Immunoprotective Efficacy of Acinetobacter baumannii Outer Membrane Protein, FilF, Predicted In silico as a Potential Vaccine Candidate

Abstract

Acinetobacter baumannii is emerging as a serious nosocomial pathogen with multidrug resistance that has made it difficult to cure and development of efficacious treatment against this pathogen is direly needed. This has led to investigate vaccine approach to prevent and treat A. baumannii infections. In this work, an outer membrane putative pilus assembly protein, FilF, was predicted as vaccine candidate by in silico analysis of A. baumannii proteome and was found to be conserved among the A. baumannii strains. It was cloned and expressed in E. coli BL21(DE3) and purified by Ni-NTA chromatography. Immunization with FilF generated high antibody titer (>64,000) and provided 50% protection against a standardized lethal dose (10(8) CFU) of A. baumannii in murine pneumonia model. FilF immunization reduced the bacterial load in lungs by 2 and 4 log cycles, 12 and 24 h post infection as compared to adjuvant control; reduced the levels of pro-inflammatory cytokines TNF-α, IL-6, IL-33, IFN-γ, and IL-1β significantly and histology of lung tissue supported the data by showing considerably reduced damage and infiltration of neutrophils in lungs. These results demonstrate the in vivo validation of immunoprotective efficacy of a protein predicted as a vaccine candidate by in silico proteomic analysis and open the possibilities for exploration of a large array of uncharacterized proteins.

Keywords: Acinetobacter baumannii; FilF; OMP; cytokines; immunoprotection; reverse vaccinology; vaccine.

Figure 1

Expression and purification of FilF protein. Lane 1, pink plus marker; lane 2, uninduced sample; lane 3, induced FilF; lane 4, purified FilF protein.

Figure 2

Total IgG antibody levels in the sera. Groups of female BALB/c mice (n = 10) were sub-cutaneously immunized with 20 μg FilF formulated with CFA/IFA at day 1, 14, and 21. Sera from adjuvant control and FilF immunized mice were collected 3 days after booster dose and IgG titer was determined. Antibody response at 14 and 21-day rose as compared to adjuvant control. ^***p < 0.001 (Adjuvant control vs. FilF immunized mice).

Figure 3

Bacterial burden in lungs. Groups of female Balb/c mice (n = 6) were immunized subcutaneously with 20 μg FilF formulated with CFA/IFA adjuvant on day 1, 14, and 21. The mice were intra-tracheally challenged with 10⁸ CFU of A. baumannii ATCC 19606 at day 29. Immunization with FilF reduced the bacterial burden by 2 and 4 log cycles in the lungs of pneumonia model mice sacrificed 12 and 24 h post infection, respectively. The data are presented as mean ± SD (n = 6). p-value was determined by the one way analysis of variance (ANOVA). ^***p < 0.001 (Adjuvant control vs. FilF immunized mice).

Figure 4

Lung histopathology. Groups of female Balb/c mice (n = 6) were immunized subcutaneously with 20 μg FilF formulated with CFA/IFA adjuvant on day 1, 14, and 21, and intra-tracheally challenged with 10⁸ CFU of A. baumannii ATCC 19606 at day 29. The mice were sacrificed at 12 and 24 h post-challenge and lungs were collected for histopathology. (A) The lung from an unimmunized uninfected mouse showing normal histological characters. (B) Unimmunized infected mouse lung showing increased inflammatory cell infiltration in the perivascular and peribronchial areas, and within the airway lumen (arrows) 12 h postinfection. (C) The lung from an immunized infected mouse showing mild inflammatory cell infiltration in the perivascular and peribronchial areas (arrows) 12 h postinfection. (D) The lung from an immunized infected mouse showing significantly reduced infiltration of inflammatory cells 24 h postinfection. H&E, Magnification 100X.

Figure 5

Cytokine levels in the sera. Groups of female Balb/c mice (n = 6) were immunized subcutaneously with 20 μg FilF formulated with CFA/IFA adjuvant on day 1, 14, and 21. The mice were intra-tracheally challenged with 10⁸ CFU A. baumannii ATCC 19606 at day 29, and sacrificed at 12 and 24 h post-challenge. The detection limit for all cytokines and chemokines is <10 pg/ml. p-value was determined by the one way analysis of variance (ANOVA). ^*p < 0.1, ^**p < 0.05, ^***p < 0.001, ^#non-significant, (Adjuvant control vs. FilF immunized mice).

Figure 6

Survival rate of mice. Groups of female Balb/c mice (n = 10) were immunized sub-cutaneously with 20 μg FilF formulated with CFA/IFA adjuvant on day 1, 14, and 21, and intra-tracheally challenged with 10⁸ CFU of A. baumannii ATCC 19606 at day 29. The survival rates of mice were recorded for seven days.