PROSAIC-19 - Prospective Longitudinal Assessment in a COVID-19 Infected Cohort

INTRODUCTION

1.1 BACKGROUND

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is a new rapidly
spreading infectious disease with no proven treatment options. The virus causes a
spectrum of disease ranging from mild coryzal symptoms to severe respiratory compromise
requiring ventilatory support. Guidance from Public Health England identifies several
groups that are at risk of severe disease including the elderly and individuals with
chronic lung disease. Additionally, there is also debate over the role of corticosteroids
with some hospital guidelines recommending their use despite WHO guidance contradicting
this due to concerns that they may impair antiviral immunity and worsen disease. The
mechanisms driving severity of disease in certain individuals infected with COVID-19 are
poorly understood. We urgently need to understand these mechanisms to facilitate rapid
development of novel effective therapies and vaccines

1. Immunosusceptibility to severe COVID-19 disease

There are several putative mechanisms through which SARS-CoV-2 could drive greater
disease severity in pre-disposed individuals. These include:

1. Impaired innate anti-viral immune responses

Following viral entry, the innate immune response to respiratory viruses involves
induction of the type I and III interferons (IFN). This initiates a cascade that
triggers expression of a range of interferon-stimulated genes expressing proteins
that act in concert to limit viral replication. Chronic lung diseases, particularly
asthma, cystic Fibrosis (CF) and COPD are associated with impaired or dysregulated
induction of interferon to commonly encountered viruses such as rhinovirus and
influenza and similar dysregulation may occur with SARS-CoV-2. Inhaled interferon-β
is currently in trials as an anti-viral therapy for asthma and COPD.

2. Exaggerated inflammatory responses to viral infection

Common respiratory viral infections such as influenza and respiratory syncyitial
virus (RSV) can promote viral pathology mediated by antibodies and T cell responses.
Early evidence suggests that severe COVID-19 disease is associated with
hyperinflammation, a feature that could be directly driven by virus burden or occur
due to independent hyperactivation of the immune system. Human coronavirus
infections including SARS-CoV can induce immunopathology in the lungs through a
number of different mechanisms including antibody enhancement of SARS-CoV infection
by human macrophages. Significant increases in CD4 and CD8 expressing
pro-inflammatory cytokines have been shown in severe SARS-CoV disease compared to
mild/moderate disease.

Accordingly, there has been considerable interest in a potential role of repurposing
existing immunosuppressive therapies for treatment of severe COVID-19. The key
mediators and pathways that drive hyperinflammation in COVID-19 are unclear and
greater elucidation of these processes will inform repurposing of existing therapies
or development of novel approaches to effective treatment. The implication of
baseline dysregulated T cell responses such as those seen in chronic lung disease
(e.g. asthma, COPD, Cystic Fibrosis) is again unclear and requires further
mechanistic understanding. As such it is, as yet, unclear whether immunosuppressive
therapies currently in clinical trials will be beneficial or harmful in these large
cohorts of susceptible patients.

3. Dysregulated interferon-dependent endothelial function to COVID-19

Acute respiratory distress syndrome (ARDS) is characterised by hydrostatic pulmonary
oedema, elevated pulmonary vascular resistance and coagulopathy, there is disruption
of endothelial function with dysregulated release of the endothelially derived
vasoactive hormones. Circulating levels of endothelin-1 are raised in patients with
ARDS[16], likely due to increased production and decreased clearance in the lungs
potentially regulated by Angiopoietin-1.

Severe SARS-CoV-2 infection is associated with cytokine storm. Raised serum levels
of a number of key inflammatory cytokines including IFN-gamma, TNF-alpha, IP10, IL-8
and IL-10 and elevated levels of IL-6 are strongly associated with mortality. IL-6
release is synergistically induced by IFN-gamma and TNF-alpha and in a positive
feedback mechanism, a low dose of IL-6 strongly enhances the cellular responses to
IFN-alpha and modulates interferon-stimulated gene expression. In an experimental
murine model, we found that infection with rhinovirus with IFN co-stimulation leads
to elevated bronchoalveolar lavage levels of IL-6 (Singanayagam et al, unpublished
observations). These data suggest that in patients with severe COVID-19, a
hyperinflammatory process ensues and we hypothesise that through dysregulated and
unchecked IFN production, key vasculo-inflammatory interferon-stimulated genes
including IP10 and ET-1 drive worse outcomes with increased parenchymal and
pulmonary vascular inflammation. This results in a dichotomy whereby initial IFN
induction is required to limit viral replication, but late unchecked production can
worsen pathophysiology. This is supported by emerging clinical data suggesting a
unique phenotype to SARS-CoV-2 critical care patients characterised by severe
hypoxia with near normal lung compliance.

4. Altered viral entry receptor expression: Recent data indicates SARS-CoV-2 utilises
angiotensin-converting enzyme (ACE)2 as a receptor for viral attachment and also the
protease TMPRSS2 which cleaves the spike(S) protein to allow fusion of the virus
with cellular membranes. ACE2 and TMPRSS2 are frequently co-expressed in the
pulmonary epithelium. A recent study also showed that ACE2 is an interferon
stimulated gene (ISG)(i.e. directly induced by the anti-viral response) and
therefore, SARS-CoV-2 might upregulate its own receptor to propagate infection.
Recent data indicates that smokers and subjects with chronic obstructive pulmonary
disease (COPD) have increased pulmonary ACE2 expression suggesting that chronic lung
disease might facilitate greater infection with SARS-CoV-2. Conversely, ACE2
expression protects against acute lung injury in the context of acid aspiration or
sepsis and therefore, if ACE2 induction by SARS-CoVs is impaired in chronic lung
disease, this could predispose to adverse outcome.

5. Increased secondary bacterial and/or fungal infections

In early case series, secondary infections have been observed more frequently in subjects
with severe COVID-19 disease. Secondary bacterial infection is a well-recognised
phenomenon following rhinovirus, influenza or respiratory syncytial virus and may be
mechanistically driven by a range of possible mechanisms including macrophage
phagocytosis or neutrophil elastase-mediated cleavage of anti-microbial peptides. These
processes may be worsened by use of corticosteroids28 and, notably, a greater proportion
of patients with secondary infections following COVID-19 had been prescribed steroids.

Invasive fungal disease consistent with aspergillosis has also been observed for both
severe acute respiratory syndrome coronavirus (SARS-CoV-2003) and Middle East Respiratory
Syndrome coronavirus (MERS-CoV), and anecdotal reports of post-mortems in patients with
COVID-19 from China suggest that Aspergillus pulmonary infections occur. These data are
early warning signs that secondary invasive fungal infections may present an increasingly
serious complication in patients affected with COVID-19 as the pandemic progresses.
Understanding the mechanistic basis for immune susceptibility to secondary
bacterial/fungal infection and an ability to prevent these secondary infections could be
a key step towards improving outcomes from the disease.

1.2 RATIONALE FOR CURRENT STUDY

In this study, the investigators will analyse blood and airway samples from individuals
with confirmed COVID-19 to understand the immune mechanisms that lead to severe disease.
We will elucidate specific mechanisms that drive more severe pathology in higher risk
individuals such as those with chronic lung disease. Through detailed immunoprofiling, we
will identify key pathways and druggable targets to exploit in future clinical
intervention studies.

Hypotheses:

1. High risk individuals including individuals with chronic lung disease have impaired
anti-viral immune responses to SARS-CoV-2, increasing virus-induced inflammation.

2. Interferon-dependent endothelial dysfunction drives pathogenicity in SARS-CoV-2
infection.

3. Susceptibility to secondary bacterial and fungal infection in chronic lung disease
is related to selective pathway defects in innate immune function.

2. STUDY OBJECTIVES

Primary objective:

1. Understand the immune mechanisms driving COVID-19 disease in patients with a history
of lung disease

Secondary objectives

1. Identify immune correlates of protection and lung pathology in SARS-CoV-2 infection.

2. Analysis of endothelial function in mild and severe COVID-19 patients and ex vivo
using virus stimulated blood-derived endothelial cells from patients with COVID-19.

3. Evaluation of changes in upper/lower airway microbiota and mycobiota occurring
during COVID-19.

4. Evaluation of ex vivo peripheral blood immune responses to viral, bacterial and
fungal stimuli in patients with COVID-19 and chronic lung disease in comparison to
patients with COVID-19 and no chronic lung disease.

5. Analysis of quality of life impact of COVID-19 infection in chronic lung disease.

3. STUDY DESIGN

Through a 24 month prospective longitudinal multicentre (Royal Brompton & Harefield NHS
Trust Chelsea and Westminster NHS Foundation Trust, and Imperial College Healthcare NHS
Trusts) observational study, the study team will analyse blood, sputum and nasal sampling
alongside non-invasive EndoPAT device testing in a total pf 230 subjects. The study team
will obtain peripheral blood, sputum, nasal lavage, brushings and nasal synthetic
absorptive matrix (SAM) samples and perform non-invasive EndoPAT testing on the day of
hospital presentation and at weekly intervals during inpatient setting and then during
outpatient visits post-discharge over a 12 month follow-up period. The study team will
collect clinical information including demographics, routine laboratory investigations,
clinical symptom scores and outcomes. Linked pseudoanonymised radiology imaging will also
be transferred for analysis.

Spontaneously expectorated sputum, nasal lavage, brushings and nasal synthetic absorptive
matrix (SAM) samples will be taken and processed as previously described and stored at
-800C for downstream analyses including ELISA/MSD, viral load measurement and microbiome
analysis. Venous blood (60mls) will be taken into Lithium Heparin tubes and PAXgene RNA
tubes and transferred to Imperial College (RBH) for processing and storage. PBMCs will be
extracted for in vitro stimulation assays and whole blood (1ml) and serum (2mls) isolated
and stored at -80oC for further analysis. PAXgene tubes will be stored at -80C for
further host genetic sequencing analysis. Further immunological analysis using yeast
surface display for serum antibody profiling, single B cell sorting to generate
monoclonal antibodies and ELISPOT to analyse CD4 and CD8 T cell ELISPOT to SARS CoV-2
peptide pools. Further deep immunological profiling using cytokine analysis, PBMC
cytokine response to viral (or viral PRR agonist), fungal stimuli, yeast surface display
for serum antibody profiling, single memory B cell expression cloning will be carried.
CD4 and CD8 T cell ELISPOT will be performed as previously described. Non-invasive
EndoPAT testing will also be performed as previously described.