Protection Against Pneumonia Induced by Vaccination with Fimbriae Subunits from Klebsiella pneumoniae

Abstract

Background: Klebsiella pneumoniae infections pose a great burden worldwide, causing high morbidity and mortality, which are worsened by the increase in multidrug-resistant strains. New therapeutic/prophylactic strategies are urgently needed to overcome antibiotic resistance and reduce the health and economic impacts of diseases caused by this pathogen. Fimbriae are important virulence factors involved in biofilm formation and adhesion to host cells. Their exposed location, conservation among clinical isolates and adjuvant properties make them interesting candidates for inclusion in protein-based vaccines. Therefore, the present work investigated the immunological potential of type 1 and 3 fimbriae subunits in a murine model of K. pneumoniae lung infection.

Methods: MrkA and FimA were produced as recombinant proteins in E. coli, purified and used to immunize mice subcutaneously. The immune responses were characterized and protection against pneumonia was evaluated after intranasal challenge.

Results: Subcutaneous immunization with recombinant FimA and MrkA induced high IgG1 production; the antibodies efficiently recognized the native proteins at the bacterial surface, promoted C3 deposition and reduced biofilm formation by K. pneumoniae in vitro. Mice vaccinated with the co-administered proteins reduced the bacterial loads in the lungs after intranasal challenge, less inflammation and reduced tissue damage.

Conclusion: The results suggest that both type 1 and type 3 fimbriae contribute to protection against K. pneumoniae lung infection, inducing antibodies that bind to the bacteria and favoring Complement deposition and clearance by the host, while inhibiting biofilm formation.

Keywords: K. pneumoniae; biofilms; fimbriae; protein vaccines.

Figure 1

Serum antibody production in mice immunized with MrkA and/or FimA. The concentration of IgG1 and IgG2a is shown for each immunization group (FimA, MrkA and FimA + MrkA). Comparisons between the groups were performed using two-way ANOVA with Sidak’s post-test. *** p < 0.0001 when comparing IgG1 × IgG2a responses. ## p < 0.01 when comparing antibody levels among different immunization groups.

Figure 2

Binding of anti-FimA and anti-MrkA antisera to K. pneumoniae. The percentages of FITC-positive cells are shown for each group. The solid lines represent bacteria incubated with control sera, while the dashed lines indicate bacteria treated with sera from vaccinated mice. (A) control x anti-FimA; (B) control x anti-MrkA; (C) control x anti-FimA + MrkA. (D) Fluorescence comparison among immunization groups. Statistical differences between control and vaccinated groups were evaluated using ANOVA with a Dunn’s post-test (*** p < 0.001) in comparison with control.

Figure 3

Antibody-mediated complement deposition. K. pneumoniae was incubated in the presence of sera from control or vaccinated mice, followed by the addition of NMS as a complement source. Detection was performed using FITC-conjugated anti-mouse C3. The percentages of FITC-positive cells are shown for each group. The solid lines represent C3 deposition on bacteria incubated with control sera, while the dashed lines indicate those treated with sera from vaccinated mice. (A) control × anti-FimA; (B) control × anti-MrkA; (C) control × anti-FimA + MrkA. Fluorescence comparison among immunization groups. (D) Fluorescence comparison among immunization groups. Statistical differences between control and vaccinated groups were evaluated using ANOVA with a Dunn’s post-test (* p < 0.05 and ** p < 0.01) in comparison with control.

Figure 4

Biofilm formation is inhibited by vaccine antibodies. (A) CFU counts of biofilm formed in the presence of sera from control and vaccinated mice. (B) Absorbance (A_600nm) of the biofilms recovered from the plate. Statistical differences were evaluated via Kruskal–Wallis test (* p < 0.05). The × symbol indicates the statistical outliers removed from the analysis.

Figure 5

Lung colonization by K. pneumoniae in vaccinated × control mice. CFU counts in the lung homogenates of control and vaccinated mice were determined 48 h after intranasal challenge with K. pneumoniae BM567. Each dot represents one animal. Statistical differences were evaluated via Kruskal–Wallis test. The × symbol indicates the statistical outliers removed from the analysis. * p < 0.05 in comparison with the control.

Figure 6

Histopathological analysis of the lung tissue from mice before and after challenge with K. pneumoniae (40× amplification). (A) Healthy unchallenged control; (B) challenged control; (C) Immunized with rFimA and challenged; (D) immunized with rMrkA and challenged; (E) immunized with rFimA + rMrkA and challenged. The red and orange arrows indicate intense and moderate inflammatory infiltrates, respectively; black arrows, vessel congestion.