. At 48 h post-infection or post-treatment, lungs and spleens were aseptically harvested, homogenized in PBS, and serial 10-fold dilutions were plated on LB agar to determine bacterial loads. After 24 h of incubation at 37 °C, colonies were counted and expressed as log_10_ CFU/mL. For histopathology, lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 µm sections. Sections were stained with hematoxylin and eosin (H&E) and examined microscopically for congestion, edema, hemorrhage, and inflammatory cell infiltration.

2.7. Cell Culture and Treatment

Primary murine peritoneal cells were harvested under sterile conditions. Briefly, 5 mL of ice-cold 20 mM PBS was injected into the peritoneal cavity and the lavage fluid was collected. Cells were centrifuged at 500 × g for 5 min, resuspended in 1× red-blood-cell lysis buffer and incubated on ice for 5 min. After a second 500 × g spin for 5 min, the cells were washed three times with PBS and finally resuspended in RPMI-1640 supplemented with 10% fetal bovine serum. Cells were plated in 12-well plates at 1 × 10^6^ cells per well. Following a 2 h adhesion period, the medium was replaced with fresh RPMI-1640 containing 10% FBS. Cells were then treated for 4 h with KPC-Pal (40.9 µg/mL), KPC-2 (27.9 µg/mL) or Pal (12.7 µg/mL). Where indicated, cells were pre-incubated for 1 h with either the TLR2 inhibitor C29 (50 µM; HY-100461, MedChemExpress, Monmouth Junction, NJ, USA) or a TLR2-neutralizing antibody (TL2.1, NB100-56722, Novus Biologicals, Littleton, Massachusetts, CO, USA).

2.8. ELISA

Serum was collected from the tail vein on day 6 after each immunization and antigen-specific IgG titers were measured by ELISA. Microtiter plates (439454, Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at 4 °C with each antigen (5 µg/mL) in 50 mM carbonate-bicarbonate buffer (pH 9.5) and blocked with 2% bovine serum albumin (BSA) at 37 °C for 2 h. Sera from the three time points were serially two-fold diluted in PBS (starting at 1:1000 after the first dose, 1:20,000 after the second, and 1:40,000 after the third) and applied as primary antibodies. HRP-conjugated goat anti-mouse IgG (AP308P, Sigma, St. Louis, MO, USA) served as the secondary antibody, and absorbance was read at 450 nm. The titer was defined as the highest dilution giving an OD ≥2.1× that of pre-immune serum. For IgG subclass analysis, sera collected after the third immunization (diluted 1:20,000) were probed with HRP-conjugated goat anti-mouse IgG1 (SA00012-1, Proteintech, Rosemont, IL, USA), IgG2a (SA00012-2, Proteintech, Rosemont, IL, USA), IgG2b (SA00012-3, Proteintech, Rosemont, IL, USA), or IgG3 (SA00012-5, Proteintech, Rosemont, IL, USA) as secondary antibodies. To assess cross-protective immunity, BALB/c mice were immunized intramuscularly on days 0, 7 and 14 with 30 µg of KPC-2, KPC-3 or KPC-33 protein adsorbed onto 0.3 mg aluminum hydroxide. Serum was collected from the tail vein 7 days after the final boost. IgG titers were measured using a 1:10,000 serum dilution as the primary antibody and HRP-conjugated goat anti-mouse IgG (Sigma) as the secondary antibody. The TMB (P0209, Beyotime, Shanghai, China) substrate was developed for 15 min, the reaction was stopped, and the absorbance was read at 450 nm using a Microplate Reader (SPECTRA MAX 190, MD, USA).

2.9. Flow Cytometry

Lungs and spleens of immunized mice were harvested, homogenized through a 70 µm nylon strainer, and resuspended in 20 mM PBS. Cell suspensions were stained with Fixable Viability Stain 700 (1:1000) to exclude dead cells. For tissue-resident memory T (TRM) cell analysis, lung cells were labeled with FITC-anti-CD3, PerCP/Cy5.5-anti-CD4, APC-anti-CD44, PE-anti-CD62L, and BV421-anti-CD69. For memory B-cell analysis, lung cells were stained with FITC-anti-CD19, APC/Cy7-anti-CD45R/B220, BV421-anti-IgD, and PE/Cy7-anti-CD80. The stained cells were then analyzed using a Flow cytometry analyzer (LSRFortessa, BD Biosciences, Franklin Lakes, NJ, USA) and the data were analyzed using FlowJo V10.

2.10. ELISpot

Murine spleens were aseptically harvested, gently pressed through a 70 µm cell strainer, and the resulting single-cell suspensions were centrifuged at 500× g for 5 min. Cells were resuspended in RPMI-1640 supplemented with 10% FBS and plated at 1 × 10^5^ cells per well on pre-coated ELISpot plates. Cells were stimulated with KPC-Pal (30 µg/mL), KPC-2 (20.5 µg/mL), Pal (9.3 µg/mL) or PMA (10 ng/mL) as a positive control, and incubated at 37 °C, 5% CO_2_ for 6 h. After removal of cells, plates were washed five times with PBS, and then incubated with detection antibodies at room temperature for 2 h. Following a second PBS wash, streptavidin-ALP (1:1000) was added for 1 h, with five additional PBS washes. BCIP/NBT substrate was applied for color development, and the reaction was stopped once distinct spots appeared. After drying, spots were quantified using an ELISpot plate reader (iSpot, AID, Straßberg, Saxony-Anhalt, Germany).

2.11. Determination of Minimum Inhibitory Concentration (MIC)

KP45147 was adjusted to 1 × 10^6^ CFU/mL and added to a 96-well plate with serial meropenem dilutions. After incubation at 37 °C for 16–18 h, the culture was 5-fold serially diluted across eight concentrations. 5 μL of each dilution was spotted onto LB agar and incubated overnight at 37 °C. Colonies were counted the following day, and the meropenem concentration yielding the desired colony count was selected.

2.12. Polyclonal Antibody Preparation

New Zealand rabbits were immunized subcutaneously on days 0, 7, and 14 with 2 mg of KPC-Pal, KPC-2, or Pal protein emulsified 1:1 (v/v) in Freund’s adjuvant. On day 21, peripheral blood was drawn, clotted at 37 °C for 30 min, and centrifuged at 4000 rpm for 10 min at 4 °C. Serum was harvested, and polyclonal antibodies were purified by Protein G affinity chromatography. Antibody purity was evaluated by SDS-PAGE.

2.13. In Vitro Bactericidal Assay

KP45147 (1 × 10^7^ CFU/mL) was incubated in LB for 6 h with polyclonal antibodies (1 µM) alone or in combination with meropenem (256 µg/mL). After incubation, cultures were plated on LB agar and surviving colonies were counted following overnight growth at 37 °C.

2.14. Opsonophagocytosis Assay

HL-60 cells were differentiated into granulocyte-like cells (10% FBS, 1% glutamine, 0.8% DMF, 88.2% RPMI-1640), harvested, and centrifuged at 1,000 rpm for 5 min. Cell pellet was washed sequentially with 1× HBSS with and without Ca^2+^/Mg^2+^, and resuspended in Opsonophagocytosis Killing Buffer (OKB) at 1 × 10^7^ cells/mL. Cells were mixed with baby rabbit complement (4:1) or heat-inactivated complement (56 °C, 30 min) as a control. In a 96-well plate, 20 µL of OKB was added to control wells and 20 µL of polyclonal antibody plus inactivated complement to test wells. KP45147 (2.5 × 10^5^ CFU/mL in OKB) was added (10 µL/well). After 1 h of shaking at room temperature (700 rpm), 50 µL of the cell-complement mixture was added and plates were incubated for 1 h at 37 °C, 5% CO_2_, shaking at 700 rpm. Reactions were stopped on ice for 20 min, then plated on LB agar and incubated overnight at 37 °C for colony counting.

2.15. Western Blot

Protein extracts from murine peritoneal macrophages stimulated with KPC-Pal or Pal were separated by SDS-PAGE and transferred to membranes. Membranes were blocked with 5% skim milk for 2 h at room temperature, and then incubated overnight at 4 °C with primary antibodies against ERK, p-ERK, p38 MAPK, p-p38 MAPK, SAPK/JNK and p-SAPK/JNK (1:1000). After three 10 min washes with TBST, membranes were incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG (1:10,000). Subsequently, the bands were developed with an ECL substrate and visualized using a chemiluminescence imaging system (ChemiDoc XRS, BIO-RAD, Hercules, CA, USA) or X-ray film.

2.16. Co-Immunoprecipitation (Co-IP)

Murine peritoneal macrophages were lysed on ice with cold IP lysis buffer for 30 min, and then centrifuged at 12,000× g for 15 min at 4 °C. A fraction of the supernatant was saved as input; the remainder was pre-cleared with Protein A/G magnetic beads for 1 h at 4 °C. Anti-Pal or anti-TLR2 antibody (1:1000) was bound to fresh Protein A/G beads for 1–2 h at 4 °C, washed, and mixed with the pre-cleared lysate overnight with rotation. After extensive washes with lysis buffer, the complexes were eluted by boiling in 1 × SDS-PAGE loading buffer at 95–100 °C for 10 min and analyzed by SDS-PAGE and Western blot.

2.17. Cytokine Detection

Cell culture supernatants from murine peritoneal macrophages stimulated with KPC-Pal or Pal were harvested. A 96-well plate was coated overnight at 4 °C with 100 µL/well of capture antibody for TNF-α (88-7324, Thermo Fisher Scientific, Waltham, MA, USA) or IL-6 (88-7064, Thermo Fisher Scientific, Waltham, MA, USA). After washing with PBST, wells were blocked with 1× Diluent for 2 h at room temperature. Standard curves (eight two-fold dilutions) and 100 µL of each test supernatant were added, followed by five PBST washes. Biotinylated detection antibody was incubated for 1 h at room temperature, then avidin-HRP for 30 min. TMB substrate was developed for 15 min, the reaction was stopped, and absorbance was read at 450 nm.

2.18. Statistical Analysis

Data are presented as mean ± SD. Analyses were performed with GraphPad Prism 9.0. Survival curves were compared by Log-rank test. Lung bacterial loads, which were non-normally distributed, were evaluated with Kruskal–Wallis test followed by Dunn’s post hoc comparisons. Histopathology scores were analyzed using the Mann–Whitney U test. Image quantification was conducted with ImageJ (version 1.54p) (NIH).

3. Results

3.1. KPC-Pal Immunization Confers Protection Against K. pneumoniae Infection

The coding sequences for KPC-Pal, KPC-2, and Pal were cloned into the pGEX-6P-1 vector, successfully expressed in E. coli BL21(DE3), and purified. SDS-PAGE confirmed bands at the expected molecular weights: KPC-Pal ≈ 40.9 kDa, KPC-2 ≈ 27.9 kDa, and Pal ≈ 12.7 kDa (Figure 1B).

To investigate the protective effect of KPC-Pal compared to the individual antigens, immunized mice were challenged with ATCC 700721, KP-YBQ, or KP45147 (Figure 1A). Survival curves (Figure 1C) showed that PBS-immunized controls suffered >90% mortality within 3 days for all strains, confirming successful establishment of the pulmonary infection model. In ATCC 700721 challenged mice, survival rates were 50% (KPC-Pal), 10% (KPC-2), 20% (Pal) and 10% (PBS), KPC-Pal conferred a significant benefit versus PBS (p < 0.05). After KP-YBQ challenge, survival rates were 50% (KPC-Pal), 30% (KPC-2), 20% (Pal) and 10% (PBS), with KPC-Pal again significantly better than PBS (p < 0.05). With KP45147, survival reached 80% (KPC-Pal), 60% (KPC-2), 10% (Pal) and 0% (PBS); the KPC-Pal group differed markedly from PBS and Pal alone (p < 0.001). Thus, the KPC-Pal fusion protein provides superior protection across K. pneumoniae strains, especially against the KPC-2 positive strain KP45147, integrating the protective properties of both KPC-2 and Pal.

To further investigate the bacterial loads of immunized mice by KPC-Pal compared to the individual antigens. Bacterial burdens in lungs and spleens were measured 48 h post-infection. As shown in Figure 1D–F, mice immunized with the KPC-Pal fusion antigen exhibited significantly lower bacterial loads in both organs compared to those immunized with either KPC-2 or Pal alone, and this advantage was particularly evident in the KP45147 infection model, indicating the protective efficacy of KPC-2. Consistently, histopathology of lungs from KP45147 challenged mice (H&E, Figure 1G) revealed severe lesions in PBS controls, including perivascular lymphatic dilation and extensive inflammatory infiltrates. All vaccinated groups showed improvement, with the greatest benefit observed in the KPC-Pal group. These findings support the bacterial load results, demonstrating that the KPC-Pal fusion elicits a stronger protective efficacy that markedly reduces pulmonary inflammation and tissue damage.

3.2. KPC-Pal Immunization Elicits a Potent Th2-Polarized Humoral Response

To evaluate the antigen-specific humoral immune response induced upon immunization, mice were immunized and serum samples were collected as shown in Figure 2A. KPC-Pal-immunized mice displayed a marked IgG rise after the first immunization, whereas the single antigens elicited no detectable response. After the second immunization, elevated IgG titers were observed for all antigens compared to PBS control, with KPC-Pal IgG titers reaching 2.56 × 10^6^, significantly higher than KPC-2 and Pal. Following the third vaccination, titers were 5.12 × 10^6^ (KPC-2), 2.56 × 10^6^ (Pal), and 1.02 × 10^7^ (KPC-Pal), confirming the superior immunogenicity of the fusion protein (Figure 2B). Specific antibodies against single antigens after each immunization showed similar results to the fusion antigen (Figure 2C,D), indicating correct folding of the KPC-2 and Pal domains in the fusion protein. Notably, KPC-2 specific IgG titers were higher in immunized sera than Pal, suggesting high immunogenicity of KPC-2.

Further, IgG subclass analysis (IgG1, IgG2a, IgG2b, IgG3) revealed that IgG1 dominated in all groups (Figure 2E), confirming a Th2-biased humoral response. Notably, the KPC-Pal fusion induced antibody levels comparable to KPC-2 and markedly higher than Pal alone, which further confirmed the high immunogenicity of KPC-2.

3.3. KPC-Pal Immunization Induces Potent T Cell Response

Mice were immunized as before, spleen and lung samples were collected as indicated in Figure 3A, the cellular immune response induced by these antigens was assessed by ELISpot. All three cytokines were increased in the antigen-immunized groups relative to the PBS control. Compared with Pal, KPC-2 induced higher IL-4 and IL-17A but lower IFN-γ, highlighting the distinct immunogenic profiles of the two antigens. Moreover, KPC-Pal generated stronger T-cell responses than either single antigen, indicating that the fusion protein successfully combines the immunogenic properties of both components to produce a mixed Th1/Th2/Th17-type response (Figure 3B,C).

Subsequent flow cytometry analysis of lung tissue on day 45 showed that KPC-Pal markedly increased tissue-resident memory T cells (CD3^+^ CD4^+^ CD44^+^ CD62L^−^ CD69^+^) compared to KPC-2 (Figure 3D) and significantly raised splenic memory B cells (CD19^+^ B220^+^ IgD^−^ CD80^+^) compared to both KPC-2 and Pal (Figure 3E). These results demonstrate that KPC-Pal simultaneously drives systemic memory B-cell responses and lung TRM formation, providing a basis for its long-lasting protective effects.

3.4. KPC-2 Vaccination Induces Cross-Protection Against Different KPC Variants

To date, over 260 KPC variants have been identified worldwide [30], to evaluate the sequence conservation of these variants, the amino-acid sequences of 21 KPC subtypes were retrieved from NCBI and aligned with Clustal Omega (version 1.2.4) (Figure S1). Only 37 mutation sites were found, mostly consisting of conservative substitutions, indicating high sequence conservation among different KPC variants. Meanwhile, a phylogenetic tree of KPC-1 to KPC-66 generated with ESPript (Figure 4B) shows that these variants are closely related, demonstrating overall strong conservation and suggesting stable enzymatic activity and key antigenic epitopes across variants.

To assess the cross-reactive immunity of a KPC-2-based vaccine, KPC-3 and KPC-33, two other most common KPC variants [30] from distinct evolutionary clusters were selected. Recombinant plasmids pGEX-6P-1-KPC-3 and pGEX-6P-1-KPC-33 were constructed and high-purity KPC-3 and KPC-33 proteins were obtained after expression and affinity-chromatography purification (Figure 4C). Mice received three immunizations with KPC-2, KPC-3, KPC-33, or KPC-Pal formulated with aluminum hydroxide, and sera were collected 7 days after the final boost (Figure 4A). ELISA of cross-reactive IgG against KPC-Pal, KPC-2, KPC-3, and KPC-33 showed that all groups had the highest titers against KPC-Pal and KPC-2, with no significant inter-group differences (Figure 4D). These consistent patterns confirm that KPC-Pal retains KPC-2 immunogenicity and elicits cross-protective antibodies against multiple KPC variants.

3.5. KPC-Pal Exhibits a Synergistic Effect with Carbapenem Antibiotics

To evaluate whether active immunization with KPC-Pal can potentiate the synergistic protective effect of carbapenem therapy. The resulting dose–response curve demonstrated a concentration-dependent bactericidal activity (Figure 5B), leading us to select 256 µg/ mL meropenem as the fixed dose for combination-treatment experiments.

Mice were immunized, treated with meropenem (0.1 mg per mouse), and challenged as outlined in Figure 5A. Seven-day survival curves showed that PBS-immunized mice receiving meropenem alone experienced over 90% mortality within five days (Figure 5C). For mice challenged with ATCC-700721, survival rates were 60% for KPC-Pal + meropenem, 30% for KPC-2 + meropenem, 40% for Pal + meropenem, and only 10% for PBS + meropenem; KPC-Pal alone yielded 50% survival, indicating an additive benefit of the combination. Similar results were observed in mice challenged with KP-YBQ. In mice challenged with KP45147, KPC-Pal + meropenem conferred 90% survival, surpassing KPC-2 + meropenem (70%), KPC-Pal alone (70%), Pal + meropenem (40%), and PBS + meropenem (0%); the difference between KPC-Pal + meropenem and PBS + meropenem was highly significant (p < 0.001). Lung bacterial burden (Figure 5D) demonstrated that PBS + meropenem mice harbored significantly higher CFU counts than any immunization + meropenem group, with the greatest reduction observed for KPC-Pal + meropenem against the highly resistant KP45147. Histopathology confirmed that KPC-Pal + meropenem markedly attenuated pulmonary tissue damage compared to all other groups (Figure 5E). These results demonstrate that KPC-Pal active immunization synergizes with meropenem to enhance survival, reduce bacterial loads, and promote lung recovery across multiple K. pneumoniae strains.

To evaluate the synergistic effect of KPC-Pal passive immunization with carbapenem antibiotics, we generated polyclonal antibodies against each antigen in New Zealand rabbits (Figure 5F). In the bactericidal assay, plate counts showed that KPC-Pal antibodies alone exerted modest killing, whereas the KPC-Pal + meropenem mixture dramatically reduced viable CFU (log_10_ CFU/mL) compared to either component alone (p < 0.0001, Figure 5G). The opsonophagocytic killing assay further demonstrated that neutrophils more efficiently cleared KP45147 when opsonized with KPC-Pal antibody plus meropenem than with antibody or drug alone (p < 0.0001, Figure 5H), indicating that the anti-KPC-2 component mediates this enhanced phagocytosis. Collectively, both active (vaccination) and passive (polyclonal antibody) immunization confirm that the KPC-Pal fusion antigen synergistically sensitizes K. pneumoniae to meropenem, markedly improving bacteria clearance.

3.6. KPC-Pal Exhibits a Self-Adjuvant Effect by Modulating TLR2 Signaling

Studies have demonstrated that Pal is a lipoprotein that acts as a ligand for the pattern-recognition receptor TLR2 [31]. To investigate whether KPC-Pal can activate the TLR2 pathway, we performed Co-IP to assess the binding of KPC-Pal and Pal protein to TLR2 (Figure 6A). A stronger band for Pal was detected in the bead fraction compared to KPC-Pal, indicating efficient binding of Pal to TLR2 and confirming its role as a natural TLR2 ligand. In contrast, the interaction was partially reduced when Pal was fused to KPC-2, possibly due to steric hindrance from the fusion. Subsequently, the phosphorylation status of key MAPK pathway components (Erk, p38, and JNK) was then examined by Western blot (Figure 6B–D). The data suggest that KPC-Pal selectively modulates MAPK inflammatory branches, with significant suppression of JNK phosphorylation while Erk and p38 phosphorylation remained unaffected.

A classic downstream consequence of TLR2 signaling is the induction of pro-inflammatory cytokines such as TNF-α and IL-6 [32]. Here, murine peritoneal macrophages were stimulated with KPC-Pal, Pal, or LPS for 1 h, 2 h, and 4 h, and the supernatants were analyzed by ELISA for TNF-α and IL-6. As shown in Figure 6E, LPS induced a strong cytokine response at 1h post-stimulation, while Pal triggered a delayed and weaker response. Although KPC-Pal retains some immunostimulatory activity, it significantly reduces Pal’s ability to activate TLR2 robustly. This moderated response may enhance the balance between immunogenicity and safety in vaccine applications by preventing immune-mediated pathology associated with excessive cytokine release. Therefore, KPC-Pal could potentially offer a mild adjuvant effect during immunization by modulating TLR2 signaling.

4. Discussion

The emergence and global spread of K. pneumoniae, especially carbapenem-resistant (CRKP) and carbapenem-resistant hypervirulent (CR-hvKP) strains, pose a significant public-health threat that requires innovative preventive and control measures beyond traditional antibiotics [33,34,35]. Prophylactic vaccines, especially those capable of targeting both bacterial resistance and virulence simultaneously, are considered a crucial strategy to disrupt the cycle of resistance development [13,36]. In line with this approach, we have developed and extensively evaluated a recombinant fusion protein vaccine, KPC-Pal, which combines the resistance enzyme KPC-2 with the conserved virulence factor Pal. Our results demonstrate that KPC-Pal induces potent and broad-spectrum immunoprotection in murine models and exhibits unique mechanisms for reversing resistance and modulating innate immunity. These findings provide new experimental support for the development of next-generation vaccines against CR-hvKP infections.

First, the high serotype diversity and rapid evolution of K. pneumoniae limit the effectiveness of single-antigen vaccines [17,18]. This study confirms the significant advantage of the dual-target fusion antigen strategy over single antigens in enhancing immune protection. KPC-Pal immunization consistently resulted in higher survival rates and improved bacterial clearance from organs compared to immunization with either KPC-2 or Pal alone when challenged with three distinct K. pneumoniae strains. The greatest benefit was observed against the highly resistant KP45147 strain, highlighting the advantage of targeting both resistance and virulence factors. The synergistic effect of targeting both KPC-2 and Pal is evident, with anti-KPC-2 antibodies neutralizing β-lactamase activity [37,38] and anti-Pal antibodies disrupting membrane integrity and colonization capacity [39]. This approach offers a promising solution to combat CR-hvKP strains [5,40]. Further research is needed to explore the efficacy of KPC-Pal against other cKP, hvKP, and CR-KP strains.

Second, we thoroughly characterized the immune response elicited by KPC-Pal. The vaccine induced a Th2-biased humoral response characterized by high-titer IgG1 antibodies, which is consistent with the Th2-promoting effect of aluminum adjuvant and indicates strong B-cell activation. Importantly, the vaccine promoted the generation of systemic memory B cells and lung-resident memory T (TRM) cells. Pulmonary TRM cells play a crucial role in providing rapid local immune responses against mucosal reinfection, enhancing protection against respiratory pathogens [41]. The expansion of TRM cells in the lungs of mice explains the rapid and effective local protection conferred by KPC-Pal [42,43]. Furthermore, splenocytes exhibited a mixed Th1/Th2/Th17 cytokine profile, demonstrating the complementary and synergistic nature of systemic and local immune responses. These findings collectively establish the immunological foundation for the broad-spectrum and long-lasting protection provided by KPC-Pal.

Third, a key finding of this study is the considerable potential of the KPC-Pal vaccine strategy to reverse bacterial resistance and act synergistically with antibiotics. Both active immunization with KPC-Pal followed by meropenem treatment and passive transfer of KPC-Pal polyclonal antibodies combined with antibiotics significantly enhanced clearance of the highly resistant KP45147 strain. In vitro assays confirmed that anti-KPC-Pal antibodies directly boosted meropenem-mediated killing and promoted neutrophil phagocytosis. These data indicate that anti-KPC-2 antibodies can bind and neutralize the KPC carbapenemase, thereby “disarming” the bacterium and restoring carbapenem activity [37,44]. This study validates the feasibility of combining a vaccine with traditional antibiotics [45], offers a novel “resistance-reversing” immunotherapeutic approach for combatting antimicrobial resistance, potentially overcoming the limitations of relying solely on new antibiotic development [7,15].

Fourth, our mechanistic investigations revealed the dual function of KPC-Pal as a vaccine antigen; it acts as both an immunogen and an intrinsic “intramolecular adjuvant.” Although Pal is a potent TLR2 agonist, the fusion protein KPC-Pal does not exhibit strong TLR2 binding. Instead, it fine-tunes downstream signaling, selectively suppressing JNK phosphorylation and attenuating the TLR2-driven surge of inflammatory cytokines such as TNF-α and IL-6. However, there may be other signaling pathways involved that we have not mentioned. Excessive inflammation is a central pathogenic factor in K. pneumoniae infection, leading to tissue damage and multi-organ failure [46]. Fusion with KPC likely alters the conformation of Pal or masks certain TLR2-binding epitopes, preserving sufficient immunogenicity while avoiding immunopathology. This offers a favorable safety-efficacy balance for clinical translation [47,48].

Finally, the high sequence conservation among various KPC subtypes and the strong cross-reactivity of KPC-2 immune sera against subtypes from different evolutionary clades (like KPC-3 and KPC-33) robustly demonstrate that a KPC-targeted vaccine strategy could possess a broad protective spectrum [49]. This cross-protective potential suggests that a KPC-based vaccine could retain efficacy against current and emerging KPC variants, enhancing its longevity and translational prospects [24,44].

5. Conclusions

This study confirms the strong protective efficacy of the KPC-Pal fusion protein as a potential vaccine against CR-hvKP. It establishes a solid mechanistic basis for its use, including diverse immune responses, synergistic antibacterial effects, and regulation of innate immunity. Future research will focus on optimizing vaccination schedules, exploring different adjuvants to balance Th1/Th2 responses, evaluating effectiveness against a wider range of clinical isolates, and understanding the molecular mechanisms underlying the modulation of TLR2 signaling by the Pal moiety. These efforts will enhance the development of vaccine-antibiotic combination strategies to address multidrug-resistant bacteria.