Directed-complement killing of Pseudomonas aeruginosa protects against lethal pneumonia

Summary

Background

Multidrug-resistant Pseudomonas aeruginosa raises major clinical concerns due to its capacity to cause a wide-array of infections in individuals with compromised immune defences and to withstand standard-of-care therapeutic treatments. Antibody-based approaches have proven to be efficient in the treatment of diverse infections. Here we propose an innovative approach harnessing the complement at the surface of bacteria for further killing.

Methods

We developed two Complement-activating Multimeric immunotherapeutic compleXes (CoMiX) targeting the bacterium through a single-chain variable fragment directed against the exopolysaccharide Psl, and carrying one of two different effector functions, Factor H Related protein 1 (FHR1) or a Fc dimer. Each CoMiX was assessed in vitro for their antibacterial activity, and further evaluated in a mouse model of acute pneumonia.

Findings

Both CoMiX-FHR1 and CoMiX-Fc effectively deposit C1q (for CoMiX-Fc), C3b, and C5b9 at the surface of multidrug-resistant clinical isolates, promoting their direct killing and/or opsonisation and subsequent phagocytosis for CoMiX-Fc (p < 0.001). Both CoMiX synergise with amikacin and protect epithelial cells against P. aeruginosa-induced cytotoxicity. Importantly, CoMiX administered intranasal to acutely infected mice significantly improve their survival (p < 0.001) by reducing local bacterial burden through the higher induction of C3b (opsonisation) and C5a (neutrophils recruitment and activation) and by decreasing lung inflammation.

Interpretation

Our proof-of-concept demonstrates the efficient, direct and indirect killing of P. aeruginosa by the complement, highlighting the therapeutic potential of CoMiX to combat multidrug-resistant bacteria.

Funding

Luxembourg National Research Fund, Ministry of Higher Education and Research of Luxembourg, COST action CA21145 EURESTOP, Institut National de la Santé et de la Recherche Médicale, and Tours University.

Research in context.

Evidence before this study

Antimicrobial resistance (AMR) is a serious and critical threat to public health, with increased prevalence of infections in hospital and the decline of antibiotic efficacy against multidrug-resistant bacteria. Among the pathogens highly resistant to current treatments, Pseudomonas aeruginosa carbapenem-resistant has been defined as a high priority for the research and development of new therapeutic strategies in the 2024 priority pathogens list of the World Health Organisation (WHO). However, despite the urgency, few promising drug candidates targeting P. aeruginosa are currently in the clinical pipeline and none has been approved by the health authorities. Monoclonal antibodies and antibody-based therapies, which have shown success in treating HIV, malaria or COVID-19 infections, are now demonstrating great promise against a broader range of infectious diseases. Evidence to find the perfect targeting system against the bacterium and exploit a mode of action not vulnerable to the usual resistance mechanisms are still needed.

Added value of this study

The complement system plays a preponderant role in host anti-bacterial defences, but remains largely unexplored as a therapeutic target. This work presents in a first part the engineering and the mode of action of Complement-activating Multimeric immunotherapeutic compleXes (CoMiX) directed against P. aeruginosa, enhancing the deposition of complement molecules at the surface of bacteria leading to their lysis and/or engulfment by macrophages and neutrophils. In a second part, preclinical studies demonstrated the efficacy of complement-activating proteins in a prophylactic and therapeutic mouse model of acute pneumonia. Importantly, our findings indicate that the protection of mice was associated with complement activation through a higher release of C3b in the bronchoalveolar lavage leading to direct killing of the bacteria, and higher production of the anaphylatoxin C5a promoting recruitment and activation of neutrophils. Overall, CoMiX administration during acute lung infection resulted in lowered lung bacterial load and the dampening of the inflammation in the lung leading to increased protection.

Implications of all the available evidence

This work demonstrates that enhancing the immune system through directed-activation of complement is a fast and promising therapeutic strategy against bacteria. This proof-of-concept lays a strong foundation for the clinical development of CoMiX against P. aeruginosa to circumvent its resistance and spare the commensal bacteria. Further studies should raise knowledge on long-term potential and efficacy against chronic infections, as well as open the way to other pathogens.

Introduction

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium responsible for life-threatening infections in immunocompromised individuals or those accommodated in intensive care units (ICU) undergoing mechanical ventilation. It exhibits a broad tropism colonising various tissues and causes a wide array of infections, including in the airways.¹ P. aeruginosa-associated lung infections are now considered a leading cause of morbidity and mortality worldwide. Reflecting its extensive multidrug-resistant, carbapenem-resistant P. aeruginosa was classified as a high priority pathogen in the updated 2024 Bacterial Priority Pathogens List (BPPL)² by the World Health Organisation (WHO), and identified as critical target for novel therapeutic development.³

Antibody-based therapies have emerged as promising alternatives to combat infections caused by multidrug-resistant Gram-negative bacteria, including P. aeruginosa.⁴ These targeted therapies offer the distinct advantage of minimising disruption of the normal bacterial flora and mitigating off-target effects. Beyond their neutralising activity, antibodies engage immune effector functions through their Fc-region, promoting pathogen clearance. Among them, antibody-dependent cellular cytotoxicity (ADCC) triggers the release of cytotoxic granules by Natural Killer (NK) cells, antibody-dependent cellular phagocytosis (ADCP) activates phagocytosis by macrophages and neutrophils, and complement-dependent cytotoxicity (CDC) leads to the opsonisation of bacteria and their direct lysis.^5,6 However, antibodies directed against P. aeruginosa have not succeeded in early clinical trials. One example is the MEDI3902, a bispecific antibody targeting both the exopolysaccharide Psl and the Type III Secretion Protein PcrV⁷ that showed promising results in preclinical and Phase I trials. However, it failed to reduce nosocomial pneumonia incidence in patients with ventilator-associated pneumonia⁸ during late-stage trials. This highlights the need for alternative strategies to overcome the current limitations of therapeutic antibodies against P. aeruginosa infections.

The complement system, a critical component of innate immunity, has been relatively underexplored as a therapeutic target for bacterial infections. Its preponderant role in host anti-bacterial defences is nevertheless well established.⁹ Thus, both complement-depleted mice and complement-deficient knock-out mice show higher susceptibility to P. aeruginosa pneumonia,¹⁰ with impaired bacterial clearance, increased lung injury and increased mortality.^11,12 In humans, recent studies have revealed the complement system as the primary defence mechanism against P. aeruginosa infection.^13,14 It operates through three activation pathways: the classical pathway (initiated by C1q binding to antibody Fc region), the mannose-binding lectin (MBL) pathway (triggered by the binding of MBL or ficolins to specific carbohydrates) and the alternative pathway (induced by spontaneous C3 hydrolysis).¹⁵ Engagement of any of these pathways leads to the cleavage of C3, and the generation of C3a (anaphylatoxin and chemotactic molecule) and C3b which opsonises target surfaces. C3b can subsequently participate to the cleavage of C5, generating C5a (chemoattractant peptide), and C5b, which drives membrane attack complex (MAC) assembly.¹⁶ The complement system can thus eradicate pathogens through a multifaceted approach: direct lysis via the MAC formation, opsonisation of microbes by C3b/iC3b facilitating their phagocytosis, and release of C3a and C5a mediating chemotaxis and immune cell activation.¹⁷

However, despite its effectiveness, P. aeruginosa has developed sophisticated mechanisms to evade complement-mediated killing.¹⁸ Increased production of exopolysaccharides and modifications in the lipopolysaccharide (LPS) O-antigen on the bacterial surface limit complement access.19, 20, 21 Enhanced secretion of proteases by the bacterium inactivate complement molecules.22, 23, 24 More pernicious, excess production of O-antigen-specific IgG2 antibodies–“inhibitory/cloaking antibodies”, can bind to the membrane of the bacterium, and shield it from other antibodies and complement molecules.²⁵ Additionally, P. aeruginosa binds host complement regulatory proteins like Factor H (FH)^26,27 which inhibits the activation of the alternative complement pathway. Interestingly, Factor H-related proteins (FHRs), which share conserved ligand and cell surface recognition domains of FH,²⁸ but lack its complement regulatory domain, may function as positive regulators of complement activation, counteracting the inhibitory effect of FH.²⁹ FHR1, the most abundant FHR in the blood, binding to various ligands of FH (e.g., C3b, pentraxins, heparin) and to FH-binding proteins of P. aeruginosa (e.g., elongation factor Tuf,²⁶ LPD,³⁰ and OprG¹⁴) has been recently shown to promote complement deposition at the surface of P. aeruginosa.¹⁴

Previously, we have developed complement-activating immunotherapeutic complexes (CoMiX) targeting HER2-positive cancer cells using the C4BP C-terminal α-chain multimerising scaffold (C4BPα) that display multivalent FHR4 moieties and elicit C3b deposition, MAC formation and CDC on cancer cells.³¹ In this study, we have generated CoMiX using the C4BPβ scaffold and single-chain variable fragments (scFv) targeting the exopolysaccharide Psl expressed on P. aeruginosa with the aim to compare the activation of classical pathway versus alternative pathway and their potency against bacteria. Complement activation on P. aeruginosa was mediated either by the Fc region for CoMiX-Fc, or by the C-terminal domain of FHR1 for CoMiX-FHR1. Both CoMiX-Fc and CoMiX-FHR1 significantly increased complement deposition and activation on the bacterial surface, resulting in direct bacterial lysis and, in the case of CoMiX-Fc, in an improved opsonisation and enhanced engulfment by macrophages and neutrophils. Remarkably, intranasal administration of targeted CoMiX provided an optimal defence against P. aeruginosa in a murine model of acute lung infection, promoting bacterial clearance through direct complement killing and indirect transient neutrophils activation, reducing lung inflammation, and significantly improving survival.

Methods

Bacterial strains

P. aeruginosa strain serotype IATS O11 (33358™), reference strain PAO1 (15692™), and PAO1-GFP (15692GFP™) were obtained from the American Type Culture Collection (ATCC, USA). The luciferase-expressing strain PAO1 (PAO1-Lux) was generated by Delphine Fouquenet at the University of Tours, France. Twenty-nine clinical isolates of P. aeruginosa were obtained from the Centre Hospitalier Régional Universitaire (CHRU) of Tours, France (protocol number 2016-003 approved by the ethics committee of the CHRU of Tours). These clinical strains were collected from expectorations of patients with cystic fibrosis (CF) or isolated on the breathing tubes of patients intubated in ICU. Antimicrobial susceptibility testing was performed on all strains. A protein lysate of Moraxella catarrhalis strain BBH18, originally isolated from the sputum of a patient with chronic obstructive pulmonary disease, was used for ELISA binding. All bacterial cells were grown in Tryptic soy broth (TSB) at 37 °C with shaking or on TSB solidified with 1.5% agar (TSA) plates. In synergy experiments, TSB was supplemented with 2 μg/mL or 10 μg/mL of amikacin (39831-55-5, Santa Cruz Biotechnology).

Production and purification of CoMiX

The recombinant plasmid DNA for the expression of CoMiX using the C4BP β chain was previously described.³² The following sequences were synthesised by ProteoGenix SAS (Schiltigheim, France): scFv anti-O11 scFv derived from Panobacumab, (patent WO2006/084758A1), human C4BP C-terminal β chain (UniProt nr. P20851.1, aa 137–252), human IgG1 H chain constant gamma (UniProt nr. P01857.1), scFv anti-Psl derived from the bispecific antibody MEDI3902 (patent WO2017/095744A1), human FHR1 CCP3-5 (UniProt nr. Q03591, aa 145–329), scFv anti-Aspergillus fumigatus directed against the Chitin ring formation 2 (Crf2) protein of A. fumigatus (MS112-IIB1, Chauvin et al., 2009) and scFv anti-M. catarrhalis. The production of all CoMiX was performed as previously reported³¹ by transfecting mycoplasma-free HEK293T/17 cells (ATCC CRL-11268, RRID: CVCL_1926) with the CoMiX-expressing plasmid using Lipofectamine 3000 (ThermoFisher Scientific, USA) according to the manufacturer's instructions. Forty-eight hours post-transfection, cells were trypsinised and seeded into 10 cm culture dishes containing complete DMEM medium and selected using 10–20 μg/mL puromycin (InvivoGen, USA).

Supernatants from clonal cultures were analysed by whole-cell ELISA. P. aeruginosa strain PAO1 or O11 were immobilised onto Maxisorp 96 well Nunc immunoplates (Thermofisher). After blocking with 4% BSA (Roth, Keerbergen, Belgium), wells were incubated with CoMiX-FHR1 or CoMiX-Fc. Bound CoMiX was detected using either an HRP-conjugated anti-His antibody (Sigma–Aldrich Cat# A7058, Overijse, Belgium, RRID: AB_258326) for CoMiX-FHR1 or an HRP-conjugated anti-Fc antibody (Abcam Cat# ab97225, Abcam, Cambridge, UK, RRID: AB_10680850) for CoMiX-Fc. Tetramethylbenzidine (TMB) (Biolegend, USA) was used as a substrate, the reaction was stopped with H₂SO₄, and the absorbance read at 450 nm using a POLARStar spectrophotometer (BMG Labtech, Germany). Clones exhibiting the highest CoMiX expression were expanded in flasks and into a 5-chamber CellSTACK® culture system (Corning Incorporated, USA) containing complete DMEM medium. CoMiX-Fc was purified with Protein G Sepharose® 4 Fast Flow resin (GE Healthcare, GE17-0618-01; 1 mL bed volume) and CoMiX-FHR1 with HisTrap HP column (Cytiva Life Sciences, Marlborough, MA, USA) using a Bio-Rad NGC™ chromatography system (Bio-Rad Laboratories, Hercules, CA, USA). After elution, CoMiX were washed of antibiotics contaminant using Amicon® Ultra centrifugal filter devices (MilliporeSigma). The final concentration was determined using a NanoDrop™ micro-volume spectrophotometer. All purified proteins were stored at −20 °C until further use.

Molecular and binding analysis of the CoMiX

The molecular pattern of the recombinant proteins was analysed by SDS/PAGE, SYPRO Ruby protein gel staining and western blotting (in non-reducing and reducing conditions using 10% β-mercaptoethanol). Samples were loaded onto 4–15% Mini-PROTEAN® Tris-Glycine Extended (TGX™) precast gels (Bio-Rad) and electrophoresed using XT MES running buffer (Bio-Rad) according to the manufacturer's instructions. Following electrophoresis, the gel was fixed in a solution of 50% methanol and 7% acetic acid (v/v) for 30 min, then stained with SYPRO Ruby protein gel stain (Thermo Fisher Scientific) overnight at 4°C, following the manufacturer's protocol. The gel was washed with 10% methanol and 7% acetic acid (v/v) for 30 min and was visualised using an Amersham™ Typhoon™ biomolecular imager (Cytiva, MA, USA) with the Cy5 filter. For western-blot, proteins from the SDS-PAGE gel were transferred to a pre-activated low-fluorescence PVDF membrane using TTB (25 mM Tris, 192 mM glycine, 0.01% (v/v) SDS, 20% (v/v) methanol, pH 8.8) blotting buffer. The membrane was blocked with 4% BSA in PBS and probed with: anti-Fc AF647 (SouthernBiotech Cat# 2048-31, Birmingham, USA, RRID: AB_2795692) for CoMiX-Fc and rabbit anti-His antibody (Bethyl Cat# A190-114A, Merelbeke, Belgium, RRID: AB_67321) followed by polyclonal goat anti-rabbit AF647 (Jackson ImmunoResearch Labs Cat# 111-607-008, Sanbio B.V., The Netherlands, RRID: AB_2632470) for CoMiX-FHR1. Protein bands were detected using an Amersham™ Typhoon™ biomolecular image (Cytiva, MA, USA).

The binding of CoMiX was confirmed on bacterial strains using whole cell ELISA. A bacterial suspension of 1 × 10⁶ colony forming units (CFU) per well was first immobilised overnight onto Nunc MaxiSorp™ 96-well flat-bottom polystyrene ELISA plates. For the binding to M. catarrhalis, 100 μL of the bacterial lysate (20 μg/mL) was immobilised the same way. After washing the plates five times with PBS containing 1% BSA, the wells were blocked with 100 μL of 5% BSA in PBS solution for 1 h at 4 °C. CoMiX and irrelevant controls were incubated for 1 h at 4 °C. Either HRP-conjugated goat anti-human IgG Fc antibody or HRP-conjugated anti-His antibody was added at a concentration of 1 μg/mL and incubated for 1 h at 4 °C. The binding was revealed by the chromogenic substrate, TMB/H₂SO₄ as described above. To test the competition between FH and CoMiX-FHR1, coated-wells of PAO1 bacteria were blocked with PBS-1% BSA, and incubated with either PBS as control or FH (20 μg/mL) and a range of concentrations of CoMiX-FHR1 (and CoMiX-Fc as control) (40 μg/mL). FH was then detected with the mouse monoclonal antibody (mAb) OX-24 (Abcam Cat# ab118820, RRID: AB_10899656), followed by an HRP-conjugated goat anti-mouse IgG (BioLegend Cat# 405306, RRID: AB_315009), and revealed by TMB/H₂SO₄. Washing steps with PBS-Tween 0.05% were included between each incubation.

C1q-, C3b-, and C5b9-deposition measurement by ELISA and microscopy

Reference strain PAO1 and clinical isolate IT 2 were immobilised onto Nunc MaxiSorp™ 96-well flat-bottom–as described above. After blocking with PBS-5% BSA, plates were incubated with serially diluted CoMiX proteins (1:3 dilutions starting at 10 μg/mL) for 1 h at 4 °C. Following washes, plates were incubated with 4% (C1q) or 2% (C3b) normal human serum (NHS) or decomplemented NHS (ΔNHS) diluted in GVB++ buffer for 30 min at 37 °C. After additional washing steps, the plates were incubated with a mAb anti-C1q (Hycult Cat# HM2382-100UG) or a mAb anti-human C3/C3b/iC3b (BioLegend Cat# 846402, RRID: AB_2572175) for 1 h at 4 °C. HRP-conjugated goat anti-mouse IgG (BioLegend Cat# 405306, RRID: AB_315009) was added at 100 ng/well and incubated for 1 h at 4 °C, revealed with TMB/H₂O₂ and read at 450 nm. To investigate the formation of MAC following C1q and C3b deposition, the same protocol was employed using 4% NHS/ΔNHS and a mAb anti-human C5b9 (Abcam Cat# ab66768, RRID: AB_1139825). ΔNHS and decomplemented mouse serum (ΔMS) were obtained by heating sera for 30 min at 56 °C under agitation and checked by ELISA for the loss of expression of complement proteins (C3b).

Complement activation was visualised on bacteria treated with CoMiX by confocal and fluorescence microscopy. Overnight culture of PAO1-GFP was grown to log-phase. After treatment with CoMiX and normal or 10% C5-deficient human in GVB++ medium, as previously described, cells were pelleted by centrifugation at 8000g for 5 min and washed with PBS. Complement deposition was detected with a mAb anti-human C3/C3b/iC3b (BioLegend Cat# 846402, RRID: AB_2572175) and a mAb anti-C5b9 (Novus Cat# NBP1-05120, RRID: AB_1522768), for MAC formation, for 2 h at room temperature followed by an anti-mouse-AF647 (Jackson ImmunoResearch Labs Cat# 115-607-003, RRID: AB_2338931) secondary antibody for 2 h. Bacterial cells were fixed with 2% paraformaldehyde (PFA) for 30 min at room temperature and mounted onto a pre-prepared agarose pad. A 1.5 × 1.6 cm Gene Frame matrix (Thermo Scientific) was adhered to a microscope slide (Thermo Fisher). The Gene Frame was then filled with 200 μl of 1% TSB-agarose gel and a glass coverslip (Menzel Gläser #1) was placed on top. Samples were analysed with a Leica SP8 confocal microscope and Leica LasX Life Sciences Software at the EuroBioimaging platform (University of Pharmacy, Strasbourg), as well as a Axio Observer and Zeiss Blue software. The chosen pictures were processed with ImageJ, to accentuate contrasts, before integrated inside the figures.

Complement-dependent killing assay

Bacterial strains PAO1 and clinical isolate IT 2 were cultured in TSB at 37 °C to an exponential growth phase and resuspended in PBS. 10⁶ CFU were incubated without NHS, with 10% (v/v) NHS, with 10% ΔNHS, with 10% mouse serum (MS) or 10% ΔMS in the presence or absence of CoMiX (30 μg/mL) or an irrelevant control for 2 h at 37 °C. Bacterial suspensions were diluted in PBS, plated onto Tryptic Soy Agar (TSA) plates, incubated overnight at 37 °C, and the number of CFU was enumerated. For the PAO1-Lux strain, an identical culture protocol was followed. The luminescence-based reporter system of PAO1-Lux was used for real-time monitoring of bacterial growth. Luminescence output (RLU) was measured every hour using the POLARStar Omega microplate reader, providing a quantitative assessment of bacterial viability and proliferation.

To measure membrane permeability, bacterial cells were grown in TSA and 10⁶ CFU were incubated with normal human serum (10%) and the different CoMiX (30 μg/mL) for 2 h. The bacterial pellet was harvested by ultracentrifugation at 4200g for 15 min and washed once with PBS. The pellet was resuspended in 100 μL of PBS containing 10 μL of Ethidium Homodimer-1 (EthD-1) (Invitrogen, United States). After 30 min of incubation, 100 μL was placed into a black 96-well plate to measure fluorescence using GloMax® Discover fluorimeter (Promega, United States).

We investigated the combined effect (synergy) of amikacin, a widely used aminoglycoside antibiotic (39831-55-5, Santa Cruz Biotechnology), and CoMiX against the reference strain PAO1 and a multidrug-resistant clinical isolate (clinical isolate IT 2). The minimum inhibitory concentration (MIC) of amikacin was determined using the broth micro dilution method as previously described (Kowalska-Krochmal, 2021). Overnight cultures of PAO1 and the clinical isolate IT 2 were grown in TSB medium until reaching the exponential growth phase. 10⁶ CFU was incubated with 10% NHS in the presence or absence of 30 μg/mL of CoMiX or irrelevant control. The samples were incubated for 2 h at 37 °C under agitation before dilution and plating on TSA agar plates. CFU were counted after 24 h. The synergy coefficient was calculated using the equation adapted from Chaudhry et al., 2017: log(C) − log(SA) − log(SB) + log(SAB), where C represents the CFU in the untreated control well, SA represents the surviving CFU after treatment with amikacin alone, SB represents the surviving CFU after treatment with the CoMiX molecules alone, and SAB represents the surviving CFU after combined treatment with amikacin and CoMiX. This equation defines whether synergy exists between amikacin and CoMiX. A synergy coefficient less than zero (<0) indicates synergy between the two treatments.

Macrophages-dependent bacterial ingestion assay

Peripheral blood mononuclear cells (PBMCs) from healthy donors (Luxembourg Red Cross, MAN_SCE_24_008) were isolated from buffy coats and differentiated into M1 macrophages using and following the manufacturer's instructions of PromoCell macrophage generation medium (PromoCell, Germany). Fresh PBMCs were plated and incubated in monocyte attachment medium (MAM) for 2 h, before removal of the floating contaminating cells. Adherent cells were washed three times with MAM, and M1-Macrophage Generation Medium DXF (containing GM-CSF and a mix of cytokines) was directly added onto the cells. Cells were incubated for 9 days at 37 °C with 5% CO2 with medium changes in between. After differentiation into M1 macrophages, cells were harvested from the flasks, counted, and dispatched into 96 wells plates in RPMI, Glutamax, 10% FBS, 1% Penicillin/Streptavidin. M1-polarised macrophages were activated by treatment with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma) for 5 days. Prior to the bacterial ingestion assay, cells were washed twice with PBS to remove any residual antibiotics, and resuspended in serum/antibiotics free RPMI medium.

PAO1 bacteria from an overnight culture were counted, fixed in PFA 2% and stained with pHrodo dye (P35372, Thermo Fisher Scientific) following the manufacturer's instructions. PBMCs M1-derived macrophages were co-incubated with pHrodo-stained bacteria at a 12:1 bacteria-to-macrophage ratio with NHS (10%), and in the presence or absence of CoMiX (15 μg/mL). Cells were incubated in the Incucyte® (Sartorius, France) and pictures under ×4 were taken every 15 min for at least 2 h. PBMCs M1-derived macrophages were also cultured on Lab-tek (Nunc, Loughborough, UK) 8 chamber slides. PAO1 bacteria were fixed from an overnight culture with ethanol PFA 2%, stained with CellTrace™ CFSE (C34554, Thermo Fisher Scientific, USA), and then added in the Lab-Tek at a 12:1 bacteria-to-macrophage ratio for 1 h in the presence of normal human serum (10%) and CoMiX (15 μg/mL). Following co-incubation, the cytoplasmic membrane of macrophages was stained for 10 min with AF647-Wheat Germ Agglutinin (WGA). Cells and bacteria were finally fixed with 2% PFA. Slides were viewed on an Axio Observer fluorescence microscopy (Zeiss, Germany). Images of the two stains (green and red) were overlaid and analysed using ImageJ.

Abiotic adhesion assay

Bacterial adherence assay for abiotic surface was performed using 96-well plates. 5 × 10⁴ CFU were inoculated with 10% NHS and 30 μg/mL of CoMiX, and then incubated for 24 h at 37 °C. After incubation, adherent bacteria were gently washed with water twice and stained with 0.01% crystal violet for 20 min at room temperature. Excess stain was removed by gentle washes with water twice. Once dried, crystals were dissolved with 250 μL of acetic acid and their absorbance was measured at 570 nm using the POLARStar Omega microplate reader.

Effect of CoMiX on BEAS-2B cells

Immortalised human bronchial epithelial BEAS-2B cells (ATCC® Cat# CRL-9609™, RRID:CVCL_0168), mycoplasma free, were cultured in Ham's F-12 medium (Gibco™) supplemented with 10% foetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% l-glutamine, and maintained at 37 °C in a humidified incubator with 5% CO₂. For infection assays, cells were detached using 0.05% trypsin–EDTA and seeded into 96-well plates at a density of 2 × 10⁴ cells/well in complete F-12 medium overnight to reach 80% confluence. Prior to infection with PAO1 in exponential growth phase, cells were washed with PBS to remove antibiotics and resuspended in antibiotic- and serum-free F-12 medium. Cells were infected with PAO1 at a multiplicity of infection (MOI) of 10:1 in presence or not of CoMiX (15 μg/mL) and 10% NHS. The plates were incubated for 2–6 h at 37 °C. Bacteria were diluted and plated for CFU counting to assess bacterial adherence. Cell culture supernatants were collected, centrifuged at 5000g for 5 min and frozen at −20 °C. Lactate dehydrogenase (LDH) activity in the supernatants was measured using the CyQUANT™ LDH Cytotoxicity Assay kit (C20300, Thermo Fisher Scientific) according to the manufacturer's instructions. LDH release was expressed as a percentage of the total LDH activity measured in the positive control wells (representing 100% cell lysis).

Antimicrobial activity of Neutrophils-Like Cells (NLCs)

The human promyeliocytic leukaemia cell line HL-60 cells (ATCC® Cat# CCL-240™, RRID:CVCL_0002), mycoplasma free, was differentiated into Neutrophils-Like Cells (NLCs) by culturing the cells in T75 flasks at a density of 1 × 10⁶ cells/mL in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) (completed with 2 mM l-Glutamine, 90% non-essential amino acids, 10% heat-inactivated foetal bovine serum, 1 U/mL penicillin, and 1 μg/mL streptomycin) and supplemented with 1.3% dimethyl sulfoxide (DMSO) for 5 days. Differentiation was verified by flow cytometry based on surface expression of the myeloid markers CD11b and CD15 upregulated during granulocytic maturation. Prior to infection with PAO1, NLCs cells were washed with PBS to remove antibiotics and DMSO and resuspended in antibiotic- and serum-free RPMI medium.

PAO1 bacteria from an overnight culture were subcultured for 2 h at 37 °C until exponential growth phase. Phagocytosis assay of live P. aeruginosa bacteria was performed as previously described.^33,34 NLCs (200,000 cells in RPMI 1640) were infected with PAO1 or PAO1-GFP for 20 min at 37 °C under agitation at a MOI of 10:1 in presence or not of CoMiX (15 μg/mL) and 2% NHS. Cells were then treated with gentamicin (67 μg/mL) for 20 min at 37 °C and washed twice in PBS to remove unbound bacteria. (1) NLCs infected with PAO1 were resuspended in H₂O for 20 min and the lysed content was diluted and plated on petri dishes. CFUs were counted after overnight incubation at 37 °C, representing the number of live bacteria phagocytosed by the NLCs. (2) NLCs infected with PAO1-GFP were fixed for 20 min with 100 μL of PFA 2%. Cells were acquired after fixation and GFP fluorescence was measured using a LSRFortessa™ Cell Analyser cytometer (BD biosciences) as a marker of phagocytosis. The mean GFP fluorescence intensity per NLCs (Gx-Mean) was also recorded representing the mean relative phagocytosis of GFP P. aeruginosa per neutrophil.

To determine the antibacterial activity of NLCs, cells were co-incubated with bacteria at a MOI of 2:1 in presence or not of CoMiX (15 μg/mL) and 10% NHS for 1 h at 37 °C under agitation. Cells were lysed with the addition of H₂O and serial dilution of the lysate were plated on petri dishes. Results were expressed as surviving bacteria compared to bacterial growth measured under the same conditions in absence of cells.

Acute pneumonia model in C57BL/6jrj (B6) mice

Adult female C57BL/6jrj (B6) mice (RRID:IMSR_RJ:C57BL-6JRJ) (7 weeks old and weighing around 20 g) were obtained from Janvier Laboratories (Le Genet Saint Isle, France). All mice were housed under specific-pathogen-free conditions at the Plateforme Scientifique et Technique Animaleries (PST-A) animal facility (Tours, France), acclimated for one week to the facilities before any experiments, and had access to food and water ad libitum. Each animal was an experimental unit, and cages housing the animal (four per cages) were randomly allocated to the different experimental groups. All animal experiments complied with the European and French legislative, regulatory and ethical requirements and the protocol was approved by the ethics committee in animal experimentation of the Centre - Val de Loire region under the referral number 7590 issued by the Ministry of Higher Education, Research and Innovation. To insure transparency for the in vivo data, we used the ARRIVE reporting guidelines.³⁵

Mice were infected with a freshly prepared inoculum. Bacteria from a frozen stock were grown overnight in 4 mL of Luria broth (LB) under agitation at 220 rpm. The following day, bacteria were transferred into fresh LB medium and grown until their mid-log phase, as measured by an optical density (OD) of about 0.4, corresponding to a titre of 2 × 10⁸ bacteria/mL. The bacterial culture was centrifuged at 3000g for 10 min, washed twice with PBS, and finally diluted to give a desired inoculum of 3 × 10⁶ bacteria/40 μL. Each inoculum was verified for accuracy by serial dilutions and direct plating on LB agar plates. Mice were anesthetised by intra-peritoneal injection of a mixture of ketamine-xylazine 1:1. Then, 40 μl of the bacterial suspension (3 × 10⁶ CFU/mice) was administrated by intranasal instillation and mice were then immediately held upright to facilitate bacterial inhalation until normal breathing resumed. In all experiments, the animals' mortality and body-weight were monitored daily for 7 days. Animals found moribund or with a body-weight loss >25% were sacrificed.

To administrate CoMiX, mice were anesthetised with 3% isoflurane. Three hours before the infectious challenge (procedure 1: prophylactic treatment) or 1 h after the infection (procedure 2: curative treatment), anti-Psl CoMiX-Fc (100 μg/animal), anti-Psl CoMiX-FHR1 (100 μg/animal), or an irrelevant CoMiX control, anti-CFS1 CoMiX-Fc (100 μg/animal), were administered by intranasal instillation as previously described (n = 8–16 mice per group). To verify the lethality of the bacterial dose, mice were treated with PBS (n = 27 mice). A boost of molecules was administered 3 h after the infection for animals following the prophylactic treatment. Photon emission of luminescent P. aeruginosa (PAO1-Lux) in the mouse was measured using the IVIS Lumina XR system (Revvity, USA), including an IVIS charge-coupled device camera coupled to the LivingImage software package (Revvity, USA). Analysis of photons was done under isoflurane inhalation anaesthesia, generating a digital false-colour photon emission image of the mouse where photons were counted using a 3-min acquisition time using the following settings: binning factor 4, Field of View: 10 cm, f1.

Bronchoalveolar lavages, lung and blood sampling, and bacterial load assay

For the preclinical study, endpoints were set at 4, 8 and 16 h after P. aeruginosa infection. Mice were euthanised using a lethal dose of pentobarbital (n = 7–8 per group, per time-point) (Exagon). Blood was collected in EDTA and non-EDTA tubes and stored at −80 °C. Bronchoalveolar lavage (BAL) was recovered by cannulating the trachea and washing sequentially the lungs twice with 1 mL of PBS at room temperature. The lavage fluid was centrifuged at 400g for 10 min at 4 °C, leaving the supernatant of the first lavage to be stored at −80 °C until analysis, and the cell pellet to be resuspended in PBS, counted in a haemocytometer chamber and used for subsequent analysis.

Lungs were perfused with 10 mL of PBS and harvested in GentleMACS C tubes (Miltenyi Biotec, Germany) containing 2 mL of RPMI medium (Invitrogen, France) for flow cytometry or GentleMACS M tubes (Miltenyi Biotec, Germany) containing 1 mL of PBS for assessment of the bacterial load. Lungs from PBS-filled tubes, containing broad-spectrum protease inhibitors (Sigma, France), were processed using a GentleMACS tissue homogeniser (Miltenyi Biotec, Germany). The homogenates were centrifuged at 800g for 10 min at 4 °C, and stored at −20 °C until further analysis. Lungs from RPMI-filled tubes were digested with 25 μg/mL of Liberase (Roche, France) and 100 μg/mL of DNAse I (Sigma, France) for 30 min at 37 °C under agitation and processed using a GentleMACS tissue homogeniser. After washes, red blood cells were eliminated using ACK Lysing Buffer (Thermofisher Scientific, France) according to manufacturer's instructions. Samples were filtered over 100 μm and 40 μm nylon mesh.

Bacterial load in BAL (before centrifugation) and lung homogenates was determined by plating ten-fold serial dilutions on LB agar plates. Plates were incubated at 37 °C, and the CFU were counted after 24 h.

Flow cytometry

Cell pellets from BAL and lung homogenates were resuspended in FACS buffer (PBS, 2% FBS, 2 mM EDTA, and 1X murine Fc-block), counted in a haemocytometer chamber, and stored at 4 °C until staining. Cells were stained in FACS buffer for 20 min at 4 °C with appropriate dilutions of the following Abs: CD45-APC-Cy7 (30-F11, RRID: AB_312980), Ly6G FITC (1A8, RRID: AB_1236488), CD11c PE (N418, RRID: AB_313776), Ly6C PeCy7 (HK1.4, RRID: AB_1732093), CD64 APC (X54-5/7.1, RRID: AB_11219205), CD11b AF700 (M1/70, RRID: AB_493705), NKp46 BV421 (29A1.4, RRID: AB_2563104), CD19 BV605 (6D5, RRID: AB_2563067), CD3 Pe-Cy7 (145-2C11, RRID: AB_312685), CD8 PE/Dazzle594 (53–6.7, RRID: AB_2564027), CD4 AF700 (GK1,5, RRID: AB_493699) from Biolegend and Siglec-F BV421 (E50-2440, RRID: AB_2722581) from Becton Dickinson as well as the LIVE/DEAD Fixable Aqua Dead Cell Staining kit (Thermofisher Scientific, France) and acquired on a CytoFLEX (Beckman–Coulter, USA) flow cytometer. Analyses were performed using Kaluza software (Beckman–Coulter, USA).

Cytokines and complement assessments

Concentration of total BAL proteins was measured using Pierce Rapid Gold BCA Protein assay kit (ThermoScientific, United States) and following the manufacturer's instructions. Secreted mediator's concentrations in BAL was assessed by a multi-array U-plex assay, simultaneously measuring IL-1β, IL-6, mKC (CXCL1), GM-CSF, MIP1, and TNFα, following the manufacturer's instructions. All incubations were performed at room temperature under agitation. Briefly, a 96 well plate was coated with a mix of capture antibodies bound to their specific linker. Samples and calibrators, diluted in assay diluent, were then added and incubated for 2 h. After washing the plates with PBS-0.05% Tween-20, a mix of detection antibodies was added and the plates incubated for an additional hour. A final washing step was performed, followed by the addition of read buffer. The plates were read using the MESO QuickPlex SQ 120MM (MSD, UT, USA). Cytokines and chemokines concentrations were determined using a curve fit model through the Meso Scale Discovery software provided with the instrument.

To assess quantitatively the amount of cleaved and activated C3 fragments (C3b, iC3b, C3c, C3a), and thus the activation of the complement cascade, sera and BAL of mice, sampled in EDTA and kept on ice to avoid ex-vivo activation, were used in a C3b mouse ELISA Kit (Hycult, Uden, The Netherlands), following the manufacturer's instructions. To measure anaphylatoxin C5a concentrations in the BAL and the lungs, we used a C5a mouse ELISA kit (#DY2150, R&D Systems, United States) following the manufacturer's instructions.

Statistical analysis

All in vitro tests were analysed based on at least three independent experiments with multiple technical replicates. Statistical differences between experimental groups were determined using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test (allowing comparison of all groups). Student's t-test was used for comparison between two groups and paired analysis was used when comparing the same donor. Group's size for in vivo mouse studies were determined beforehand using power analysis to ensure sufficient statistical power. Log-rank test was used for survival analysis, and a Kruskal–Wallis test followed by Dunn's post-test (allowing comparison of all pair of groups) helped to compare the effect of each molecule to the irrelevant molecule control. The positive/negative bacterial-positive lung images were analysed with a Chi-square test for direct comparisons between anti-Psl CoMiX and the irrelevant control. Association between two experimental results was analysed by the Pearson's correlation coefficient. All tests were performed with GraphPad Prism version 10 for Windows (GraphPad Software, San Diego, CA, USA). All data are presented as individual data with the bars representing mean ± standard error of the mean (SEM). A p-value inferior to 0.05 was considered statistically significant (NS non significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Role of funders

The funding sources had no role in study design, data collection, data analysis, data interpretation, or writing of the report.

Results

Anti-Psl CoMiX exhibit a broad recognition of P. aeruginosa clinical isolates

We first engineered two distinct CoMiX targeting P. aeruginosa (Fig. 1) using two different scFv recognising either the O11 serotype LPS or the exopolysaccharide Psl (in red) and the C-terminal dimerisation domain of the β-chain of the C4b-binding protein (C4BPβ, in grey) to facilitate dimerisation. CoMiX-Fc (Fig. 1a) employs the Fc domain of an IgG1 antibody (in blue) to activate the classical complement pathway and Fc receptor-mediated cellular responses. Conversely, CoMiX-FHR1 (Fig. 1b) utilises the C-terminal domains (CCP3-5, in green) of FHR1 to engage the alternative pathway. The molecular pattern of both molecules was verified by SYPRO and Western blot (Fig. 1c and d).

Fig. 1.

Schematic representation and characterisation of CoMiX-Fc (a) and CoMiX-FHR1 (b) proteins. The dimerisation scaffold derived from the β-chain of C4BP is used to combine the targeting function of the scFv recognising P. aeruginosa, to either an Fc-region (CoMiX-Fc, a) or the CCP3-5 domains of Factor H-related protein 1 (CoMiX-FHR1, b). Both proteins were purified with protein G affinity columns for CoMiX-Fc and a Nickel column binding to the His-tag at the C-terminal end of CoMiX-FHR1. (c) SYPRO Ruby Staining: 3 μg of CoMiX-Fc and CoMiX-FHR1 proteins were loaded onto 4–15% SDS-PAGE gels and stained with SYPRO Ruby. (d) Western Blotting: Proteins, in non-reduced (NR) and reduced (R) conditions, from the SDS-PAGE gel were transferred to a low-fluorescence PVDF membrane and probed with specific fluorochrome-conjugated antibodies: anti-Fc AF647 for CoMiX-Fc and anti-His AF647 for CoMiX-FHR1. Protein bands from the two techniques were detected using an Amersham Typhoon scanner. The predicted molecular weight of CoMiX-Fc is approximately 122 kDa in normal conditions and 61 kDa when reduced, while CoMiX-FHR1 has a smaller size of around 107 kDa in normal conditions, and 53 kDa when reduced.

We assessed the targeting potential of the scFv fragments by comparing the binding affinities of the anti-Psl CoMiX-Fc and the anti-O11 CoMix-Fc to a clinically relevant panel of 26 bacterial strains (Supplementary Figure S1). These strains, isolated from patients with cystic fibrosis (n = 11) and intubation tubes of patients in intensive care unit (ICU) (n = 15), included multidrug-resistant isolates (Supplementary Table S1) reflecting a critical challenge in the clinic. The anti-O11 scFv bound to only 20% of the strains, suggesting a low prevalence of the O11 serotype in this population of patients. In contrast, the anti-Psl scFv displayed significantly broader targeting. It recognised 45% of CF strains and, remarkably, 100% of the strains isolated from intubation tubes of patients in ICU. Overall, anti-Psl scFv bound to 82% of all strains, and was selected for further therapeutic evaluation.

To confirm the specificity of the anti-Psl CoMix-Fc and CoMiX-FHR1, we tested their binding to one reference strain of P. aeruginosa, PAO1 (Fig. 2a) and to a multidrug-resistant clinical isolate IT 2 isolated from the bronchial aspiration of a patient in ICU (Fig. 2b). Two irrelevant CoMiX, one with each effector function and containing a scFv targeting A. fumigatus³⁶ were also produced and tested in parallel: no binding was observed for either of these irrelevant controls but both anti-Psl CoMiX displayed a significant strong binding to both strains (p < 0.0001, one-way ANOVA and Tukey's post-hoc test). Additionally, dose-dependent binding of molecules (starting concentration: 10 μg/mL to 40 ng/mL, 1:3 dilutions) for each strain was also confirmed (Supplementary Figure S2a–c). To verify their specificity intra-order, we produced a second CoMiX control targeting the Gram- bacteria, M. catarrhalis. Those were unable to bind to either P. aeruginosa strains (Fig. 2a and b). Similarly, neither anti-Psl CoMiX (targeting P. aeruginosa) nor the anti-Aspergillus CoMiX were able to bind to a lysate of M. catarrhalis (Fig. 2c). Altogether, we confirmed the specificity of binding of anti-Psl CoMiX to P. aeruginosa through its scFv moiety. Both anti-Aspergillus CoMiX were carried through the study as controls of the characteristics and mechanisms of action of anti-P. aeruginosa CoMiX.

Fig. 2.

Anti-Psl CoMiX bind specifically to P. aeruginosa, but not to other bacterial strains, with CoMiX-FHR1 competing with FH. The binding of anti-P. aeruginosa CoMiX, irrelevant anti-A. fumigatus CoMiX, and irrelevant anti-M. catarrhalis CoMiX to reference strain PAO1 (a), clinical isolate IT 2 (b), and Gram-bacterial strain M. catarrhalis(c) was analysed by whole-cell ELISA. Immobilised bacteria (1 × 10⁷ CFU/mL) or bacterial lysate (20 μg/mL) were incubated with 10 μg/mL of CoMiX and irrelevant controls. Bound CoMiX was detected using specific antibodies: anti-His for CoMiX-FHR1 and anti-Fc for CoMiX-Fc. CoMiX targeting the second bacterial strain and the fungi were used as negative control. Data are presented as the mean values ± SEM. Results correspond to two-three pooled independent experiments (2–3 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. ∗∗∗∗p < 0.0001. To investigate the competition between FH and CoMiX-FHR1, recombinant FH (20 μg/mL) and increasing concentrations of CoMiX-FHR1 (and CoMiX-Fc as control) were added to PAO1 bacteria. The binding of FH to bacterial cells was detected with the FH-specific monoclonal antibody OX-24 (d). Data are presented as the mean values ± SEM. Results correspond to two-three pooled independent experiments (3 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test: it indicates significant differences for the binding of FH in absence versus presence of CoMiX-FHR1. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

To validate that CoMiX-FHR1 was able to disrupt FH binding to P. aeruginosa, regardless of the presence of both the scaffold and targeting system of the molecule, we conducted a competition ELISA between recombinant FH and CoMiX-FHR1. A reduction of bound FH to the bacteria when in presence of CoMiX-FHR1, but not CoMiX-Fc (around 40% for an initial CoMiX-FHR1 concentration of 40 μg/mL), was observed in a dose-dependent manner (Fig. 2d), indicating that CoMiX-FHR1 competes with FH, and could be used to improve host-immunity against P. aeruginosa.

CoMiX significantly increase C1q (for CoMiX-Fc), C3b-, and C5b9-deposition

We next determined if CoMiX were able to induce the deposition of complement on top of the bacterium. We first verified that the detection was specific to complement molecules, through the utilisation of decomplemented normal human serum (ΔNHS) using whole cell ELISA and fluorescence microscopy imaging (Fig. 3). While only CoMiX-Fc deposited C1q on the bacterium (p < 0.001, one-way ANOVA and Tukey's post-hoc test) (Fig. 3a), both CoMiX-Fc and CoMiX-FHR1 significantly enhanced C3b deposition (p < 0.0001, one-way ANOVA and Tukey's post-hoc test) on both the reference strain PAO1 and the clinical isolate IT 2 as compared to all controls (Fig. 3b). CoMiX-Fc induced a stronger C3b deposition increase on PAO1 (64%, 2.4-fold) compared to CoMiX-FHR1 (40%, 1.6-fold) as well as for the clinical isolate, with CoMiX-Fc (90%, 3.9-fold) displaying a slightly greater effect than CoMiX-FHR1 (51%, 2.1-fold). For MAC assembly (Fig. 3c), CoMiX-Fc increased C5b9 deposition by 120% (2.2-fold) and 92% (1.9-fold) on PAO1 and the clinical isolate, respectively. CoMiX-FHR1 induced a 163% (2.6-fold) increase on PAO1 and a 128% (2.3-fold) increase on the clinical isolate. Similarly, we further confirmed a dose-dependent complement activation at lower CoMiX concentrations as in the binding assay (Supplementary Figure S2d–f). Our observation by confocal and fluorescence microscopy imaging confirmed qualitatively the activation of the complement: we observed on the surface of GFP-expressing PAO1 bacteria a higher red fluorescence signal (C3b and C5b9) in presence of CoMiX indicating an increase deposition of complement when compared to the control (Fig. 3d and e). The absence of co-localisation between green (bacterial cells) and red (complement) indicates that the cells surrounded by the complement are dead or in an apoptotic state, with compromised membranes, and a disappearance of GFP fluorescence, suggesting a complement-dependent killing mechanism for CoMiX. Our data support the specific activation of the classical complement pathway by CoMiX-Fc and of the alternative pathway by CoMiX-FHR1.

Fig. 3.

Anti-Psl CoMiX enhance C1q deposition (for CoMiX-Fc), C3b opsonisation and the formation of membrane attack complex (C5b9/MAC) on P. aeruginosa. Immobilised bacterial cells of the P. aeruginosa reference strain PAO1 and the clinical isolate IT 2 were incubated with 10 μg/mL of CoMiX, before the addition of either 2% (C3b) or 4% (C1q, C5b9/MAC) of normal human serum (NHS) or heat-inactivated human serum (ΔNHS) in GVB++ at 37 °C for 30 min. Complement deposition at the bacterial surface was measured by ELISA, using (a) an anti-human C1q mAb, (b) an anti-human C3/C3b/iC3b mAb, and (c) an anti-human C5b9 mAb, followed by an HRP-conjugated anti-mouse IgG mAb. Data are presented as the mean values ± SEM. Results correspond to three pooled independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. GFP-expressing PAO1 bacteria were incubated with 10% C5-deficient serum (C3b) or normal human serum (C5b9/MAC) at 37 °C for 1 h, in the presence or absence of 10 μg/mL of CoMiX. Bacteria were then incubated with either a goat anti-human C3b or goat anti-human C5b9 antibody, followed by a secondary AF647-conjugated anti-goat mAb. Once stained, bacteria were mounted onto an agarose gel pad, visualised on a confocal Leica Sp8 microscope (C3b), or on a wide field Axio observer microscope (C5b), and analysed by ImageJ to detect C3 cleavage product (C3b, iC3b, and C3c) deposition (d) or C5b9 deposition (e). Two to four fields have been acquired for each conditions. Representative images of the deposition are presented here. Green = GFP-expressing PAO1, red = C3b or C5b9 deposition by anti-goat AF647. Scale bar = 5 μm; 10 μm.

CoMiX enhance the complement-mediated killing of P. aeruginosa and have a synergistic effect with amikacin

We further investigated the direct killing efficacy of CoMiX-Fc and CoMiX-FHR1 (30 μg/mL) against P. aeruginosa strains PAO1 and the multidrug-resistant clinical isolate IT 2 in the presence or absence of human serum, first by CFU counting and then using a luminescent bacterial strain (Fig. 4). In the absence of serum, or with heat inactivated serum (decomplemented serum-ΔNHS), no CoMiX exhibited a killing effect, with bacterial growth similar to the free-molecule control and the irrelevant CoMiX (Fig. 4a and b). However, in presence of normal human serum, we observed, after 2 h, a significant reduction of bacterial growth for both strains when treated with CoMiX molecules compared to the non-treated and their respective irrelevant control groups, resulting in an approximate 40% and 35% decrease in CFU for CoMiX-Fc and CoMiX-FHR1, respectively (Fig. 4a). The degree of killing for the clinical strain IT2 was lower than for the PAO1 strain, illustrating bacteria variability to resist complement killing.³⁷ Consistently with the CFU data, both CoMiX treatment also resulted in around 40% reduction in relative luminescence units (RLU) when using a PAO1-Luciferase strain, indicating decreased activity and bacterial growth (Fig. 4b). To decipher whether mouse serum would be efficient in lysing P. aeruginosa, and thereafter evaluate CoMiX in a mice model of infection, we checked the potency of mouse serum in vitro. While both CoMiX were able to potentiate significantly complement-dependent killing of the reference strain PAO1 (p < 0.001, one-way ANOVA and Tukey's post-hoc test), only CoMiX-Fc was able to have an effect on the growth of the clinical isolate suggesting the difficulty for mouse serum to kill the bacteria P. aeruginosa (Fig. 4c). The absence of killing effect of CoMiX when in presence of heat-inactivated decomplemented mouse serum (MS) confirmed that this effect was indeed complement dependent. Because MAC formation produces pores at the surface of the bacteria, we further examined the effect of CoMiX on the membrane permeability of P. aeruginosa through the release of ethidium homodimer-1. After 2 h treatment of bacteria, the anti-Psl CoMiX, and not irrelevant controls, increased bacterial fluorescence for both reference and clinical strains (p < 0.001, one-way ANOVA and Tukey's post-hoc test), confirming that CoMiX significantly increase the membrane permeability of these strains, affecting the integrity of the bacterial cell wall, through MAC formation upon complement activation (Fig. 4d).

Fig. 4.

CoMiX enhance complement-mediated killing of P. aeruginosa and display synergy with amikacin. (a) Bacterial reference strain PAO1, and clinical isolate IT 2 were incubated without serum (w/o NHS), with 10% normal human serum (NHS) or with 10% decomplemented serum (ΔNHS) in the presence or absence of 30 μg/mL CoMiX or irrelevant control for 2 h at 37 °C. Bacteria were plated and CFU were enumerated to assess bacterial viability. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (2–3 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (b) A mutated PAO1 bacterial strain with a luminescence-based reporter system (PAO1-Lux) was used in the same protocol to monitor in real-time bacterial growth. The luminescence of bacteria (RLU) was measured after 2 h of incubation on a POLARStar Omega microplate reader, as it is known that luminescence of bacteria correlates well with its concentration. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (2–3 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. ∗∗∗∗p < 0.0001. (c) The complement potency of mouse serum (MS) and decomplemented MS (ΔMS) was assessed using the same protocol as in a. Bacteria were plated and CFU were enumerated to assess bacterial viability. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (2–3 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (d) Following the same protocol as in a, the membrane permeabilisation of both PAO1 and clinical isolate IT 2 was quantified after 2 h through ethidium homodimer fluorescence, measured on a GloMax® Discover (Ex = 520 nm, Em = 580–640 nm). Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (3 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. ∗∗p < 0.01; ∗∗∗∗p < 0.0001. (e) To assess the potential of CoMiX in combination with the antibiotic amikacin, both PAO1 and clinical isolate IT 2 were incubated with 10% NHS in the presence or absence of 30 μg/mL of CoMiX or irrelevant control, as well as various sub-MIC concentrations of amikacin (2 μg/mL for PAO1, 10 μg/mL for clinical isolate IT 2) for 2 h at 37 °C. Bacteria were plated, and CFU were enumerated to assess bacterial viability. A synergy was admitted when the synergy coefficient (log(C) − log(SA) − log(SB) + log(SAB) < 0 (where C is CFU without treatment, SA is CFU with amikacin only, SB is CFU with CoMiX only, and SAB is CFU with both)), was negative (S). Data are presented as synergy coefficient calculated from the mean of three independent experiments (3 replicates per experiment).

To evaluate the potential for synergistic interactions, we combined CoMiX-Fc and CoMiX-FHR1 with the aminoglycoside antibiotic amikacin against the P. aeruginosa strain PAO1 and the clinical isolate IT 2 (Fig. 4e). This clinical isolate exhibited inherent resistance to amikacin, requiring a higher MIC (40 μg/mL) compared to the reference strain PAO1 (8 μg/mL). Using sub-MIC concentrations of amikacin, we calculated a synergy coefficient, as described by Chaudhry et al.,³⁸ and observed the strongest synergy between the combinatory treatment at 2 μg/mL of amikacin for PAO1 and 10 μg/mL of amikacin for clinical isolate IT 2. Interestingly, CoMiX-Fc consistently exhibited a more negative synergy coefficient compared to CoMiX-FHR1 for both strains (PAO1: −0.061 vs −0.052; clinical isolate IT 2: −0.027 vs −0.002), suggesting a potentially stronger synergistic effect with amikacin in reducing P. aeruginosa growth. The irrelevant CoMiX control combined with amikacin displayed no synergy (positive coefficient values), indicating the specificity of the antibacterial effect of CoMiX-Fc and CoMiX-FHR1.

CoMiX-Fc enhances phagocytosis of P. aeruginosa by macrophages and more importantly by neutrophil-like cells

Beyond their complement-dependent lysis of bacteria, we investigated the effects of CoMiX on the phagocytosis of P. aeruginosa mediated by the host innate immune cells. First, we assessed as an indirect estimation of phagocytosis the engulfment of bacteria by human PBMCs-derived macrophages through the use of pHrodo-stained bacteria. This staining only release fluorescence in an acidic environment, such as when reaching the endosome and lysosome compartments. Using the Incucyte®, we were able to watch over time the engulfment of bacteria when reaching those compartments. When looking at the percentage of pHrodo positive cells, we only observed an insignificant tendency of higher phagocytic cells number in presence of CoMiX-Fc (Fig. 5a). However, macrophages incubated with both bacteria and the CoMiX-Fc tended to have a higher mean fluorescence intensity (MFI) than the controls (serum alone and irrelevant CoMiX), already after 30 min, suggesting that more bacteria per cells were ingested (Fig. 5b). This trend was confirmed after 1 h of co-culture: CoMiX-Fc treatment significantly increased the intensity of pHrodo in positive macrophages compared to the untreated group, but also to the irrelevant control (p < 0.05, paired one-way ANOVA and Tukey's post-hoc test) (Fig. 5b and c). CoMiX-FHR1 did not display a higher effect than the control suggesting the inability of CoMiX-FHR1 to increase the opsonisation of bacteria for phagocytosis. Fluorescent microscopy was used to confirm and visualise the CoMiX-Fc mediated engulfment of bacteria by PBMCs-derived macrophages. Only a small amount of bacteria was detected in the cells (1–2 bacteria/cells) indicating either a rapid degradation of the bacteria when engulfed by the macrophages, or that CoMiX-Fc preferentially select MAC formation and direct lysis of bacteria as a mechanism of action (Fig. 5d).

Fig. 5.

CoMiX-Fc enhances significantly phagocytosis of P. aeruginosa by PBMCs-derived M1 macrophages and more importantly by Neutrophils-Like Cells, nevertheless, both CoMiX slightly improve NLCs-dependent antimicrobial activity against the bacteria. PMA-activated M1 macrophages, from four different healthy donors, were co-incubated with pHrodo-stained P. aeruginosa (PAO1) at a 12:1 bacteria-to-cell ratio with 10% NHS and in the presence or absence of 15 μg/mL CoMiX-Fc, CoMiX-FHR1 or CoMiX-irrelevant. Engulfment of bacteria was assessed after 30 min (left panels) and 1 h (right panels) by real-time Incucyte® microscope (a–c). The percentage of pHrodo positive cells (a) and the intensity of pHrodo rationalised over the surface of cells and calculated as integrated intensity (b) were recorded. Data are presented as the mean values ± SEM. Results correspond to two independent experiments with macrophages from 4 healthy donors (three technical replicates per donor). Statistical analysis was performed using paired one-way ANOVA (to smooth inter-donor variability), followed by Tukey's post-hoc test: ∗p < 0.05; ∗∗p < 0.01. (c) Representative incucyte images for the phagocytosis induced by serum and CoMiX-Fc over time. (d) Fluorescence microscopy image obtained on a wide field Axio Observer Z1, and treated on ImageJ of phagocytosed P. aeruginosa bacteria by PBMCs-derived M1 macrophages in presence of 10% serum and 15 μg/mL CoMiX-Fc. Red = wheat germ agglutinin Alexa-647, staining carbohydates residues of macrophages membranes; green = CellTrace™ CFSE stained P. aeruginosa bacteria. Scale bar = 25 μm. P. aeruginosa PAO1 strain and PAO1-GFP strain were co-cultured with NLCs at a 10:1 bacteria-to-cell ratio, treated with 2% NHS and CoMiX (15 μg/mL) and incubated for 20 min at 37 °C under agitation (200 rpm). After washes and treatment with gentamicin to eliminate non-phagocytosed bacteria from the cells samples were (1) fixed and read by flow cytometry. (e) Gating of NLCs with FSC and SSC to eliminate cell debris and free bacteria from the analysis (left graph). Representative histogram describing the total population of NLCs, and composed of GFP negative cells and GFP positive cells (right graph). To assess phagocytosis, the percentage of GFP positive cells (f) and the mean fluorescence (g) were acquired. Data are presented as the mean values ± SEM. Results correspond to three pooled experiments (2 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test: ∗∗∗∗p < 0.0001. (h) After washes and treatment with gentamicin, cells were also (2) lysed by Triton X-100, diluted in PBS and plated onto petri dishes. CFUs, corresponding to phagocytosed bacteria were counted in duplicates. Data are presented as the mean values ± SEM. Results correspond to three pooled independent experiments (2 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test: ∗∗∗∗p < 0.0001. To assess NLCs-dependent killing, P. aeruginosa PAO1 strain at a MOI of 2:1 was co-cultured with NLCs, treated with 10% NHS and CoMiX (15 μg/mL) for 1 h, plated on petri dishes to count the final bacterial CFUs in duplicates (i). Conditions which were not co-cultured with NLCs were used as control (100% cell survival). Results are expressed as surviving bacteria compared to bacterial growth under the same conditions in the absence of NLCs. Data are presented as the mean values ± SEM. Results correspond to three pooled independent experiments (2 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test: ∗p < 0.05; ∗∗p < 0.01.

Neutrophils also play a primordial role in the elimination of P. aeruginosa during infection using phagocytosis or by secreting enzymes and cytotoxic peptides.³⁹ We used DMSO-differentiated HL-60 human leukaemic cells as neutrophil like cells (NLCs) known for their strong phagocytic activity.⁴⁰ We co-cultured for 20 min PAO1 expressing constitutively GFP with the activated NLCs and NHS in presence or absence of CoMiX, and measured the cells with internalised fluorescent bacteria through flow cytometry (Fig. 5e). CoMiX-Fc induced a two-fold increase of GFP-positive neutrophils in comparison to its respective controls reaching 60% of phagocytic NLCs (p < 0.0001, one-way ANOVA and Tukey's post-hoc test) (Fig. 5f). This increase of two-fold phagocytic activity was also confirmed when analysing the MFI representing the overall phagocytic activity of neutrophils in comparison to all controls (Fig. 5g). These results were confirmed by plating the bacterial content of lysed NLCs which demonstrates an increase number of bacteria phagocytosed in presence of CoMiX-Fc (p < 0.001, one-way ANOVA and Tukey's post-hoc test) (Fig. 5h). Interestingly, and as observed for macrophages, CoMiX-FHR1 was not able to increase the phagocytic activity of NLCs (Fig. 5f–h). To investigate whether the enhanced phagocytosis lead to the killing of bacteria, we co-incubated NLCs with P. aeruginosa in presence of CoMiX and NHS for 1 h. After lysis, the total content of cells was plated: CoMiX-Fc slightly reduced bacterial growth as compared to the incubation of the bacteria with only NLCs (to 59.5% vs 74%, respectively) (Fig. 5i). Interestingly, CoMiX-FHR1, unable to increase NLCs phagocytic activity also reduced bacterial growth (to 59.6%) compared to its respective controls (74% and 69.7%), albeit slightly less than CoMiX-Fc (Fig. 5i). Taken together our results indicate that while CoMiX-Fc is able to increase the phagocytic activity of innate immune cells through the involvement of its Fc-fragment, both CoMiX can induce NLC-dependent killing of the bacteria via the activation of other neutrophilic defence mechanisms.

CoMiX modulate bronchial epithelial cell cytotoxicity and inflammation during P. aeruginosa infection

We next investigated the anti-bacterial adhesion potential and protective function of CoMiX on host cells. In presence of both CoMiX-Fc and CoMiX-FHR1 (and serum), we observed a reduction up to 40% of adherence of the bacteria after 2 h (Supplementary Figure S3a), suggesting that CoMiX may inhibit initial attachment of P. aeruginosa on abiotic surface, such as medical devices. After confirming no intrinsic cytotoxicity of CoMiX on human bronchial epithelial BEAS-2B by LDH release (Supplementary Figure S3b), we investigated CoMiX activity on infected epithelial cells pathologically affected by P. aeruginosa infection. Interestingly, while CoMiX had no effect on the adherence of P. aeruginosa to the epithelial cell line (Supplementary Figure S3c), CoMiX-Fc administration decreased substantially epithelial cell cytotoxicity caused by the bacteria (LDH release reduced by 47%) (Supplementary Figure S3d). These data imply that only CoMiX-Fc is able to mitigate PAO1's detrimental effects on BEAS-2B cells through Fc receptors engagement and not CoMiX-FHR1.

CoMiX protect mice against an acute lung infection by facilitating the clearance of P. aeruginosa

To initially assess the efficacy of our molecules, we employed a prophylactic mouse model as described in Fig. 6a. Eight-week-old female C57BL/6J mice were intranasal challenged with a lethal dose of bioluminescent PAO1 (PAO1-lux) to establish acute lung infection resulting in more than 85% of mice death between 2 and 3 days post-infection (Supplementary Figure S4). The mice were divided into three treatment groups: CoMiX-Fc, CoMiX-FHR1, or an irrelevant CoMiX control (anti-Aspergillus CoMiX-Fc). Treatments, consisting of 100 μg/mice of molecules diluted in PBS, were administered 3 h prior to the infection (T_-3), followed by a boost administered 3 h post-infection (p.i.) (T₃) (Fig. 6a). Survival was monitored daily throughout the experimental period (Fig. 6b). Body weight was also measured daily to evaluate the impact of the treatments on host health (Fig. 6c).

Fig. 6.

Prophylactic treatment with anti-Psl CoMiX protects mice from lethal P. aeruginosa lung infection. (a) Eight-week-old female C57BL/6J mice were infected intranasal (i.n.) with a lethal dose (3 × 10⁶ CFU) of luciferase-expressing PAO1 strain. Mice were treated with anti-Psl CoMiX-Fc, anti-Psl CoMiX-FHR1 or an irrelevant CoMiX control 3 h before and after intranasal infection with luciferase-positive P. aeruginosa PAO1. Survival (b) and body weight (c) were assessed daily for six days following infection. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (n = 8–16 mice per group). Statistical analysis was performed using the log-rank test (Mantel–Cox). ∗∗∗∗p < 0.0001. (d, e) Lung infection was assessed by visualising P. aeruginosa-associated luminescence emission in live animals at 24 h and 48 h p.i. One representative mouse image for the time-point 24 h and for each group is displayed. Data are presented as a percentage of mice with negative versus positive signal for bacteria. The data were analysed using a Chi-square test for direct comparisons between anti-Psl CoMiX and the irrelevant control (∗p < 0.05; ∗∗p < 0.01).

Administration of CoMiX significantly enhanced the survival of mice compared to control groups (p < 0.0001, log-rank test Mantel–Cox) (Fig. 6b). While no control mice (Irr. CoMiX group) survived beyond day 2 p.i., all anti-Psl CoMiX-treated mice survived until this point. By day 6, 62.5% of mice treated with CoMiX-Fc and 87.5% of mice treated with CoMiX-FHR1 were alive and exhibited weight gain from day 2 onwards (Fig. 6c). Bioluminescence imaging of the luciferase-expressing P. aeruginosa PAO1 strain within the lungs and nasal cavity, using the IVIS Lumina system, was performed 24 and 48 h p.i. to assess bacterial burden (Fig. 6d and e). 24 h p.i. only 43.75% of mice in the CoMiX-Fc treatment group and 27.27% of mice in the CoMiX-FHR1 treatment group exhibited lung or nasal cavity luciferase activity, indicating reduced bacterial colonisation compared to the control group, where all mice were luciferase-positive (p < 0.01, Chi-square test).

We next assessed CoMiX potency in a therapeutic model (Fig. 7) where molecules were administered 1 h p.i. (T₁). All groups received a single intranasal dose of 100 μg of their respective CoMiX molecule (Fig. 7a). Treatment with both anti-Psl CoMiX significantly improved survival rates compared to the control group (Fig. 7b). 48 h p.i., 50% of control mice treated with the irrelevant CoMiX succumbed to infection. In contrast, all mice treated with CoMiX-Fc or CoMiX-FHR1 survived up to this point, demonstrating the protective efficacy of CoMiX. By the study endpoint (day 7) only 31% of mice were still alive in the irrelevant control group (potentially due to the fluctuation in the bacterial load reaching the lungs), while all mice were alive in the CoMiX-Fc treatment group and 93% survived in the CoMiX-FHR1 group. Body-weight changes were similar between all groups overtime, even if we noted that the group treated with the irrelevant CoMiX had a slightly slower recovery (Fig. 7c). Compared to the irrelevant control group, both CoMiX treatments resulted in a significant reduction in bacterial burden (Fig. 7d and e). 24 h p.i., only 31.25% and 50% of mice treated with CoMiX-Fc and CoMiX-FHR1, respectively, displayed detectable bioluminescence, indicating a substantial decrease in bacterial colonisation compared to the control group, where 93% of mice were luciferase-positive (p < 0.01, Chi-square test). 48 h p.i., the percentage of luciferase-positive mice remained lower in the CoMiX-treated groups (25% for CoMiX-Fc, 43.75% for CoMiX-FHR1) compared to the control group (66.67%) (p < 0.01, Chi-square test).

Fig. 7.

CoMiX protect mice from acute lung infection of P. aeruginosa. (a) Eight-week-old female C57BL/6J mice were infected intranasal (i.n.) with a lethal dose (3 × 10⁶ CFU) of luciferase-expressing PAO1 strain. Mice were treated 1 h later with 100 μg of CoMiX-Fc, CoMiX-FHR1 or an irrelevant CoMiX via i.n. administration. Survival (b) and body weight (c) were monitored for 7 days after infection. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (n = 16 mice per group). Statistical analysis was performed using the log-rank test (Mantel–Cox). ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (d, e) Lung infection was assessed by visualising P. aeruginosa-associated luminescence emission in live animals at 24 h and 48 h p.i. One representative mouse image for the time-point 24 h and for each group is displayed. Data are presented as a percentage of mice with negative versus positive signal for bacteria. The data were analysed using a Chi-square test for direct comparisons between anti-Psl CoMiX and the irrelevant control (∗∗p < 0.01; ∗∗∗p < 0.001).

CoMiX enhance neutralisation of bacteria in vivo through complement activation and killing

We investigated the mechanisms underlying the protective effect promoted by CoMiX against P. aeruginosa acute infection in the therapeutic animal protocol. First, when looking at representative lungs from each group at 16 h p.i., macroscopic differences could be observed between the groups. Mice treated with the irrelevant CoMiX had fully haemorrhagic lungs, a typical presentation of severe lung injury due to P. aeruginosa infection⁴¹ whereas mice treated with either CoMiX-Fc or CoMiX-FHR1 had lungs with a lesser injury, a reduced stage of haemorrhage, and clear visible healthy areas (Fig. 8a). In agreement with the bacteria luminescence inside the lungs, we confirmed that mice treated with the irrelevant control presented the higher bacterial load in both the bronchoalveolar lavage (BAL) (6 log CFU/mL) and the lungs (7 log CFU/mL) from 4 h, only accentuated at 16 h (Fig. 8b and c). In contrast, the therapeutic administration of both CoMiX-Fc and CoMiX-FHR1 decreased significantly this bacterial load, with a reduction of one log of the CFU in the BAL and the lungs (p < 0.01, Kruskal–Wallis test and Dunn's post-test). The rapid and strong decrease in the number of CFU (already at 4 h p.i.) suggests, at least in part, a direct killing effect of CoMiX as observed in vitro. We detected at 16 h p.i. an increase of total proteins in the BAL in both CoMiX-Fc and CoMiX-FHR1-treated groups (Fig. 8d), which might originate, in part, from the higher concentration of cleaved C3 fragments observed in the blood and BAL as compared to the mice that received the irrelevant control (p < 0.01, Kruskal–Wallis test and Dunn's post-test) (Fig. 8e and f). This increased complement activation was maintained locally in the BAL even at 16 p.i., but was already downregulated at the same time-point in the blood suggesting a local and specific activation of CoMiX. Higher concentrations of cleaved C3 molecules were correlated with a decreased bacterial load in the BAL for the anti-Psl CoMiX-treated mice (p < 0.001, Pearson correlation test) (Fig. 8g). Overall, our results suggest that the protection of mice against an acute P. aeruginosa infection, mediated by CoMiX, depends at least partly on their complement-dependent killing activity, and a fast-disappearing effect causing less damage to the lungs.

Fig. 8.

Therapeutic administration of CoMiX in vivo results in enhanced bacterial clearance through local and systemic complement activation. Mice were infected/treated as described in Fig. 7a, sacrificed at 4 h, 8 h and 16 h p.i., and lungs, BAL and blood were collected for analysis. (a) Lungs were first perfused for a visual assessment of inflammation. Bacterial load in BAL (b) and lungs (c) were determined after serial dilution and plating on Petri dishes. Total protein was measured by BCA in the BAL (d). To assess the activation of the complement cascade, the concentration of activated fragments of the mouse complement protein C3 was determined by ELISA, systemically in the serum (e) and locally in the BAL (f). Concentration of local C3 activated fragments was correlated with the BAL bacterial load (g). The concentration of mouse complement protein C5a was determined by ELISA locally in the BAL (h) and the lungs (i). All data are shown as individual values and quoted as the mean values ± SEM and the results correspond to one experiment per time-point (n = 7–8 mice per group). Unless otherwise stated, all statistical analyses were performed using Kruskal–Wallis test followed by a Dunn's post-test for comparisons between the groups, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Correlation between experimental variable were statistically analysed, and interpreted in regards to their Pearson correlation coefficient (ρ) and p-value.

We also observed a transient increase of the complement anaphylatoxin C5a locally in the lungs (4 h p.i.) and released in the BAL (16 h p.i.) for both CoMiX-Fc and CoMiX-FHR1 compared to the irrelevant control (p < 0.01, Kruskal–Wallis test and Dunn's post-test) (Fig. 8h and i). Since C5a is known to activate immune cells, especially neutrophils to facilitate the clearance of the infection, we further analysed the cellular immune response stimulated by CoMiX.

CoMiX lung protection against P. aeruginosa is associated with transient neutrophil activation and improved control of lung inflammation

First, we measured the total number of cells and specific immune populations of leukocytes (CD45+) in the airways by flow cytometry (Fig. 9a). We observed that the mice receiving CoMiX-Fc and CoMiX-FHR1 had significantly fewer cells in their BAL than the mice treated with the irrelevant control, observed at 4 (tendency), 8 and 16 h p.i. (p < 0.01, Kruskal–Wallis test and Dunn's post-test). In a similar way, CoMiX-Fc and CoMiX-FHR1 mice groups had fewer alveolar macrophages (Fig. 9b) and eosinophils (Fig. 9c) as compared to the irrelevant group (p < 0.05, p < 0.01, Kruskal–Wallis test and Dunn's post-test).

Fig. 9.

CoMiX in vivo protection is associated with transient neutrophil upregulation and improved control of lung inflammation. Mice were infected/treated as described in Fig. 7a, sacrificed at 4 h, 8 h and 16 h p.i., and lungs and BAL were collected for analysis. As a reflection of lung inflammation, immune cell populations were assessed by flow cytometry. The absolute number of (a) leukocytes (CD45⁺ cells), (b) alveolar macrophages (CD45⁺ SiglecF⁺ CD11b⁻), (c) eosinophils (CD45⁺ SiglecF⁺ CD11c⁺), and (d) neutrophils (CD45⁺ SiglecF⁻ Ly6G⁺ CD11b⁺) were quantified in the BAL. (e) CD11b expression on neutrophils was measured to assess their activation. (f) Concentration of local anaphylatoxin C5a was correlated with neutrophil number in the BAL at 8 h p.i. The production of the cytokines and chemokines (g) IL-6, (h) TNF-α, and (i) CXCL-1 were quantified in the BAL by a MSD assay. All data are shown as individual values and quoted as the mean values ± SEM and the results correspond to one experiment per time-point (n = 7–8 mice per group). Unless otherwise stated, all statistical analyses were performed using Kruskal–Wallis test followed by a Dunn's post-test for comparisons between the groups, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. IL = interleukin; TNF = tumour necrosis factor. Correlation between experimental variable were statistically analysed, and interpreted in regards to their Pearson correlation coefficient (ρ) and p-value.

Interestingly, when looking at the neutrophils population, primarily involved in anti-P. aeruginosa immune defences, two phases were apparent. In the first phase, at 8 h p.i., within the acute phase of the infection, neutrophils were found in high number in the protected mice (CoMiX-Fc and CoMiX-FHR1) compared to the mice treated with the irrelevant control (Fig. 9d). Furthermore, they showed an increased neutrophil surface CD11b expression, a known marker involved in the adhesion and migration of leukocytes during the process of diapedesis during infection, suggesting an active recruitment from the circulation⁴² (Fig. 9e). This recruitment of neutrophils in the BAL was positively correlated with the higher production of the anaphylatoxin C5a, shown in Fig. 8, induced by the CoMiX-Fc and CoMiX-FHR1 treatment (Fig. 9f) (p = 0.0002, Pearson correlation test). In a second phase, at 16 h p.i., in parallel of the decrease of the bacterial load in the lungs, neutrophils were fewer in the BAL (with no more different production of C5a between the groups) even if they were still more activated than the control group (p < 0.05 for CoMiX-Fc vs irrelevant and p = 0.0647 for CoMiX-FHR1 vs irrelevant, Kruskal–Wallis test and Dunn's post-test). Those results suggest that CoMiX participate in the early defence and clearing of the infection via at least two modes of action: direct complement-dependent killing of bacteria, and indirect and transient induction of neutrophils migration and activation. Once the infection is under control, limited neutrophils proliferation in the lungs might occur to avoid an over excessive local inflammatory response, often associated with poor outcomes during P. aeruginosa acute lung infection. Remarkably, while most of innate immunity seems to be modulated by CoMiX therapy, no changes were observed in the number of adaptive cells in lungs, for B lymphocytes (Supplementary Figure S5b), T Lymphocytes (Supplementary Figure S5c and d) nor in the number of NK cells, found at the frontiers between innate and adaptive immunity (Supplementary Figure S5e).

Finally, we investigated the release of pro-inflammatory cytokines and chemokines associated with innate immunity in cell-free BAL. At 4 h p.i., animals that received either CoMiX-Fc or CoMiX-FHR1, had significantly lower levels, compared to mice that received the irrelevant control (p < 0.05 and p < 0.01, Kruskal–Wallis test and Dunn's post-test), of the cytokines IL-6, TNF-α (Fig. 9g and h), and to a lesser extent IL-1β and GM-CSF (Supplementary Figure S5f and g). We observed the same pattern of expression for the chemokines KC/CXCL1 (Fig. 9i) and MIP1α (Supplementary Figure S5h). At a later time-point, 16 h p.i., the levels of pro-inflammatory mediators were similar for all groups of mice in the BAL (Fig. 9g–i). This suggests that the rapid complement-dependent killing of P. aeruginosa infection was enough to lessen the expression of pro-inflammatory mediators, resulting in early resolution of the acute local inflammation.

Overall, protection of the mice induced by the therapeutic administration of CoMiX may be attributable to both the direct and fast complement-dependent killing of the bacteria and the indirect rapid and transient activation of neutrophils, resolving rapidly the acute infection and thus limiting global inflammation of the lungs and its deleterious effects.

Discussion

In the last decade, the emergence of multidrug-resistance in P. aeruginosa bacterium and the increased prevalence of nosocomial infections has made the treatment of P. aeruginosa highly challenging. Those infections can range from severe acute pneumonia to persistent chronic infections, such as those observed in patients with CF. The decline in antibiotic efficacy, and sometimes their complete failure against multidrug-resistant strains, highlights the urgent need for novel therapeutic strategies.^3,43 Antibody-based therapies, which have shown success in treating HIV, malaria or COVID-19 infections, are now demonstrating great promise against a broader range of infectious diseases.⁴⁴

In this work, we engineered and evaluated two anti-bacterial CoMiX, an innovative class of complement-activating immunotherapeutic complexes, intended for fast and directed-killing of multidrug-resistant P. aeruginosa. Unlike current complement-targeting therapies, which aim to inhibit complement activity to mitigate its deleterious effects in diseases such as the anti-neutrophilic cytoplasmic antibodies–associated vasculitis (AAV),⁴⁵ we, and others⁴⁶ seek to enhance complement activity selectively. This strategy, inspired by findings in cancer cell models, involves blocking complement inhibitors on target cells to prevent undesired inflammation while promoting cell death.³¹

Both CoMiX constructs utilise the C-terminal dimerisation domain of the C4BP, a scaffold long recognised as a valuable tool for protein engineering and synthetic biology.47, 48, 49 Our group pioneered its application in developing therapeutic agents against HER2-positive breast cancer³¹ and HIV infection.³² CoMiX-Fc was designed to activate the classical pathway through Fc receptor engagement on immune cells and binding to C1q molecule. In contrast, CoMiX-FHR1 enhances the alternative pathway by a novel mechanism, recently described,¹⁴ where FHR1 disrupts FH-mediated regulation of C3b degradation and C3 convertase decay, ultimately leading to increased C3 deposition on P. aeruginosa. These findings align with previous reports demonstrating FHR1's role as a competitive inhibitor of FH in various contexts, including interactions with Group A Streptococcus,⁵⁰ Plasmodium falciparum,²⁹ and DNA and dead cells.⁵¹ Our data bring compelling evidence that FHR1 promotes opsonisation by limiting FH-dependent complement inhibition, offering a promising avenue for addressing multidrug-resistant P. aeruginosa infections.

The main challenge in developing targeted therapies against infectious diseases is the selection of the appropriate target. The expression of a specific target at the surface of a pathogen can differ widely between strains, disease contexts, and even infection sites. For instance, Enterobacteraciae preferentially present fimbriae in the urinary tract but express capsular polysaccharides in the blood.⁵² Therefore, past efforts to target P. aeruginosa with antibodies have shown initial promise in preclinical and early clinical studies but failed to demonstrate consistent efficacy in large-scale trials. A prime example is KB001-A, an antibody targeting the Type III Secretion System (T3SS) protein PcrV. While it reduced pneumonia incidence in patients mechanically ventilated colonised with P. aeruginosa, it was ineffective in patients with CF, probably due to reduced expression of T3SS in the isolated bacterial strains.^53,54 Here, we initially compared the binding of two scFvs (anti-O11 and anti-Psl) in CoMiX to a diverse panel of clinical isolates. The Psl-targeting scFv demonstrated broad recognition (82%), including multidrug-resistant strains with particularly high efficacy against non-CF isolates (96%). In contrast, the anti-O11 scFv showed limited efficacy, reflecting the lower prevalence (20%) of the O11 serotype, which was nevertheless tested in clinical trials under the name of Panobacumab.⁵⁵ Ultimately, Psl is an attractive target due to its ability to hinder P. aeruginosa infections and to prevent bacterial attachment and biofilm formation.^56,57

CoMiX-Fc (activating the classical complement pathway), and not CoMiX-FHR1 (triggering the alternate complement pathway), facilitated the deposition of complement component C1q on the surface of P. aeruginosa.¹⁴ Nevertheless, activation of CoMiX respective complement pathways converged to the formation of the C3 convertases (C4bC2b vs C3bBb), leading to the enhance deposition of C3b on the surface of the bacterium and the increased accumulation of C5b9, thus contributing to enhanced opsonisation and bacterial lysis by MAC formation. Our in vitro data suggest that complement-dependent bacterial lysis, through cell wall permeabilisation, occurs quickly after treatment but diminishes rapidly after 6 h with regain growth of the bacteria. This effect is entirely dependent on the complement component of the serum: lack of killing activity in absence of serum and in presence of decomplemented serum. Thus, our results indicate a bacteriostatic activity of CoMiX, likely due to the depletion of both complement components and convertase enzymes, supplied only at the beginning of the infection, caused by their progressive consumption. As reported by others, infection resolution can deplete substantial amounts of complement, leading to bacterial resurgence in the absence of “replenishment”.⁵⁸ Fortunately, this limitation can be overcome in vivo where complement pools are continuously replenished. Interestingly, while mouse serum is known to have limited cytotoxicity and killing activity, even against serum-sensitive bacterial strains,^59,60 CoMiX can increase significantly this activity, although never achieving the effect of normal human serum. The same increase of activity was also observed in 2019 when Cruz et al. developed a novel therapeutic antibody targeting C1q for efficient lysis of Staphylococcus aureus. The results of their in vitro experiments with human serum were transferable to a mice model of infection, with a similar protective effect as ours.⁶¹

CoMiX displayed a synergistic activity with the aminoglycoside antibiotic amikacin against both reference and clinical P. aeruginosa isolates. Since CoMiX and antibiotics act through different mechanisms, this synergistic interaction could enhance their respective effects while enabling lower therapeutic doses. Furthermore, as their pharmacokinetics would likely differ, their ratio and concentrations in blood and at the infection sites should vary overtime, reducing selective pressure on the bacteria and potentially slowing resistance emergence.⁶² Testing combinations of CoMiX with other antipseudomonal antibiotics (e.g., broad-spectrum penicillins, cephalosporins, etc …) could offer new perspectives for improved therapeutic efficacy and resistance management.⁶³

In a cytotoxicity assay, we observed no significant detrimental effects of CoMiX on human bronchial epithelial cells, suggesting a favourable safety profile. Notably, CoMiX-Fc but not CoMiX-FHR1 reduced cytotoxicity of BEAS-2B cells challenged by P. aeruginosa infection. This difference could be explained to the engagement of FcR and FcRn, the latter being expressed in epithelial cells and known to contribute to local immune protection against bacterial and viral infections through its transport functions across epithelial cells.⁶⁴ The activation of FcRn by CoMiX-Fc may limit tissue damages caused by bacterial infection while the FcR activation could induce a higher secretion of anti-microbial peptides, as shown in mouse models of infection with Citrobacter rodentium, Clostridioides difficile, and Helicobacter species.⁶⁵

Beyond their lysis activity, CoMiX could possibly enhance the phagocytic activity of immune cells, through an increase of complement opsonisation of bacteria. Enhanced opsonisation could happen via the activation of phagocytic cells by the liaison between CoMiX-Fc and FcγR present at the surface of the macrophages and neutrophils, or through the well described axis integrin CD11b/CD18 (=complement receptor 3 (CR3))-iC3b for CoMiX-FHR1.^66,67 In our in vitro 2D co-cultures, we observed that anti-Psl CoMiX-Fc, and not CoMiX-FHR1, was able to enhance the engulfment of bacteria and the phagocytic activity of human PBMCs-derived macrophages and NLCs, probably through the engagement of the Fc fragment to the FcγR present at the surface of the cells. However, CoMiX-FHR1 showed similar efficiency than CoMiX-Fc for the binding, the deposition of the complement or the in vivo protection. Remarkably, while only CoMiX-Fc was able to enhance phagocytosis of NLCs, both CoMiX were able to slightly increase the overall anti-microbial activity of the cells. Apart from phagocytosis, neutrophils secrete extracellular enzymes, such as serine proteases, able to eliminate pathogens (e.g., cleavage of P. aeruginosa bacterial membrane OprF), or generate reactive oxygen species (ROS) able to kill the bacteria.³⁹ While differentiated HL-60 are a good model to study phagocytosis (similar to primary neutrophils) they do not harbour the entire set of cytotoxic granules, and have a reduced capacity to generate ROS.⁶⁸ This could explain the weak NLCs-dependent killing observed with HL-60 cells. Overall, our results indicate that both CoMiX might activate alternate killing mechanisms of neutrophils, which will need to be further investigated using primary neutrophils and/or neutropenic mice.

In vivo studies conducted in a murine model of acute P. aeruginosa lung infection demonstrated the therapeutic potential of CoMiX. Both prophylactic and curative treatments significantly enhanced survival rates and reduced bacterial burden compared to the irrelevant control groups. While CoMiX-FHR1 exhibited slightly superior efficacy in the prophylactic model, the opposite trend was observed in the curative model probably reflecting the variability inherent to the animal model. Variations in infection severity between the control groups in the prophylactic and curative treatments could be attributed to several factors such as true bacterial load in the lung, host susceptibility, and experimental conditions (e.g., additional anaesthesia for the prophylactic treatment).⁶⁹ In the curative protocol, local bacterial burden reduction was correlated with higher concentrations of C3b, confirming the critical role of the complement system in the host defence against pathogens. Complement activation was detected rapidly at the site of infection in the BAL as well as in the blood. Furthermore, the control anti-Asp CoMiX-Fc, also with an Fc fragment, show no sign of complement activation, increased phagocytosis and protection of the mice, highlighting the role of targeted-complement activation when bound to the bacteria as compared to the activation of FcγR. Importantly, no severe side effects were observed during the 7 days of CoMiX treatment, confirming the safety of targeted complement activation over this timeframe. The reduction of complement systemically to near normal levels after 16 h implied a transient activation, effectively avoiding chronic complement activation, and thus detrimental side-effects.

Importantly, a shared mechanism of anti-P. aeruginosa immunity, and general lung infection, involves improved cell activation and rapid neutrophil recruitment to the site of the infection to phagocytose and kill the bacteria.⁷⁰ While at the early time-point, we observed no differences between the groups, at 8 h p.i., CoMiX administration induced an increased recruitment of neutrophils, with a higher expression of CD11b at their surface labelling them as activated. This recruitment is transient and was reduced at 16 h p.i. in the BAL. Upregulation of CR3, involved in the adhesion to endothelial cells, indicates the readiness of immune cells to engage in the processes of migration (e.g., diapedesis), and to recognise the complement component iC3b, leading to phagocytosis of opsonised particles.⁶⁷ While those processes involve CR3 integrin as a whole, CD11b subunit is responsible for the interaction with the complement molecules and is sufficient to support the firm adhesion and spreading of neutrophils.⁷¹ During infection CD11b high neutrophils are the cells responsible for the uptake of Escherichia coli bacteria.⁷² While in vitro the direct complement-dependent killing only tally for a significant but limited reduction of bacterial growth, in vivo this effect should be completed with a higher recruitment of neutrophils. Complement activation by CoMiX led to increased production of the anaphylatoxin C5a, associated to neutrophils migration and activation. While this recruitment is needed for the initial control of the infection, an excessive recruitment of immune cells to the lungs can lead to local inflammation associated with tissue injury, chronic immune cells activation and a poor outcome.^73,74 The action of the complement is tightly regulated: its down-regulation might have further limited costly neutrophils recruitment, thereby preventing excessive local inflammation and contributing to the rapid resolution of the infection, and favoured mouse survival. Interestingly, when looking at their absolute numbers, all other cell populations are decreased (e.g., alveolar macrophages) or non-implicated (e.g., NK cells, lymphocytes) aligning with the observed decrease in pro-inflammatory mediators in the BAL of mice treated with CoMiX.

The overall efficacy of CoMiX-FHR1 and CoMiX-Fc was comparable, highlighting similar promising therapeutic potential. CoMiX-FHR1, and its engagement to the alternative pathway could be an additional advantage as this pathway preferentially acts as a protective mechanism during critical illness, more robust compared to the sole reliance on the classical pathway.⁷⁵ We however acknowledge that we focused our in vivo study on the efficacy of the complement activation by CoMiX. Based on the potential of both CoMiX to recruit and activate other immune cells, further investigation will be required to understand how complement-activated neutrophils kill the bacterium and the different mechanisms stimulated by CoMiX-Fc as compared to CoMiX-FHR1. It will be notably interesting to look into CD63, associated with the release of antimicrobial proteins, and the intracellular expression of myeloperoxidase, involved in the oxidative burst, a mechanism of neutrophil-mediated pathogen killing. To complete the overview of the mechanisms of action of CoMiX, it would be also meaningful to look further into the activation state of these cells at a longer time post-infection (e.g., ADCC by NK cells or antibody secretion by B lymphocytes).

Besides the development of antibiotic resistance, one of the main challenges with antibiotics is their broad-spectrum activity, which can be lethal for beneficial bacteria, as those found in the microbiota.⁷⁶ The use of targeted therapies (e.g., therapeutic antibodies) has already shown its potential in protecting the microbiota. We showed here that CoMiX are specific to P. aeruginosa by testing the binding and the killing of CoMiX to irrelevant bacterial and fungi strains suggesting that CoMiX should be safer for the microbiota than antibiotics.

Beyond their practicality in treating acute infections, CoMiX also hold promise against P. aeruginosa, a persistent bacterium forming biofilms and causing chronic infections. Biofilms develop as 3D structures within a protective microenvironment making them difficult to target with conventional therapies. Targeting the exopolysaccharide Psl, a conserved element of the extracellular polymeric substances, has the potential to disrupt biofilm formation.⁷⁷ We showed that CoMiX reduce the adherence of bacteria to abiotic surface, an initial stage of biofilm formation on prosthesis. However, once biofilms are fully formed, access to complement and surface proteins, becomes limited, and may be challenging for CoMiX to act.⁷⁸ Combining CoMiX with antibiotics or other biofilm inhibitors could help overcome the protective barriers formed by biofilms and restore complement functionality.⁷⁹

Ultimately, the biggest strength of CoMiX lies in their molecular design, which allows for the customisation of both targeting and effector functions. This versatility enables the development of highly specific therapeutic agents for a wide range of diseases. Beyond airways infections, CoMiX-based antibacterial therapies may also be useful in protecting immuno-suppressed or immuno-deficient individuals, reducing the toxicity associated with high antibiotic doses, and mitigating healthcare-associated infection.

Long-term effect of CoMiX on the immune system still need to be addressed, and could offer new insights on the use of CoMiX as prophylaxis agents (e.g., vaccines). Other antibody-based constructs have proven effective in prevention, particularly as a measure before surgery to reduce the risk of infection in patients immunocompromised or in ICUs to prevent healthcare-associated infections.⁸⁰ Since many licenced vaccines elicit the production of neutralising antibodies, and complement can enhance their neutralising effects, it seems logical to explore complement induction as a new tool to improve vaccination efficacy,⁸¹ especially since complement activation can also regulate and enhance the adaptive immune system, playing a critical role in generating long-term immunity.^82,83 Recombinant antibody therapeutics hold significant potential for addressing a wide range of infectious diseases and can be cost-effective when targeted toward high-risk individuals.⁸⁴ Although the high cost of antibody-based drugs remains a limitation, advances in production methods and manufacturing are expected to drive those costs down.⁸⁵

In conclusion, this proof-of-concept demonstrates the efficient, directed killing of P. aeruginosa by the complement, through complement-dependent lysis of the bacteria and indirect transient neutrophils recruitment. This study lays a strong foundation for the clinical development of CoMiX as a therapeutic strategy against bacterial infections. Future studies should aim to raise compelling evidence of CoMiX's mode of action long-term potential, including pharmacokinetics studies, analysis of off-target effects, and evaluation of their activity against chronic infections. These efforts will be instrumental in enhancing CoMiX's safety profile and optimising their efficacy in treating P. aeruginosa infections in humans.

Contributors

Conceptualisation, A.P, X.D, C.S.D, G.D, M.S.T; methodology, A.P, B.Brandus, G.I, C.R, J.Y.S, D.F, B.Briard, A.C, M.S.T, G.D, Y.M, P.R, L.R, J.Z, X.D, C.S.D; validation, A.P, B.Brandus, G.I, J.Y.S, C.S.D, G.D; analysis, A.P, B.Brandus, G.I, C.R, J.Y.S, D.F, B.Briard, A.C, M.S.T, G.D, Y.M, P.R, L.R, J.Z, X.D, C.S.D; data curation and verification, A.P, B.Brandus, C.S.D; writing-original draft preparation, A.P, B.Brandus, C.S.D; writing-review and editing, A.P, B.Brandus, G.I, C.R, J.Y.S, D.F, B.Briard, M.S.T, G.D, Y.M, P.R, L.R, J.Z, X.D, C.S.D; resources, C.S.D, G.I, J.Y.S, A.P, J.Z, X.D, M.S.T; funding acquisition, C.S.D, G.D, M.S.T. All authors have read and approved the final version of the manuscript.

Data sharing statement

The raw data generated in this study and supporting the conclusions of this article are available upon request from the corresponding author.

Declaration of interests

A patent application has been filed for CoMiX (LIH-023-PCT WO2023281120) by the inventors (B.Brandus, J.Z, X.D, C.S.D). The authors have declared that no other conflict of interest exists.