Anti-Acinetobacter Baumannii single-chain variable fragments provide therapeutic efficacy in an immunocompromised mouse pneumonia model

Abstract

Background: The emergence of carbapenem-resistant and extensively drug-resistant (XDR) Acinetobacter baumannii as well as inadequate effective antibiotics calls for an urgent effort to find new antibacterial agents. The therapeutic efficacy of two human scFvs, EB211 and EB279, showing growth inhibitory activity against A. baumannii in vitro, was investigated in immunocompromised mice with A. baumannii pneumonia.

Results: The data revealed that infected mice treated with EB211, EB279, and a combination of the two scFvs showed better survival, reduced bacterial load in the lungs, and no marked pathological abnormalities in the kidneys, liver, and lungs when compared to the control groups receiving normal saline or an irrelevant scFv.

Conclusions: The results from this study suggest that the scFvs with direct growth inhibitory activity could offer promising results in the treatment of pneumonia caused by XDR A. baumannii.

Keywords: Acinetobacter baumannii; Bactericidal antibodies; Immunotherapy; Pneumonia; scFv.

Fig. 1

EB211 and EB279 caused no toxicity to the kidneys and liver of mice. No noticeable histopathological changes were observed in the kidneys and liver of mice injected intraperitoneally twice daily with EB211 (12 mg/kg), EB279 (12 mg/kg), or a cocktail of the two scFvs (CKT; 6 mg/kg of each) for 72 h, similar to mice injected intraperitoneally twice daily with colistimethate sodium (CMS; 30 mg/kg) or normal saline (NS). Green arrowheads: sinusoids, V: central venules, Yellow arrowheads: hepatocytes, Yellow arrows: renal tubule and renal corpuscle

Fig. 2

Administration of EB211 and EB279 led to an increased survival rate and a reduced bacterial burden in the lungs of immunocompromised mice with A. baumannii pneumonia. Immunocompromised mice were inoculated intranasally with XDR A. baumannii A.b.56 at a 50% lethal dose (LD₅₀; 4×10⁷ CFU per mouse). Immunocompromised infected mice were administered intraperitoneally twice daily with EB211 (12 mg/kg), EB279 (12 mg/kg), a cocktail of the two scFvs (CKT; 6 mg/kg of each), an irrelevant scFv (MEH158; an S. aureus-specific scFv) (12 mg/kg), colistimethate sodium (CMS; 30 mg/kg), or normal saline (NS), or once daily with EB211 (12 mg/kg) (Q24H) two hours after inoculation for 72 h. (A) The survival rate of mice (n = 8) monitored daily for up to seven days after infection. The log-rank test was used to compare Kaplan-Meier survival curves. *p < 0.05 for EB211, EB279, CKT, or CMS versus NS. The results represent three independent experiments. (B) Bacterial burden in the lungs of immunocompromised infected mice (n = 6) administered with the scFvs, CMS, or NS after 24, 48, and 72 h of infection. The results represent the mean ± SEM of three independent experiments. *p < 0.05 for EB211 (Q24H) versus NS; **p < 0.01 for EB211, EB211 (Q24H), EB279, CKT, or CMS versus NS.

Fig. 3

Administration of EB211 and EB279 prevented severe tissue damage in the kidneys of immunocompromised mice with A. baumannii pneumonia. Immunocompromised mice were inoculated intranasally with XDR A. baumannii A.b.56 at a 50% lethal dose (LD₅₀; 4×10⁷ CFU per mouse). Immunocompromised infected mice were administered intraperitoneally twice daily with EB211 (12 mg/kg), EB279 (12 mg/kg), a cocktail of the two scFvs (CKT; 6 mg/kg of each), an irrelevant scFv (MEH158; an S. aureus-specific scFv) (12 mg/kg), colistimethate sodium (CMS; 30 mg/kg), or normal saline (NS), or once daily with EB211 (12 mg/kg) (Q24H) two hours after inoculation for 72 h. Healthy (H), immunocompromised uninfected (IU), and immunocompromised infected (Iinf) mice were killed at 72 h of infection, and the kidneys were collected. Tissue sections were stained with hematoxylin-eosin and microscopically evaluated for histopathological alterations. Tubular degeneration and necrosis, and infiltration of inflammatory cells (leukocytes) into the interstitial tissue surrounding the tubules in the cortex and medulla were the marked pathological symptoms in Iinf mice treated twice daily with NS, MEH158, or CMS, or once daily with EB211 (Q24H). After 72 h of infection, no severe pathological signs were detected in the kidneys of Iinf mice treated twice daily with EB211, EB279, or CKT for 72 h. Black arrowheads: tubular necrosis, Green arrows: diminished and distorted glomeruli, White arrows: infiltration of inflammatory cells (leukocytes), White arrowheads: cytoplasmic vacuolation and pale-staining plus fragmented cytoplasm

Fig. 4

Administration of EB211 and EB279 prevented severe tissue damage in the liver of immunocompromised mice with A. baumannii pneumonia. Immunocompromised mice were inoculated intranasally with XDR A. baumannii A.b.56 at a 50% lethal dose (LD₅₀; 4×10⁷ CFU per mouse). Immunocompromised infected mice were administered intraperitoneally twice daily with EB211 (12 mg/kg), EB279 (12 mg/kg), a cocktail of the two scFvs (CKT; 6 mg/kg of each), an irrelevant scFv (MEH158; an S. aureus-specific scFv) (12 mg/kg), colistimethate sodium (CMS; 30 mg/kg), or normal saline (NS), or once daily with EB211 (12 mg/kg) (Q24H) two hours after inoculation for 72 h. Healthy (H), immunocompromised uninfected (IU), and immunocompromised infected (Iinf) mice were killed at 72 h of infection, and the liver were collected. Tissue sections were stained with hematoxylin-eosin and microscopically evaluated for histopathological alterations. Bacterial communities and infiltration of inflammatory cells (neutrophils and mononuclear cells) along with spotty necrosis‌ were observed in the liver of Iinf mice treated twice daily with NS or MEH158. After 72 h of infection, no severe pathological signs were detected in the liver of Iinf mice treated twice daily with EB211, EB279, or CKT for 72 h. Black arrows: bacterial foci, Black arrowheads: necrosis of hepatocytes, Red arrowheads: bi-nucleated cells, White arrows: infiltration of inflammatory cells (mononuclear cells) along with spotty necrosis, White arrowheads: micro and macro vesicles

Fig. 5

Administration of EB211 and EB279 prevented severe tissue damage in the lungs of immunocompromised mice with A. baumannii pneumonia. Immunocompromised mice were inoculated intranasally with XDR A. baumannii A.b.56 at a 50% lethal dose (4×10⁷ CFU per mouse). Immunocompromised infected mice were administered intraperitoneally twice daily with EB211 (12 mg/kg), EB279 (12 mg/kg), a cocktail of the two scFvs (CKT; 6 mg/kg of each), an irrelevant scFv (MEH158; an S. aureus-specific scFv) (12 mg/kg), colistimethate sodium (CMS; 30 mg/kg), or normal saline (NS), or once daily with EB211 (12 mg/kg) (Q24H) two hours after inoculation for 72 h. (A) and (B) Healthy (H), immunocompromised uninfected (IU), and immunocompromised infected (Iinf) mice were killed at 72 h of infection, and the lungs were collected. Tissue sections were stained with hematoxylin-eosin and microscopically evaluated for histopathological alterations. Bacterial communities in perivascular areas, infiltration of inflammatory cells (neutrophils and mononuclear cells) in the perivascular and peribronchial areas, parenchymal necrosis, necrosis of bronchial epithelial cells, edema of perivascular spaces, and hemorrhage in the alveoli and interstitium were detected in the lungs of Iinf mice treated twice daily with NS or MEH158. After 72 h of infection, no severe pathological signs were detected in the lungs of Iinf mice treated twice daily with EB211, EB279, or CKT for 72 h. Black arrows: bacterial foci, Black arrowheads: necrosis of bronchial epithelial cells, Black asterisks: edema of perivascular spaces, Red arrow: macrophage accumulation in the perivascular and peribronchial areas, White arrows: infiltration of inflammatory cells (neutrophils and mononuclear cells) in the perivascular and peribronchial areas, and parenchymal necrosis, White asterisks: areas of hemorrhage within alveoli and interstitium