Pathfinder: Determining the efficacy of plasmapheresis as a treatment for patients with chronic Pseudomonas infections and inhibitory antibodies

Chronic Pseudomonas aeruginosa lung infections occur in patients suffering from cystic fibrosis (CF), non-CF bronchiectasis and chronic obstructive pulmonary disorder (COPD). Such infections lead to chronic inflammation, deteriorating lung function and increased morbidity and mortality. Once colonisation is established, Pseudomonas is almost impossible to remove by current methods. Methods to ameliorate infection would positively impact on patient quality of life and longevity.

Antibody normally protects against infection. However, recently we identified that some patients with chronic Pseudomonas lung infections had a type of antibody that actually protects their colonising bacteria from killing by the immune system. These inhibitory antibodies were found to prevent both serum and cell killing of patient's Pseudomonas cognate strains. The inhibitory antibodies were identified as IgG2 specific to lipopolysaccharide, specifically the O-antigen component. We hypothesise that these antibodies exert their inhibitory effect either by depositing complement away from the bacterial membrane preventing membrane attack complex insertion or by blocking access of protective antibody. Importantly, we found patients with high titres of inhibitory antibodies had worse lung function than patients infected with Pseudomonas who had normal serum killing.

Based on our data, we opted for a radical treatment programme for one critically ill patient who had inhibitory antibodies. Using plasmapheresis we removed all serum protein, including the inhibitory IgG2, and replaced it with IVIg that we had demonstrated could mediate serum killing of the patient's isolate. The patient improved dramatically after plasmapheresis and Pseudomonas remained undetectable in the sputum for a significant period of time. A second critically ill patient has recently undergone a similar treatment regime with similar success. Patients receiving this treatment required fewer days in hospital, fewer treatment interventions such as intravenous antibiotics and reported a much reduced level of symptoms. Excitingly, this offers a novel treatment for critically ill patients with inhibitory antibodies, however many vital questions remain unanswered.

This project is vital to determine the clinical relevance of inhibitory antibodies in patients with chronic Pseudomonas lung infections and to identify whether plasma exchange is a viable treatment option for this novel problem. The project will do this using two approaches; first, by determining the prevalence of inhibitory antibodies in a large cohort of patients with both CF and non-CF bronchiectasis. This will determine the scale of the problem as well as thoroughly investigate the link between inhibitory antibodies and health degradation. Secondly, the project will investigate the treatment option by conducting a longitudinal study on patients currently being treated by plasma exchange. We will identify if previous plasma exchange treatments were successful due to removal of inhibitory antibodies alone, or if other factors were at play. We will investigate whether increases in IgG2 correlate with reappearance of the strain and worsening disease. We will also use next-generation sequencing to determine if strain replacement is being driven by plasmapheresis, as this would support the theory that the removal of inhibitory antibody is having a positive impact on the ability of the immune system to control infection post plasma exchange.

Finally, this project aims to optimise a diagnostic test for detecting the presence of inhibitory antibodies.

Chronic P. aeruginosa lung infections are found in patients suffering from cystic fibrosis or bronchiectasis and once P. aeruginosa colonisation is established, it is almost impossible to remove by current methods Recently we investigated patients with bronchiectasis and chronic P. aeruginosa infections. Serum was taken from each patient and tested for its ability to kill the patients colonising Pseudomonas strain. This identified a subset of patients whose sera was unable to kill their colonising strain due to a blocking factor in their serum. This inhibitory factor was identified as high titres of IgG2 antibody specific to P. aeruginosa O-antigen. The results suggested that the IgG2 bound the O-antigen (a target distal from the cell surface) where it either held complement away from the bacterial membrane, or physically blocked access of protective antibodies. Crucially, patients with impaired serum killing had worse lung function than patients with normal serum killing. Two critically ill patients with this IgG2 were treated with plasmapheresis in an attempt to remove the inhibitory antibody. Both patients had immediate benefit from the treatment. Although significant, many vital questions remain unanswered; 1) Is plasmapheresis a viable treatment option for patients with inhibitory antibodies? 2) How does having inhibitory antibodies impact on clinical status? 3) What is the prevalence of the inhibitory antibodies in patients with Pseudomonas lung infections? 4) Is plasmapheresis driving strain replacement? 5) Are other antibody subtypes involved in immune-killing inhibition? These answers will have far-reaching implications for monitering and treatment of lung infections. This project aims to answer these questions by combining our unique access to clinical samples with the techniques already established in our previous work coupled with next-gen genome sequencing.

Who will benefit from this research?

Pseudomonas aeruginosa is a ubiquitous environmental pathogen and is one of the three major causes of opportunistic human infections. P. aeruginosa is found in an estimated 10-20% of all hospital acquired infections, most likely due to its high resistance to antibiotics and disinfectants that eliminate other environmental bacteria. It is implicated in respiratory infections, urinary tract infections, gastrointestinal infections, keratitis, otitis media, and bacteraemia. However, the greatest burden to society by far is P. aeruginosa chronic infection in the lung. infections are found in patients suffering from cystic fibrosis, bronchiectasis or chronic obstructive pulmonary disease and once P. aeruginosa colonisation is established, it is almost impossible to remove by current methods. In all diseases chronic long-term colonisation of the lungs is possible due to abnormal airway epithelia. In all cases, infection with P. aeruginosa is the predominant cause of morbidity and mortality in these patients. Patients with chronic P. aeruginosa infection are also a great burden on the NHS with most patients regularly assessed and the prescription of nebulised or I.V. antibiotic treatment (tobramycin or colomycin). Despite this, many patients will have multiple exacerbation periods a year, leading to further treatment.

Output from this project will have direct benefits for patients with chronic P. aeruginosa colonisation. Indeed, two patients with colonisation and inhibitory antibodies have already been treated with plasmapheresis in an attempt to restore antibody-specific killing of the colonising strain. Data produced from tracking these patients in the future will be of vital importance for treatment of other patients going forward. The project also aims to optimise a quick, easy diagnostic method to determine if a patient is producing inhibitory antibodies. This will help both clinicians and their patients manage the infection. Finally, the project aims to solidify the link between inhibitory antibodies and clinical severity of disease. This will directly benefit both clinicians and patients by identifying novel causes of a patients' health degrading.

This research will also directly benefit the medical sector. Output from this research will aid in the creation of new diagnostics to test for and track the production of inhibitory antibodies in patients, informing the treatment options. Removing or overcoming inhibitory antibodies in these patients will allow their own immune system to tackle the infection, perhaps leading to less exacerbation events a year, lower antibiotic usage and thus a lower burden on the NHS. Finally, the research will also shed light on the prevalence of inhibitory antibodies in chronic lung infections, opening up avenues for further research and potential novel treatment options for patients with these infections.