Identification and Evaluation of Recombinant Outer Membrane Proteins as Vaccine Candidates Against Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae found in the normal flora of the human oral and intestinal tract mainly causes hospital-acquired infections but can also cause community-acquired infections. To date, most clinical trials of vaccines against K. pneumoniae have ended in failure. Furthermore, no single conserved protein has been identified as an antigen candidate to accelerate vaccine development. In this study, we identified five outer membrane proteins of K. pneumoniae, namely, Kpn_Omp001, Kpn_Omp002, Kpn_Omp003, Kpn_Omp004, and Kpn_Omp005, by using reliable second-generation proteomics and bioinformatics. Mice vaccinated with these five KOMPs elicited significantly higher antigen-specific IgG, IgG1, and IgG2a. However, only Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 were able to induce a protective immune response with two K. pneumoniae infection models. These protective effects were accompanied by the involvement of different immune responses induced by KOMPs, which included KOMPs-specific IFN-γ-, IL4-, and IL17A-mediated immune responses. These findings indicate that Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 are three potential Th1, Th2, and Th17 candidate antigens, which could be developed into multivalent and serotype-independent vaccines against K. pneumoniae infection.

Keywords: Klebsiella pneumoniae; outer membrane proteins; proteomics and bioinformatics; serotype-independent vaccines; vaccine.

Figure 1

Vaccine antigen gene analysis. (A) The workflow for selecting potential antigens combining proteomics with bioinformatics analysis. After “shaving” directly digested the surface protein, process the enzyme digested samples for LC-MS/MS to determine the cut-down proteins. Sequence conservation analysis predicted potential antigens. (B) Sequence conservation analysis. Comparing the genomic information of 389 Klebsiella pneumoniae strains obtained a conserved core gene database, which contains 562 proteins. The database was then analyzed from the structure information. The predicted surface proteins or secreted proteins were aligned with mass spectral data to exclude non-specific mass spectral data, resulting in the most potential antigens. (C) Conservative analysis of KOMPs. The amino acid sequence of these KOMPs (Kpn_Omp001, Kpn_Omp002, Kpn_Omp003, Kpn_Omp004, and Kpn_Omp005) identity ranges from 96% to 100%.

Figure 2

Specific antibody responses in immunized mice. (A) Balb/c mice were immunized with the five KOMPs individually or PBS, and antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). (B) Specific IgG/IgG1/IgG2a antibody responses in mouse sera were collected at 7 days after the third vaccination of the five KOMPs. The experiment was repeated at least twice.

Figure 3

Murine bacteremia model. (A) Balb/c mice were immunized with the KOMPs individually or PBS. Seven days after the second booster vaccination, different doses of K pneumoniae 260 in 100 μl of PBS were injected intravenously into mice. (B) The survival rate of murine bacteremia model was observed every 12 h for 14 days. *p < 0.05, ***p < 0.001. ns, p > 0.05.

Figure 4

Murine pneumonia model. (A) Balb/c mice were immunized with the KOMPs individually or PBS. Seven days after the second booster vaccination, different doses of K pneumoniae 260 in 50 μl of PBS were injected intranasally into mice. (B) The survival rate of murine pneumonia model was observed for 144 h. *p < 0.05.

Figure 5

Bacterial load assay. (A, B) Balb/c mice were immunized with the Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 individually or PBS, and they were injected with 5 × 10⁸ CFU of K pneumoniae 260 in 7 days after the second booster vaccination. The lungs, kidneys, and spleens were collected 4 days after the sub-lethal challenge and homogenized. CFUs were enumerated following serial diluting and plating on BHI agar. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

ELISPOT assay and opsonophagocytosis killing assay. (A) Balb/c mice were immunized with the Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 individually or PBS, and mice were sacrificed 7 days after the second booster vaccination. After euthanasia, spleens were collected and a single suspension of splenocytes was obtained for cytokine-specific enzyme-linked Immunospot assay (ELISPOT assay), and the serum was isolated for the opsonophagocytosis killing assay. (B) Interferon gamma (IFN-γ), interleukin 4 (IL-4), and interleukin 17A (IL-17A)-producing splenocytes from vaccinated or control mice were analyzed using ELISPOT assay. SI: Immunized mice stimulated with KOMP, UI: Unstimulated immunized mice, SM: Mock mice stimulated with KOMP. (C) The opsonophagocytosis killing assay. The bacteria were incubated with heat-activated mouse antiserum against different KOMPs at 4°C for 20 min, differentiated HL-60 cells and rabbit complement co-incubated at 37°C for 1 h with agitation at 600 rpm, and samples were plated on BHI agar plates for CFU enumeration. **p < 0.01, ***p < 0.001.