RATIONALE:Cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) are characterized by neutrophilic airway inflammation but differ in clinical features. OBJECTIVES:We investigated relationships of pulmonary and systemic inflammatory markers with functional and structural lung disease. METHODS:Systemic (CRP, IgG, IL-6, and IL-8) and sputum (calprotectin, IL-6, and IL-8) markers were measured at baseline. Relationships were explored with spirometry, lung clearance index and lung computed tomography (CT) scores at baseline and with spirometry 10 years later. RESULTS:42 patients (21 CF, 21 PCD) of similar age (median CF 27 y [12-59], PCD 27 y [11-62]) and lung function (median ppFEV1CF 59% [40-99], PCD 67% [30-101]) were enrolled in 2009. Systemic inflammation was significantly higher in CF (Neutrophils, p < 0.05, IL-6 and 8, p < 0.01). Conversely, sputum IL-6 and 8 were higher in PCD (p < 0.01). In CF, sputum IL-6 counter-intuitively correlated with better CT scores (r = -0.51/p < 0.05) at baseline and with better spirometry (r = 0.58, p < 0.05) 10 years later. Conversely, high sputum IL-6 at baseline was associated with worse lung function in PCD (r = 0.81/p = 0.001) after 10 years. CONCLUSION:This study emphasizes the differences of functional and structural aspects in both diseases. Markers that predict long-term outcome in PCD and in CF were identified. The most striking and unanticipated finding was that sputum IL-6 correlated with better CT scores and lung function in CF. This paradox merits further research but challenges whether sputum IL-6 in CF is always a bad prognostic indicator.

Vaccines are central to the strategy to control antimicrobial resistant (AMR) bacterial infections; one multidrug resistant pathogen of particular concern is Acinetobacter baumannii. In this study we identified two novel A. baumannii antigens using mass spectrometry and phage expression: Oxa23 and PAL. These genes are highly conserved between different isolates of A. baumannii and recognised by convalescent human sera. We explored their protective immunity using two different vaccine platforms, recombinant outer membrane vesicles (rOMV) and mRNA. RNA vaccine immunised mice had significantly reduced bacterial load in their lower airways following challenge with carbapenem resistant A. baumannii, with Oxa23 providing better protection than PAL. We then compared routes of delivery and RNA vaccine platforms, demonstrating that intranasally delivery of mRNA encoding OXA-23 (formulated with GL67A) significantly reduced disease severity and enhanced bacterial clearance. These studies validate in silico identified antigens through challenge studies and novel mucosal vaccine delivery approaches.

Rationale and objective Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CFTR modulators offer significant improvements, but similar to 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimised for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial. Methods Air-liquid interface cultures of primary human bronchial epithelial cells (HBECs) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalised human lung epithelial cell line mimicking class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies. Results rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence <= 10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalisation assay. Conclusions The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.

Aspergillus fumigatus (Af) is a major mould pathogen found ubiquitously in the air. It commonly infects the airways of people with cystic fibrosis (CF) leading to Aspergillus bronchitis or allergic bronchopulmonary aspergillosis. Resident alveolar macrophages and recruited neutrophils are important first lines of defence for clearance of Af in the lung. However, their contribution to the inflammatory phenotype in CF during Af infection is not well understood. Here, utilising CFTR deficient mice we describe a hyperinflammatory phenotype in both acute and allergic murine models of pulmonary aspergillosis. We show that during aspergillosis, CFTR deficiency leads to increased alveolar macrophage death and persistent inflammation of the airways in CF, accompanied by impaired fungal control. Utilising CFTR deficient murine cells and primary human CF cells we show that at a cellular level there is increased activation of NFκB and NFAT in response to Af which, as in in vivo models, is associated with increased cell death and reduced fungal control. Taken together, these studies indicate that CFTR deficiency promotes increased activation of inflammatory pathways, the induction of macrophage cell death and reduced fungal control contributing to the hyper-inflammatory of pulmonary aspergillosis phenotypes in CF.

Aspergillus fumigatus (Af) is a major mould pathogen found ubiquitously in the air. It commonly infects the airways of people with cystic fibrosis (CF) leading to Aspergillus bronchitis or allergic bronchopulmonary aspergillosis. Resident alveolar macrophages and recruited neutrophils are important first lines of defence for clearance of Af in the lung. However, their contribution to the inflammatory phenotype in CF during Af infection is not well understood. Here, utilising CFTR deficient mice we describe a hyperinflammatory phenotype in both acute and allergic murine models of pulmonary aspergillosis. We show that during aspergillosis, CFTR deficiency leads to increased alveolar macrophage death and persistent inflammation of the airways in CF, accompanied by impaired fungal control. Utilising CFTR deficient murine cells and primary human CF cells we show that at a cellular level there is increased activation of NFκB and NFAT in response to Af which, as in in vivo models, is associated with increased cell death and reduced fungal control. Taken together, these studies indicate that CFTR deficiency promotes increased activation of inflammatory pathways, the induction of macrophage cell death and reduced fungal control contributing to the hyper-inflammatory of pulmonary aspergillosis phenotypes in CF. ### Competing Interest Statement The authors have declared no competing interest.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Although CF is a multiorgan disease, the leading causes of morbidity and mortality are related to progressive lung disease. Current understanding of the effects of the broad spectrum of CFTR mutations on CFTR function has allowed for the development of CFTR modulator therapies. Despite the remarkable impact that these therapies have had, there remains a significant proportion of people with CF (estimated at 10-15% of the global CF population) who are genetically ineligible for, or intolerant of, current CFTR-targeting therapies and whose therapeutic needs remain unmet. Inhaled genetic therapies offer the prospect of addressing the unmet pulmonary treatment need in people with CF, with several approaches, including gene addition therapy (the focus of this review), RNA-based therapies, antisense oligonucleotides, and gene editing, being explored. Various nonviral and viral vectors have been investigated for CF gene addition therapy for mutation-agnostic restoration of CFTR function in the lungs. Lentiviral vectors offer the prospect of highly efficient and long-lasting gene expression, and the potential to be safely and, in contrast to other commonly used viral vectors, effectively redosed. A third-generation lentiviral vector pseudotyped with Sendai virus F and HN envelope proteins (rSIV.F/HN) has been developed for the treatment of CF. Promising preclinical results support the progression of this vector carrying a full-length CFTR transgene (BI 3720931) into a first-in-human clinical trial expected to begin in 2024.

Objectives: We have developed a lentiviral vector platform pseudotyped with the Sendai virus F and HN envelope proteins (rSIV.F/HN), including the clinical candidate BI 3720931 for cystic fibrosis (CF) gene therapy. We have previously demonstrated restoration of CFTR function in CF-patient bronchial epithelial cell air–liquid interface cultures and intestinal organoids, as well as efficient and persistent in vivo transduction of murine airways. We have now assessed transduction efficiency and acute toxicology in non-human primates (NHPs). Methods: Male cynomolgus monkeys received a single dose of aerosolised rSIV.F/HN vector expressing green fluorescent protein (GFP) (4.2e9 transduction units in ~2 ml) or placebo via an endotracheal tube (n = 3/group). Approximately 25% of the dose (mass median aerodynamic diameter 3.9 µm) was deposited in the lungs. Toxicology was assessed by histopathology, clinical pathology, cytokine levels and changes in body and organ weight, and transduction efficiency was quantified by GFP immunohistochemistry and vector-specific mRNA 7 days post-dosing. Results: There were no vector-related clinical observations, mortality, or changes in body or organ weight. Clinical pathology and cytokine analyses were unremarkable. A minimal mixed-cell centriacinar inflammation was observed in 1/3 active treated animals. Airway epithelial cell transduction efficiency was 9–12% and vector-specific mRNA levels were ~45x higher than endogenous CFTR mRNA levels. Conclusions: This study confirms and extends our previous findings of rSIV.F/HN-based in vivo gene transfer in mice to NHPs. Using a relatively low vector dose, NHPs demonstrated transduction efficiency in the range of values likely to relate to clinical benefit, without evidence of toxicity. Transduction efficiency and toxicology in animals treated with a higher dose is currently being analysed. These data, together with our previous murine data, support further progression of BI 3720931 towards the clinic.

Objectives: Cystic fibrosis (CF) & CFTR-related disorders (CFTR-RD) can be difficult to diagnose. NPD & extended CFTR genetics can improve the diagnostic yield when sweat Cl- and genotyping are inconclusive. We report the diagnostic outcomes of our DCFD service. Methods: All patients referred (2017-2022) who had NPD were included in this retrospective study. Every patient was reviewed at a consensus meeting to assign them into diagnostic categories. We then explored the diagnostic discriminatory value of sweat Cl- & NPD using Kruskal-Wallis testing. Results: 122 patients (median[range] age 38.9y[7.3-75.1]) with a median sweat Cl- 39 (9.5-89.5)mmol/L. Diagnostic outcomes: CF, 24%(n=29); CFTR-RD, 22%(27); not-CF/CFTR-RD, 47%(58); inconclusive,7%(8). 2 CFTR variants were identified in 97% & 96% of CF & CFTR-RD patients, respectively. Of these, more were disease-causing in the CF(58%) v CFTR-RD(46%) group. Sweat Cl- could not discriminate between the groups whereas NPD could (table). Conclusions: Our DCFD service clarified the diagnosis in 93% of patients. NPD played a pivotal role in discriminating between groups.

Bacteriophages (phages) are antimicrobials with resurgent interest that are being investigated for the treatment of antibiotic refractory infection, including for Pseudomonas aeruginosa (Pa) lung infection in cystic fibrosis (CF). In vitro work supports the use of this therapy in planktonic and biofilm culture models; however, consistent data are lacking for efficacy across different clinical Pa strains, culture models, and in combination with antibiotics in clinical use. We first examined the efficacy of a 4-phage cocktail as an adjunct to our CF centre's first-line systemic combination antibiotic therapy (ceftazidime + tobramycin) for 16 different clinical Pa strains and then determined subinhibitory interactions for a subset of these strains with each antibiotic in planktonic and biofilm culture. When a 4-phage cocktail (4 × 108 PFU/mL) was added to a ceftazidime-tobramycin combination (ceftazidime 16 mg/mL + tobramycin 8 mg/mL), we observed a 1.7-fold and 1.3-fold reduction in biofilm biomass and cell viability, respectively. The four most antibiotic resistant strains in biofilm were very susceptible to phage treatment. When subinhibitory concentrations of antibiotics and phages were investigated, we observed additivity/synergy as well as antagonism/inhibition of effect that varied across the clinical strains and culture model. In general, more additivity was seen with the phage-ceftazidime combination than with phage-tobramycin, particularly in biofilm culture, where no instances of additivity were seen when phages were combined with tobramycin. The fact that different bacterial strains were susceptible to phage treatment when compared to standard antibiotics is promising and these results may be relevant to ongoing clinical trials exploring the use of phages, in particular in the selection of subjects for clinical trials.

The International Committee on Mental Health in CF advise screening with PHQ-8 or 9 for depression and GAD-7 for anxiety due to the higher levels of depression and anxiety reported by CF caregivers. There is no accepted minimal clinically important difference (MCID) for these measures in CF (general population, ≥3 for PHQ-8 & ≥4 for GAD-7). We aimed to explore variability of total scores and severity over 6 months and explore change using the MCID for the general population in CF caregivers experiencing standard care as part of CLIMB-CF.