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.