Patrolling Alveolar Macrophages Conceal Bacteria from the Immune System to Maintain Homeostasis.

During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing in vivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P. aeruginosa and S. aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.

In vivo mRNA expression of a multi-mechanistic mAb combination protects against Staphylococcus aureus infection.

Single monoclonal antibodies (mAbs) can be expressed in vivo through gene delivery of their mRNA formulated with lipid nanoparticles (LNPs). However, delivery of a mAb combination could be challenging due to the risk of heavy and light variable chain mispairing. We evaluated the pharmacokinetics of a three mAb combination against Staphylococcus aureus first in single chain variable fragment scFv-Fc and then in immunoglobulin G 1 (IgG1) format in mice. Intravenous delivery of each mRNA/LNP or the trio (1 mg/kg each) induced functional antibody expression after 24 h (10-100 µg/mL) with 64%-78% cognate-chain paired IgG expression after 3 days, and an absence of non-cognate chain pairing for scFv-Fc. We did not observe reduced neutralizing activity for each mAb compared with the level of expression of chain-paired mAbs. Delivery of the trio mRNA protected mice in an S. aureus-induced dermonecrosis model. Intravenous administration of the three mRNA in non-human primates achieved peak serum IgG levels ranging between 2.9 and 13.7 µg/mL with a half-life of 11.8-15.4 days. These results suggest nucleic acid delivery of mAb combinations holds promise and may be a viable option to streamline the development of therapeutic antibodies.

A Bispecific Monoclonal Antibody Targeting Psl and PcrV Enhances Neutrophil-Mediated Killing of Pseudomonas aeruginosa in Patients with Bronchiectasis.

Rationale: Pseudomonas aeruginosa infection is associated with worse outcomes in bronchiectasis. Impaired neutrophil antimicrobial responses contribute to bacterial persistence. Gremubamab is a bivalent, bispecific monoclonal antibody targeting Psl exopolysaccharide and the type 3 secretion system component PcrV. Objectives: This study evaluated the efficacy of gremubamab to enhance killing of P. aeruginosa by neutrophils from patients with bronchiectasis and to prevent P. aeruginosa-associated cytotoxicity. Methods: P. aeruginosa isolates from a global bronchiectasis cohort (n = 100) underwent whole-genome sequencing to determine target prevalence. Functional activity of gremubamab against selected isolates was tested in vitro and in vivo. Patients with bronchiectasis (n = 11) and control subjects (n = 10) were enrolled, and the effect of gremubamab in peripheral blood neutrophil opsonophagocytic killing (OPK) assays against P. aeruginosa was evaluated. Serum antibody titers to Psl and PcrV were determined (n = 30; 19 chronic P. aeruginosa infection, 11 no known P. aeruginosa infection), as was the effect of gremubamab treatment in OPK and anti-cytotoxic activity assays. Measurements and Main Results: Psl and PcrV were conserved in isolates from chronically infected patients with bronchiectasis. Seventy-three of 100 isolates had a full psl locus, and 99 of 100 contained the pcrV gene, with 20 distinct full-length PcrV protein subtypes identified. PcrV subtypes were successfully bound by gremubamab and the monoclonal antibody-mediated potent protective activity against tested isolates. Gremubamab increased bronchiectasis patient neutrophil-mediated OPK (+34.6 ± 8.1%) and phagocytosis (+70.0 ± 48.8%), similar to effects observed in neutrophils from control subjects (OPK, +30.1 ± 7.6%). No evidence of competition between gremubamab and endogenous antibodies was found, with protection against P. aeruginosa-induced cytotoxicity and enhanced OPK demonstrated with and without addition of patient serum. Conclusions: Gremubamab enhanced bronchiectasis patient neutrophil phagocytosis and killing of P. aeruginosa and reduced virulence.

Efficacy of a Pseudomonas aeruginosa serogroup O9 vaccine.

There are currently no approved vaccines against the opportunistic pathogen Pseudomonas aeruginosa. Among vaccine targets, the lipopolysaccharide (LPS) O antigen of P. aeruginosa is the most immunodominant protective candidate. There are 20 different O antigens composed of different repeat sugar structures conferring serogroup specificity, and 10 are found most frequently in infection. Thus, one approach to combat infection by P. aeruginosa could be to generate immunity with a vaccine cocktail that includes all these serogroups. Serogroup O9 is 1 of the 10 serogroups commonly found in infection, but it has never been developed into a vaccine, due in part to the acid-labile nature of the O9 polysaccharide. Our laboratory has previously shown that intranasal administration of an attenuated Salmonella strain expressing the P. aeruginosa serogroup O11 LPS O antigen was effective in clearing bacteria and preventing mortality in mice following intranasal challenge with serogroup O11 P. aeruginosa. Consequently, we set out to develop a P. aeruginosa serogroup O9 vaccine using a similar approach. Here, we show that Salmonella expressing serogroup O9 triggered an antibody-mediated immune response following intranasal administration to mice and that it conferred protection from P. aeruginosa serogroup O9 in a murine model of acute pneumonia.

Multicomponent Pseudomonas aeruginosa Vaccines Eliciting Th17 Cells and Functional Antibody Responses Confer Enhanced Protection against Experimental Acute Pneumonia in Mice.

The Gram-negative pathogen Pseudomonas aeruginosa is a common cause of pneumonia in hospitalized patients. Its increasing antibiotic resistance and widespread occurrence present a pressing need for vaccines. We previously showed that a P. aeruginosa type III secretion system protein, PopB, elicits a strong Th17 response in mice after intranasal (IN) immunization and confers antibody-independent protection against pneumonia in mice. In the current study, we evaluated the immunogenicity and protective efficacy in mice of the combination of PopB (purified with its chaperone protein PcrH) and OprF/I, an outer membrane hybrid fusion protein, compared with immunization with the proteins individually either by the intranasal (IN) or subcutaneous (SC) routes. Our results show that after vaccination, a Th17 recall response from splenocytes was detected only in mice vaccinated with PopB/PcrH, either alone or in combination with OprF/I. Mice immunized with the combination of PopB/PcrH and OprF/I had enhanced protection in an acute lethal P. aeruginosa pneumonia model, regardless of vaccine route, compared with mice vaccinated with either alone or adjuvant control. Immunization generated IgG titers against the vaccine proteins and whole P. aeruginosa cells. Interestingly, none of these antisera had opsonophagocytic killing activity, but antisera from mice immunized with vaccines containing OprF/I, had the ability to block IFN-γ binding to OprF/I, a known virulence mechanism. Hence, vaccines combining PopB/PcrH with OprF/I that elicit functional antibodies lead to a broadly and potently protective vaccine against P. aeruginosa pulmonary infections.

Antivirulence Bispecific Monoclonal Antibody-Mediated Protection against Pseudomonas aeruginosa Ventilator-Associated Pneumonia in a Rabbit Model.

Ventilator-associated pneumonia is an important clinical manifestation of the nosocomial pathogen Pseudomonas aeruginosa. We characterized the correlates of protection with MEDI3902, a bispecific human IgG1 monoclonal antibody that targets the P. aeruginosa type 3 secretion system PcrV protein and the Psl exopolysaccharide, in a rabbit model of ventilator-associated pneumonia using lung-protective, low-tidal-volume mechanical ventilation. Rabbits infused with MEDI3902 prophylactically were protected, whereas those pretreated with irrelevant isotype-matched control IgG (c-IgG) succumbed between 12 and 44 h postinfection (100% survival [8/8 rabbits] versus 0% survival [8/8 rabbits]; P < 0.01 by log rank test). Lungs from rabbits pretreated with c-IgG, but not those pretreated with MEDI3902, had bilateral, multifocal areas of marked necrosis, hemorrhage, neutrophilic inflammatory infiltrate, and diffuse fibrinous edema in alveolar spaces. All rabbits pretreated with c-IgG developed worsening bacteremia that peaked at the time of death, whereas only 38% of rabbits pretreated with MEDI3902 (3/8 rabbits) developed such high-grade bacteremia (two-sided Fisher's exact test, P = 0.026). Biomarkers associated with acute respiratory distress syndrome were evaluated longitudinally in blood samples collected every 2 to 4 h to assess systemic pathophysiological changes in rabbits pretreated with MEDI3902 or c-IgG. Biomarkers were sharply increased or decreased in rabbits pretreated with c-IgG but not those pretreated with MEDI3902, including the ratio of arterial oxygen partial pressure to the fraction of inspired oxygen of <300, hypercapnia or hypocapnia, severe lactic acidosis, leukopenia, and neutropenia. Cytokines and chemokines associated with acute respiratory distress syndrome were significantly downregulated in lungs from rabbits pretreated with MEDI3902, compared with c-IgG. These results suggest that MEDI3902 prophylaxis could have potential clinical utility for decreasing the severity of P. aeruginosa ventilator-associated pneumonia.

Safety, efficacy, and pharmacokinetics of gremubamab (MEDI3902), an anti-Pseudomonas aeruginosa bispecific human monoclonal antibody, in P. aeruginosa-colonised, mechanically ventilated intensive care unit patients: a randomised controlled trial.

BACKGROUND: Ventilator-associated pneumonia caused by Pseudomonas aeruginosa (PA) in hospitalised patients is associated with high mortality. The effectiveness of the bivalent, bispecific mAb MEDI3902 (gremubamab) in preventing PA nosocomial pneumonia was assessed in PA-colonised mechanically ventilated subjects. METHODS: EVADE (NCT02696902) was a phase 2, randomised, parallel-group, double-blind, placebo-controlled study in Europe, Turkey, Israel, and the USA. Subjects ≥ 18 years old, mechanically ventilated, tracheally colonised with PA, and without new-onset pneumonia, were randomised (1:1:1) to MEDI3902 500, 1500 mg (single intravenous dose), or placebo. The primary efficacy endpoint was the incidence of nosocomial PA pneumonia through 21 days post-dose in MEDI3902 1500 mg versus placebo, determined by an independent adjudication committee. RESULTS: Even if the initial sample size was not reached because of low recruitment, 188 subjects were randomised (MEDI3902 500/1500 mg: n = 16/87; placebo: n = 85) between 13 April 2016 and 17 October 2019. Out of these, 184 were dosed (MEDI3902 500/1500 mg: n = 16/85; placebo: n = 83), comprising the modified intent-to-treat set. Enrolment in the 500 mg arm was discontinued due to pharmacokinetic data demonstrating low MEDI3902 serum concentrations. Subsequently, enrolled subjects were randomised (1:1) to MEDI3902 1500 mg or placebo. PA pneumonia was confirmed in 22.4% (n = 19/85) of MEDI3902 1500 mg recipients and in 18.1% (n = 15/83) of placebo recipients (relative risk reduction [RRR]: - 23.7%; 80% confidence interval [CI] - 83.8%, 16.8%; p = 0.49). At 21 days post-1500 mg dose, the mean (standard deviation) serum MEDI3902 concentration was 9.46 (7.91) µg/mL, with 80.6% (n = 58/72) subjects achieving concentrations > 1.7 µg/mL, a level associated with improved outcome in animal models. Treatment-emergent adverse event incidence was similar between groups. CONCLUSIONS: The bivalent, bispecific monoclonal antibody MEDI3902 (gremubamab) did not reduce PA nosocomial pneumonia incidence in PA-colonised mechanically ventilated subjects. Trial registration Registered on Clinicaltrials.gov ( NCT02696902 ) on 11th February 2016 and on EudraCT ( 2015-001706-34 ) on 7th March 2016.

Treatment Efficacy of MEDI3902 in Pseudomonas aeruginosa Bloodstream Infection and Acute Pneumonia Rabbit Models.

Pseudomonas aeruginosa is a challenge for clinicians due to increasing drug resistance and dwindling treatment options. We report on the activity of MEDI3902, an antibody targeting type 3 secretion protein PcrV and Psl exopolysaccharide, in rabbit bloodstream and lung infection models. MEDI3902 prophylaxis or treatment was protective in both acute models and exhibited enhanced activity when combined with a subtherapeutic dose of meropenem. These findings further support MEDI3902 for the prevention or treatment of serious P. aeruginosa infections.

Humanised effector-null FcγRIIA antibody inhibits immune complex-mediated proinflammatory responses.

OBJECTIVE: Immune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development. METHODS: VIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates. RESULTS: We generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies. CONCLUSIONS: VIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases.

The Neutrophilic Response to Pseudomonas Damages the Airway Barrier, Promoting Infection by Klebsiella pneumoniae.

Pseudomonas aeruginosa and Klebsiella pneumoniae are two common gram-negative pathogens that are associated with bacterial pneumonia and can often be isolated from the same patient. We used a mixed-pathogen pneumonia infection model in which mice were infected with sublethal concentrations of P. aeruginosa and K. pneumoniae, resulting in significant lethality, outgrowth of both bacteria in the lung, and systemic dissemination of K. pneumoniae. Inflammation, induced by P. aeruginosa activation of Toll-like receptor 5, results in prolonged neutrophil recruitment to the lung and increased levels of neutrophil elastase in the airway, resulting in lung damage and epithelial barrier dysfunction. Live P. aeruginosa was not required to potentiate K. pneumoniae infection, and flagellin alone was sufficient to induce lethality when delivered along with Klebsiella. Prophylaxis with an anti-Toll-like receptor 5 antibody or Sivelestat, a neutrophil elastase inhibitor, reduced neutrophil influx, inflammation, and mortality. Furthermore, pathogen-specific monoclonal antibodies targeting P. aeruginosa or K. pneumoniae prevented the outgrowth of both bacteria and reduced host inflammation and lethality. These findings suggest that coinfection with P. aeruginosa may enable the outgrowth and dissemination of K. pneumoniae, and that a pathogen- or host-specific prophylactic approach targeting P. aeruginosa may prevent or limit the severity of such infections by reducing neutrophil-induced lung damage.

MEDI3902 Correlates of Protection against Severe Pseudomonas aeruginosa Pneumonia in a Rabbit Acute Pneumonia Model.

Pseudomonas aeruginosa is among the most formidable antibiotic-resistant pathogens and is a leading cause of hospital-associated infections. With dwindling options for antibiotic-resistant infections, a new paradigm for treatment and disease resolution is required. MEDI3902, a bispecific antibody targeting the P. aeruginosa type III secretion (T3S) protein PcrV and Psl exopolysaccharide, was previously shown to mediate potent protective activity in murine infection models. With the current challenges associated with the clinical development of narrow-spectrum agents, robust preclinical efficacy data in multiple animal species are desirable. Here, we sought to develop a rabbit P. aeruginosa acute pneumonia model to further evaluate the activity of MEDI3902 intervention. In the rabbit model of acute pneumonia, prophylaxis with MEDI3902 exhibited potent dose-dependent protection, whereas those receiving control IgG developed fatal hemorrhagic necrotizing pneumonia between 12 and 54 h after infection. Blood biomarkers (e.g., partial pressure of oxygen [pO2], partial pressure of carbon dioxide [pCO2], base excess, lactate, and creatinine) were grossly deranged for the vast majority of control IgG-treated animals but remained within normal limits for MEDI3902-treated animals. In addition, MEDI3902-treated animals exhibited a profound reduction in P. aeruginosa organ burden and a marked reduction in the expression of proinflammatory mediators from lung tissue, which correlated with reduced lung histopathology. These results confirm that targeting PcrV and Psl via MEDI3902 is a promising candidate for immunotherapy against P. aeruginosa pneumonia.

Pseudomonas aeruginosa Bacteremic Patients Exhibit Nonprotective Antibody Titers Against Therapeutic Antibody Targets PcrV and Psl Exopolysaccharide.

BACKGROUND: The type 3 secretion protein PcrV and Psl exopolysaccharide are promising therapeutic antibody targets against Pseudomonas aeruginosa. We examined P. aeruginosa bloodstream infection (BSI) isolates for the ability to express PcrV and Psl and evaluated corresponding patient serum for active titers to these targets. METHODS: We identified 114 patients with acute P. aeruginosa BSI; 56 cases were accompanied by acute sera. Serum was evaluated for PcrV- and Psl-specific immunoglobulin G (IgG) and for cytotoxicity and opsonophagocytosis. Isolates were evaluated for susceptibility to antibiotics, expression of PcrV and Psl, and susceptibility to the anti-PcrV/Psl bispecific antibody and clinical candidate MEDI3902. RESULTS: In-hospital mortality for patients with P. aeruginosa BSI was 39%. A total of 26% of isolates were resistant to ≥3 antibiotic classes. Although PcrV and/or Psl were detected in 99% of isolates, a majority of patients lacked active titers to PcrV (100%) and Psl (98%). In addition, MEDI3902 was active against all tested isolates. CONCLUSIONS: A vast majority of P. aeruginosa BSI isolates express PcrV and Psl; however, patient sera most often lacked IgG and functionally active responses to these targets. These results suggest that therapies directed at PcrV and Psl could be a promising approach for combating P. aeruginosa bloodstream infections.

Synthetic Enterobacterial Common Antigen (ECA) for the Development of a Universal Immunotherapy for Drug-Resistant Enterobacteriaceae.

All Enterobacteriaceae express a polysaccharide known as enterobacterial common antigen (ECA), which is an attractive target for the development of universally acting immunotherapies. The first chemical synthesis of ECA-derived oligosaccharides for the development of such therapies is described. A number of synthetic challenges had to be addressed, including the development of concise synthetic procedures for unusual monosaccharides, the selection of appropriate orthogonal protecting groups, the development of stereoselective glycosylation methods, appropriate timing for the introduction of the carboxylic acid groups on the ManpNAcA moieties, and the selection of appropriate conditions for the reduction of multiple azido moieties. The synthetic compounds were employed to uncover immunodominant moieties of ECA. Furthermore, a monoclonal antibody (mAb) was developed that binds to ECA and can selectively recognize a wide range of Enterobacteriaceae species.

A novel anti-PcrV antibody providing enhanced protection against Pseudomonas aeruginosa in multiple animal infection models.

Pseudomonas aeruginosa is a major cause of hospital-acquired infections, particularly in mechanically ventilated patients, and it is the leading cause of death in cystic fibrosis patients. A key virulence factor associated with disease severity is the P. aeruginosa type III secretion system (T3SS), which injects bacterial toxins directly into the cytoplasm of host cells. The PcrV protein, located at the tip of the T3SS injectisome complex, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In an effort to identify a highly potent and protective monoclonal antibody (MAb) that inhibits the T3SS, we generated and characterized a panel of novel anti-PcrV MAbs. Interestingly, some MAbs exhibiting potent inhibition of T3SS in vitro failed to provide protection in a mouse model of P. aeruginosa infection, suggesting that effective in vivo inhibition of T3SS with anti-PcrV MAbs is epitope dependent. V2L2MD, while not the most potent MAb as assessed by in vitro cytotoxicity inhibition assays, provided strong prophylactic protection in several murine infection models and a postinfection therapeutic model. V2L2MD mediated significantly (P < 0.0001) better in vivo protection than that provided by a comparator antibody, MAb166, a well-characterized anti-PcrV MAb and the progenitor of a clinical candidate, KB001-A. The results described here support further development of a V2L2MD-containing immunotherapeutic and may suggest even greater potential than was previously recognized for the prevention and treatment of P. aeruginosa infections in high-risk populations.

Epitope mapping of monoclonal antibodies using synthetic oligosaccharides uncovers novel aspects of immune recognition of the Psl exopolysaccharide of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that can cause life-threatening infections in critically ill and cystic fibrosis patients. The Psl exopolysaccharide of P. aeruginosa offers an attractive serotype-independent antigen for the development of immunotherapies. Here, the first chemical synthesis of a panel of oligosaccharides derived from the exopolysaccharide of P. aeruginosa by a synthetic strategy that efficiently deals with the stereoselective installation of several ß-mannosides and the formation of a mannoside that is extended by saccharide moieties at C-1, C-2, and C-3 in a crowded 1,2,3-cis configuration is described. The approach was employed to prepare tetra-, penta-, and hexa- and decasaccharide part structures. The compounds were employed to define the epitope requirements of several functionally active monoclonal antibodies (mAbs) that can bind three distinct epitopes of Psl (class I, II, and III). The class II mAb reacted potently with each oligosaccharide indicating its epitope resides within the tetrasaccharide and does not require the branched mannoside of Psl. The class III antibody did not bind the tetra- or pentasaccharide; however, it did react potently with the hexasaccharide and weakly with the decasaccharide, suggesting a terminal glucoside is required for optimal binding. Unexpectedly, the class I mAb did not bind any of the oligosaccharides indicating that Psl contains a yet to be elucidated sub-stoichiometric isoform. This study demonstrates that functional activity of a mAb does not only depend on the avidity of binding but also on the location of an epitope within a bacterial polysaccharide. The results also provide a strong impetus to analyze further the structure of Psl to identify the class I epitope, that is expected to provide an attractive target for the development of a synthetic vaccine for P. aeruginosa.

Differential in vivo labeling with barcoded antibodies allows for simultaneous transcriptomic profiling of airway, lung tissue and intravascular immune cells.

Single-cell RNA sequencing (scRNA-seq) is the state-of-the-art approach to study transcriptomic signatures in individual cells in respiratory health and disease. However, classical scRNA-seq approaches provide no spatial information and are performed using either bronchoalveolar lavage fluid (BAL) or lung single cell suspensions to assess transcript levels in airway and tissue immune cells, respectively. Herein we describe a simple method to simultaneously characterize transcriptomic features of airway, lung parenchymal and intravascular immune cells based on differential in vivo labeling with barcoded antibodies. In addition to gaining basic spatial information, this approach allows for direct comparison of cells within different anatomical compartments. Furthermore, this method provides a time- and cost-effective alternative to classical scRNA-seq where lung and BAL samples are processed individually, reducing animal and reagent use. We demonstrate the feasibility of this approach in a preclinical mouse model of bacterial lung infection comparing airway, parenchymal and vasculature neutrophils early after infection.

Efficacy of a Pseudomonas aeruginosa Serogroup O9 Vaccine.

There are currently no approved vaccines against the opportunistic pathogen Pseudomonas aeruginosa. Among vaccine targets, the lipopolysaccharide (LPS) O antigen of P. aeruginosa is the most immunodominant protective candidate. There are twenty different O antigens composed of different repeat sugars structures conferring serogroup specificity, and ten are found most frequently in infection. Thus, one approach to combat infection by P. aeruginosa could be to generate immunity with a vaccine cocktail that includes all these serogroups. Serogroup O9 is one of the ten serogroups commonly found in infection, but it has never been developed into a vaccine, likely due, in part, to the acid labile nature of the O9 polysaccharide. Our laboratory has previously shown that intranasal administration of an attenuated Salmonella strain expressing the P. aeruginosa serogroup O11 LPS O antigen was effective in clearing and preventing mortality in mice following intranasal challenge with serogroup O11 P. aeruginosa. Consequently, we set out to develop a P. aeruginosa serogroup O9 vaccine using a similar approach. Here we show that Salmonella expressing serogroup O9 triggered an antibody-mediated immune response following intranasal administration to mice and that it conferred protection from P. aeruginosa serogroup O9 in a murine model of acute pneumonia.

Early diagnostic BioMARKers in exacerbations of chronic obstructive pulmonary disease: protocol of the exploratory, prospective, longitudinal, single-centre, observational MARKED study.

INTRODUCTION: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) play a pivotal role in the burden and progressive course of chronic obstructive pulmonary disease (COPD). As such, disease management is predominantly based on the prevention of these episodes of acute worsening of respiratory symptoms. However, to date, personalised prediction and early and accurate diagnosis of AECOPD remain unsuccessful. Therefore, the current study was designed to explore which frequently measured biomarkers can predict an AECOPD and/or respiratory infection in patients with COPD. Moreover, the study aims to increase our understanding of the heterogeneity of AECOPD as well as the role of microbial composition and hostmicrobiome interactions to elucidate new disease biology in COPD. METHODS AND ANALYSIS: The 'Early diagnostic BioMARKers in Exacerbations of COPD' study is an exploratory, prospective, longitudinal, single-centre, observational study with 8-week follow-up enrolling up to 150 patients with COPD admitted to inpatient pulmonary rehabilitation at Ciro (Horn, the Netherlands). Respiratory symptoms, vitals, spirometry and nasopharyngeal, venous blood, spontaneous sputum and stool samples will be frequently collected for exploratory biomarker analysis, longitudinal characterisation of AECOPD (ie, clinical, functional and microbial) and to identify host-microbiome interactions. Genomic sequencing will be performed to identify mutations associated with increased risk of AECOPD and microbial infections. Predictors of time-to-first AECOPD will be modelled using Cox proportional hazards' regression. Multiomic analyses will provide a novel integration tool to generate predictive models and testable hypotheses about disease causation and predictors of disease progression. ETHICS AND DISSEMINATION: This protocol was approved by the Medical Research Ethics Committees United (MEC-U), Nieuwegein, the Netherlands (NL71364.100.19). TRIAL REGISTRATION NUMBER: NCT05315674.

A protein-free vaccine stimulates innate immunity and protects against nosocomial pathogens.

Traditional vaccines are difficult to deploy against the diverse antimicrobial-resistant, nosocomial pathogens that cause health care-associated infections. We developed a protein-free vaccine composed of aluminum hydroxide, monophosphoryl lipid A, and fungal mannan that improved survival and reduced bacterial burden of mice with invasive blood or lung infections caused by methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, extended-spectrum beta-lactamase-expressing Escherichia coli, and carbapenem-resistant strains of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The vaccine also conferred protection against the fungi Rhizopus delemar and Candida albicans. Efficacy was apparent by 24 hours and lasted for up to 28 days after a single vaccine dose, with a second dose restoring efficacy. The vaccine acted through stimulation of the innate, rather than the adaptive, immune system, as demonstrated by efficacy in the absence of lymphocytes that were abrogated by macrophage depletion. A role for macrophages was further supported by the finding that vaccination induced macrophage epigenetic alterations that modulated phagocytosis and the inflammatory response to infection. Together, these data show that this protein-free vaccine is a promising strategy to prevent deadly antimicrobial-resistant health care-associated infections.

Development and validation of a rabbit model of Pseudomonas aeruginosa non-ventilated pneumonia for preclinical drug development.

Background: New drugs targeting antimicrobial resistant pathogens, including Pseudomonas aeruginosa, have been challenging to evaluate in clinical trials, particularly for the non-ventilated hospital-acquired pneumonia and ventilator-associated pneumonia indications. Development of new antibacterial drugs is facilitated by preclinical animal models that could predict clinical efficacy in patients with these infections. Methods: We report here an FDA-funded study to develop a rabbit model of non-ventilated pneumonia with Pseudomonas aeruginosa by determining the extent to which the natural history of animal disease reproduced human pathophysiology and conducting validation studies to evaluate whether humanized dosing regimens of two antibiotics, meropenem and tobramycin, can halt or reverse disease progression. Results: In a rabbit model of non-ventilated pneumonia, endobronchial challenge with live P. aeruginosa strain 6206, but not with UV-killed Pa6206, caused acute respiratory distress syndrome, as evidenced by acute lung inflammation, pulmonary edema, hemorrhage, severe hypoxemia, hyperlactatemia, neutropenia, thrombocytopenia, and hypoglycemia, which preceded respiratory failure and death. Pa6206 increased >100-fold in the lungs and then disseminated from there to infect distal organs, including spleen and kidneys. At 5 h post-infection, 67% of Pa6206-challenged rabbits had PaO2 <60 mmHg, corresponding to a clinical cut-off when oxygen therapy would be required. When administered at 5 h post-infection, humanized dosing regimens of tobramycin and meropenem reduced mortality to 17-33%, compared to 100% for saline-treated rabbits (P<0.001 by log-rank tests). For meropenem which exhibits time-dependent bactericidal activity, rabbits treated with a humanized meropenem dosing regimen of 80 mg/kg q2h for 24 h achieved 100% T>MIC, resulting in 75% microbiological clearance rate of Pa6206 from the lungs. For tobramycin which exhibits concentration-dependent killing, rabbits treated with a humanized tobramycin dosing regimen of 8 mg/kg q8h for 24 h achieved Cmax/MIC of 9.8 ± 1.4 at 60 min post-dose, resulting in 50% lung microbiological clearance rate. In contrast, rabbits treated with a single tobramycin dose of 2.5 mg/kg had Cmax/MIC of 7.8 ± 0.8 and 8% (1/12) microbiological clearance rate, indicating that this rabbit model can detect dose-response effects. Conclusion: The rabbit model may be used to help predict clinical efficacy of new antibacterial drugs for the treatment of non-ventilated P. aeruginosa pneumonia.