Due to the inappropriate use of antibiotics, antibiotic resistance poses a significant threat to human health globally. Antibiotic-resistant bacterial infections cause more than 700,000 deaths worldwide each year. The death toll will increase to 10 million by 2050 if effective strategies are not developed [1]. The World Health Organization (WHO) has called on countries around the world to attach great importance to global bacterial resistance and infection as a major threat to human health [2]. In 2017, WHO announced 12 strains of resistant bacteria that pose a major threat to human health, nine of which are Gram-negative bacteria (GNB). Multi-drug-resistant (MDR) GNB infections are among the leading causes of death in immunosuppressed patients, especially in intensive care units (ICUs) [3,4]. With the continuous emergence of MDR strains, there is an urgent need for the development of alternative biological agents or drugs for MDR bacterial infections.

Acinetobacter baumannii is a common opportunistic pathogen. It has become a significant cause of nosocomial infections, which cause hospital-acquired pneumonia, particularly in immunocompromised and/or post-surgical patients. The mortality rate can be as high as 43% in immunocompromised patients in the ICU due to antibiotic resistance [5]. Reports from drug resistance monitoring of A. baumannii from 2005 to 2014 showed that its drug resistance rate reached 75% in ICU patients [6].

MDR A. baumannii refers to strains that are resistant to three or more antibacterial drugs, extensivelydrug-resistant (XDR) A. baumannii refers to strains that are sensitive to one or two antibacterial drugs, and pan-drug-resistant (PDR) A. baumannii refers to strains that are resistant to all drugs [7]. Drug-resistant A. baumannii infections are becoming extremely difficult to treat because of the increasing incidence of MDR, XDR and PDR strains. MDR A. baumannii was identified as one of the six leading pathogens with high mortality, causing 50,000–100,000 deaths in 2019 [8]. In 2017, WHO added carbapenem-resistant A. baumannii to its list of priority bacteria that urgently require the development of new antibiotics [9]. However, the development of new antibiotics lags behind the drug resistance of bacteria [10,11]. Developing new biological antibacterial molecules to address substantial unmet medical need is particularly important.

Antimicrobial peptides are crucial components of innate immunity against bacterial invasions. The bactericidal permeability-increasing protein (BPI) is a cationic, neutrophil/epithelial-cell-derived antimicrobial protein with potent lipopolysaccharide (LPS)-binding/bactericidal activity, and plays crucial roles in innate immunity against GNB infection [12], [13], [14], [15]. The N-terminal domain of BPI can bind LPS, mediate antimicrobial cytotoxicity, neutralize LPS, and block LPS-induced inflammation. The C-terminal domain of BPI functions in opsonization, which is required for BPI-dependent phagocytosis of GNB by phagocytes [16,17]. The authors’ previous study showed that patients with typical bacterial pneumonia had higher BPI levels as an inflammatory marker [18]. BPI-deficient mice had increased serum LPS levels and aggravated acute colitis [19].These make BPI an attractive agent to prevent and/or treat sepsis. The recombinant full-length human BPI protein expressed from a replication-deficient adenoviral vector was reported to be a therapeutic agent that could effectively neutralize LPS, markedly reduce the production of pro-inflammatory cytokines, and improve the survival of mice suffering from lethal septic shock elicited by LPS in vitro and in vivo [20]. A recombinant N-terminal bioactive fragment of BPI (rBPI21) was demonstrated to be a safe and effective approach to neutralize LPS and sterilize GNB, and also significantly reduced morbidities and improved serious complications among children with severe meningococcaemia in phase III clinical trials [21,22]. The BPI21/LL-37 fusion protein also enhanced bacterial clearance and LPS neutralization [23].

However, the high cost and short half-life (<45 min) of recombinant proteins limits their clinical application, resulting in disappointing outcomes in phase I/II/III clinical trials [21,24]. In order to prolong their half-life and enhance their bactericidal activity, a chimeric BPI23-Fcγ fusion protein comprising the functional N-terminal domain of human BPI (amino acids 1–199) and the Fc segment of human immunoglobulin G1 (IgG1) was expressed using replication-deficient adenoviral vector 5 (Ad5). The authors’ previous studies showed that the BPI23-Fcγ protein not only had the bactericidal activity of BPI, but also had the long half-life and enhanced opsonophagocytosis of IgG1. Recombinant Ad5-BPI23-Fcγ1 or adeno-associated virus type 2-BPI23-Fcγ1 virus protected against lethal endotoxaemia and systemic/lethal Escherichia coli/Klebsiella pneumoniae challenge [25], [26], [27], [28], [29]. However, it is not yet known whether recombinant chimeric BPI23-Fcγ fusion proteins can protect against the challenge of pneumonia induced by MDR A. baumannii infection, and if so, their mechanisms of action.

This study explored the potential roles and mechanisms of a recombinant chimeric BPI23-Fcγ protein expressed by replication-deficient Ad5 on the challenge of MDR A. baumannii infection. The chimeric BPI23-Fcγ protein was extensively expressed and secreted in vitro and in vivo. Ad5-BPI23-Fcγ or recombinant protein significantly improved the survival of mice infected with a minimal lethal dose (MLD) of MDR A. baumannii or Klebsiella pneumoniae. BPI23-Fcγ protected lung pathology by reducing lung/blood/liver bacterial load, enhancing bactericidal activity, and synergically elevating the phagocytic activity of neutrophils in vitro and in vivo. Ad5-BPI23-Fcγ treatment increased the proportion and number of neutrophils in peripheral blood and lung, decreased pro-inflammatory cytokines, and promoted bone marrow (BM) neutrophil maturation. These results demonstrated that chimeric BPI23-Fcγ may be a promising drug candidate for the treatment of MDR A. baumannii.