Identification of Autoantigens in EGPA and Severe Eosinophilic Asthma

Conditions

Churg-Strauss Syndrome, Eosinophilic Asthma, Eosinophilic Esophagitis, Eosinophilic Pneumonia

Brief summary

In this project the investigators will look for auto-antibodies to relevant proteins both in native form and importantly in post-translationally modified forms. Potential modified auto-antigens are eosinophil proteins (analogous to the cytoplasmic neutrophil proteins identified in vasculitides such as Granulomatosis with Polyangiitis (formerly known as Wegener's granulomatosis) and alternatively structural proteins such as collagen V. As well as advancing the understanding of asthma pathology, identifying a serum auto-antibody that could then be used as a clinical blood test, analogous to anti-cyclic citrullinated peptide (CCP) antibodies in rheumatoid arthritis, may revolutionise diagnosis of severe eosinophilic asthma and Eosinophilic Granulomatosis with Polyangiitis (EGPA). There is a considerable burden of undiagnosed severe eosinophilic asthma in part due to difficulties in definitive diagnosis and a diagnostic blood test would help diagnose these patients, allowing them to receive necessary treatment.

Detailed description

The investigators will approach the research question with parallel agnostic and targeted approaches. In the agnostic approach the presence of auto-antibodies in patient serum and sputum to inactive and activated eosinophils, with and without post-translational modification, will be examined by indirect immunofluorescence. In the targeted approach the investigators will examine by enzyme-linked immunoassay (ELISA) the presence/absence of antibodies to pre-selected candidate eosinophil and base membrane proteins both in native form and post-translationally modified. Proteins to examine will be chosen based on literature review (e.g. eosinophil peroxidase and collagen V) and eosinophil-specific proteins identified by FANTOM5 (Functional Annotation of the Mouse/Mammalian Genome) geneset analysis (FANTOM Consortium et al. 2014). Both blood and sputum samples from highly-characterised patients with severe eosinophilic asthma and/or EGPA will be examined given the possibility of compartment-specific immune responses. Once candidate auto-antigens have been identified in the selected group of patients with severe eosinophilic asthma and EGPA, the investigators will then examine their prevalence in serum samples from a wider selection of patients with eosinophilic airways diseases including mild-to-moderate asthma, severe eosinophilic asthma, EGPA, nasal polyposis and eosinophilic chronic obstructive pulmonary disease (COPD) as well as healthy controls. Length of disease, atopy, presence/absence nasal polyps, gender, age will be examined as co-variates. Correlations with highest blood eosinophil counts, requirement for oral corticosteroids and presence of other auto-antibodies, e.g. anti-MPO (myeloperoxidase) ANCA (anti-neutrophil cytoplasmic antibody), will be examined. In particular the investigators will look for the presence of novel autoantibodies in specific patient subsets: i) ANCA negative, ii) ANCA positive by immunofluorescence but negative for anti-MPO and anti-PR3 (proteinase-3) antibodies, iii) ANA (anti-nuclear antibody) positive but ANCA and extractable nuclear antigen (ENA) negative; since patients in all three groups may have novel, as yet undetermined autoantibodies. ROC (receiver operator characteristic curve) AUC (area under curve) analyses will be conducted to ascertain the predictive value of blood auto-antibodies for diagnosis of eosinophilic airways disease.

Ilaria Esposito, Ioanna Kontra, Chiara Giacomassi, Sotiria Manou-Stathopoulou, James Brown et al. (12 authors)

Frontiers in Immunology2023-06-022 citations

10.3389/fimmu.2023.1164941

Interventions

DIAGNOSTIC_TESTAutoantibody ELISA

Serum will be tested for novel autoantibodies against candidate auto-antigens by ELISA, such as those previously identified as of importance (e.g. eosinophil peroxidase, EPX) and also novel candidate proteins specific to eosinophils as identified by FANTOM5 geneset analysis.

DIAGNOSTIC_TESTGranulocyte Immunofluorescence

The presence of auto-antibodies in patient serum to inactive and activated neutrophils / eosinophils, with and without post-translational modification, will be examined by indirect immunofluorescence.

Study design

Observational model

CASE_CONTROL

Time perspective

PROSPECTIVE

Eligibility

Sex/Gender

ALL

Age

18 Years to No maximum

Healthy volunteers

Yes

Inclusion criteria

* Severe Eosinophilic Asthma (with multi-disciplinary diagnosis as per ERS/ATS Criteria, blood eosinophils ≥ 0.3 x109/L on inhaled corticosteroids); or * EGPA (as per American College of Rheumatology (ACR) Criteria); or * Eosinophilic COPD (post-bronchodilator FEV1/FVC \< 70% predicted, absence of bronchodilator reversibility, \> 20 pack year smoking history, no history of asthma, blood eosinophils ≥ 0.3 x109/L); or * Eosinophilic oesophagitis (with diagnostic histology); or * Granulomatosis with Polyangiitis (GPA, formerly called Wegener's) (as per American College of Rheumatology (ACR) Criteria)

Exclusion criteria

* Known Pregnancy * Anaemia * Hepatitis B Virus, Hepatitis C Virus or HIV infection * Donation of more than 240mls blood in the last sixteen weeks (four months) to any other research study or as a donation to the National Blood Transfusion Service * Rituximab, plasmapharesis or polyclonal immunoglobulin infusion (ever)

Design outcomes

Primary

Measure	Time frame	Description
Number and Percentage of Patients With a Positive Autoantibody ELISA	Baseline, at study entry	Serum tested for novel autoantibodies against candidate auto-antigens by ELISA. Outcome: Positive OD by ELISA (serum samples) to autoantigen panel (MPO, Collagen V, TREM1, IL1R2).

Secondary

Measure	Time frame	Description
Number and Percentage of Patients With Positive Granulocyte Immunofluorescence	Baseline, at study entry	FITC anti-IgG immunofluorescence with slides of unstimulated/PMA-stimulated neutrophils (serum samples)

Countries

United Kingdom

Participant flow

Participants by arm

Arm	Count
Severe Eosinophilic Asthma Patients with eosinophilic asthma meeting ERS/ATS criteria for severe asthma.	63
Healthy Controls Healthy patients with no chronic lung condition.	30
EGPA Patients with asthma meeting criteria for a diagnosis of EGPA (eosinophilic granulomatosis with polyangiitis)	17
Other Respiratory Conditions Mild-to-moderate asthma, other vasculitides, eosinophilic COPD, and/or eosinophilic oesophagitis	10
Total	120

Baseline characteristics

Characteristic	Severe Eosinophilic Asthma	Healthy Controls	EGPA	Other Respiratory Conditions	Total
Age, Categorical <=18 years	0 Participants	0 Participants	0 Participants	0 Participants	0 Participants
Age, Categorical >=65 years	9 Participants	1 Participants	4 Participants	5 Participants	19 Participants
Age, Categorical Between 18 and 65 years	54 Participants	28 Participants	13 Participants	5 Participants	100 Participants
Age, Continuous	54.4 years STANDARD_DEVIATION 12.3	42.0 years STANDARD_DEVIATION 14.1	56.6 years STANDARD_DEVIATION 14.2	63.1 years STANDARD_DEVIATION 9.7	52.4 years STANDARD_DEVIATION 14.2
Race/Ethnicity, Customized Ethnicity Asian	16 Participants	9 Participants	0 Participants	4 Participants	29 Participants
Race/Ethnicity, Customized Ethnicity Black	8 Participants	0 Participants	2 Participants	0 Participants	10 Participants
Race/Ethnicity, Customized Ethnicity Other / Declined / Not Available	8 Participants	5 Participants	3 Participants	0 Participants	16 Participants
Race/Ethnicity, Customized Ethnicity White	31 Participants	16 Participants	12 Participants	6 Participants	65 Participants
Region of Enrollment United Kingdom	63 participants	30 participants	17 participants	10 participants	120 participants
Sex: Female, Male Female	29 Participants	16 Participants	7 Participants	4 Participants	56 Participants
Sex: Female, Male Male	34 Participants	14 Participants	10 Participants	6 Participants	64 Participants

Adverse events

Event type	EG000 affected / at risk	EG001 affected / at risk	EG002 affected / at risk	EG003 affected / at risk
deaths Total, all-cause mortality	0 / 63	0 / 30	0 / 17	0 / 9
other Total, other adverse events	0 / 63	0 / 30	0 / 17	0 / 9
serious Total, serious adverse events	0 / 63	0 / 30	0 / 17	0 / 9

Outcome results

Primary

Number and Percentage of Patients With a Positive Autoantibody ELISA

Serum tested for novel autoantibodies against candidate auto-antigens by ELISA. Outcome: Positive OD by ELISA (serum samples) to autoantigen panel (MPO, Collagen V, TREM1, IL1R2).