MAbs are novel biologic agents that can exterminate pathogens [34–37]. Of note, mAbs can enhance the efficacy of existing antibiotics (e.g. synergistic or additive effect) and minimize the adverse effects of toxic antibiotics (e.g. colistin) [37], or improve drug delivery to target sites (e.g. DSTA4637S comprised of a monoclonal THIOMAB™ IgG1 specific to S. aureus β-O-linked N-acetylglucosamine conjugated with a rifamycin analogue [dmDNA31]) [36, 38]. In this regard, several mAbs targeting A. baumannii have been developed, including anti-capsule mAbs [21, 39, 40], anti-OmpA and anti-Omp34 egg yolk antibodies (IgYs) [23], anti-OmpA mAbs [41], anti-siderophore-specific receptors (Iron Regulated Outer Membrane Proteins [IROMPs]) mAbs [34], and AR-401 (a fully human mAb; Aridis Pharmaceuticals, Inc.). There are controversial results about the efficacy of anti-capsule mAbs (13D6, C8, and 8E3 mAbs) against A. baumannii infection [21, 39, 40]. Two mAbs, 13D6 (an IgM specific to K1 capsular polysaccharide) [39] and C8 (an IgG1 specific to K4 capsular polysaccharide) [21], showed antibacterial activity in vitro and in vivo models of A. baumannii infections. In contrast, mAb 8E3, a mouse IgG3 specific to K2 capsular polysaccharide, exacerbated the disease in mice with pneumonia caused by A. baumannii strain AB899 [40]. Wang-Lin et al. indicated that antibody-dependent enhancement (ADE) of infection, previously reported for viruses such as Ebola virus [42], human immunodeficiency virus type 1 (HIV-1) [43, 44], dengue virus [45], and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [46], and “the sink effect” (binding of capsule-specific mAbs to shed capsule), led to the loss of therapeutic activity of mAb 8E3, followed by an increased bacterial burden in the blood, lungs, and spleen, and a low survival rate in infected mice [40]. Moreover, Jahangiri et al. reported a low survival rate in immunocompromised mice with A. baumannii pneumonia treated with a combination of anti-OmpA and Omp34 IgYs (particularly at a high dose) at four hours post-infection compared to immunocompromised infected mice receiving anti-OmpA IgY or anti-Omp34 IgY alone [23]. They hypothesized that some similarity between OmpA and Omp34 proteins with murine proteins and subsequent binding of anti-OmpA and anti-Omp34 IgYs to mouse proteins (as decoys) led to the ADE of A. baumannii infection in immunocompromised mice [23]. Of note, Goel et al. indicated that five mouse IgM mAbs targeting IROMPs, involved in the uptake of siderophore-iron complex, inhibited the growth of A. baumannii cells in vitro by two mechanisms, including the augmentation of opsonization and the interference with the biological activity of IROMPs (direct bactericidal activity) [34]. In any case, the ADE of bacterial infection might be observed with antibody-based therapy [36], exacerbating the disease in patients. In contrast, antibody fragments such as bactericidal scFvs [27, 30, 31, 47, 48], having remarkable features including target-specific binding, small size, great tissue penetration, phagocytes- and complement-independent antimicrobial activity (particularly in immunocompromised patients), and subsequently, no ADE effect (Fc-related unwanted events), can be substantial alternatives to conventional mAbs [25, 30, 49, 50]. Aiming to isolate bactericidal scFvs specific to A. baumannii, two scFvs, EB211 and EB279, with unique sequences and significant binding ability to A. baumannii, were identified [29]. Of note, we demonstrated that EB211 and EB279 elicited an antibacterial effect against A. baumannii [29] possibly through the disruption in the outer membrane, followed by the interference with the activity of OprD and TonB, respectively [33].
Off-target toxicity is one of the major causes impeding the evaluation of many therapeutics in clinical trials due to a group of various unwanted events in patients [51]. As abovementioned, Jahangiri et al. showed the similarity between peptides derived from OmpA and Omp34 proteins with some peptides of Mus musculus proteins (as decoys), resulting in the inefficacy of the combination anti-OmpA and anti-Omp34 IgYs in the treatment of mice with A. baumannii pneumonia [23]. In this regard, we investigated the off-target effect of both scFvs in vivo. The histopathological examination of the kidneys and liver of mice receiving 12 mg/kg of EB211, EB279, or a cocktail of two scFvs twice daily for 72 hours indicated no inflammation or tissue damage, confirming the safety of the scFvs in healthy mice.
The therapeutic activity of EB211, EB279, and a cocktail of two scFvs was investigated in immunocompromised mice intranasally inoculated with an XDR A. baumannii strain. EB211, EB279, and a cocktail of two scFvs conferred complete protection against lethal pneumonia. Besides, immunocompromised infected mice administered twice daily with EB211, EB279, or a cocktail of two scFvs for 72 hours showed a lower bacterial load in the lungs and no marked histopathological abnormalities in the kidneys, liver, and lungs after 72 hours of infection compared to immunocompromised infected mice administered twice daily with NS or an irrelevant scFv. A few studies assessed the protective efficacy of anti-A. baumannii mAbs in the murine model of pneumonia [21, 23, 40]. Nielsen et al. assessed the therapeutic efficacy of mAb C8, targeting the K4 capsular polysaccharide of A. baumannii, in a murine model of aspiration pneumonia [21]. Mice intratracheally infected with an XDR A. baumannii (strain HUMC1) received mAb C8 (5 µg/mouse/intravenously or 50 µg/mouse/intraperitoneally) immediately or four hours after infection [21]. At both doses and routes of administration, mAb C8 exhibited significant protective activity in infected mice after seven days. Moreover, the administration of mAb C8 (50 µg/mouse/intraperitoneally) led to a decreased bacterial burden in the blood and lungs and no pathological symptoms in the lungs of infected mice compared to infected mice receiving an isotype control mAb (50 µg/mouse/intraperitoneally) at 24 hours of infection [21]. Nonetheless, Wang-Lin et al. reported that the administration of mAb 8E3 (against the K2 capsular polysaccharide) (50 or 200 µg/g/intraperitoneally) immediately after intratracheal inoculation with A. baumannii strain AB899 caused increased disease severity (100% mortality at 48 hours of infection) [40]. Additionally, a higher bacterial load in the blood, lungs, and spleen was observed in 8E3-treated mice than PBS-treated mice at 24 hours of infection. Based on the findings, mAb 8E3 could not confer protection in infected mice [40]. In another study, Jahangiri et al. evaluated the therapeutic efficacy of a group of IgYs specific to OmpA, Omp34, and the whole inactivated cells of A. baumannii in immunocompromised mice intranasally inoculated with A. baumannii strain AbI101 [23]. All mice were intranasally administered with IgYs at four hours after inoculation. Based on the results, IgYs against OmpA and the whole inactivated cells had significant therapeutic activity and decreased the mortality by 25% in infected mice (75% survival) at eight days after inoculation [23]. In contrast, the combination of anti-OmpA and anti-Omp34 IgYs exacerbated the disease in mice due to the ADE effect, as previously mentioned [23].