Pathogenesis of viral infection in exacerbations of airways disease

Andrew I. Ritchie,1,2,3 Hugo A. Farne,1,2,3 Aran Singanayagam,1,2,3 David J. Jackson,1,2,3 Patrick

Mallia,1,2,3 and Sebastian L. Johnston.1,2,3*

1Airway Disease Infection Section, National Heart and Lung Institute, Imperial College,

London, UK.

2MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK.

3Imperial College Healthcare NHS Trust, London, UK.

Corresponding author: Professor Sebastian L Johnston, Airway Disease Infection Section,

National Heart and Lung Institute, Imperial College London, Norfolk Place, London W2 1PG,

United Kingdom. s.johnston@imperial.ac.uk. Tel: +442075943764, fax: +442072628913.

Author contributionsWriting of manuscript and critical review of content: All authors.

Word Count: 4858

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Abstract

Chronic airways diseases are a significant cause of morbidity and mortality worldwide with

prevalence predicted to increase in the future. Respiratory viruses are the most common

cause of acute pulmonary infection and there is clear evidence of their role in acute

exacerbations of inflammatory airways diseases such as asthma and chronic obstructive

pulmonary disease. Studies have reported impaired host responses to virus infection in

these diseases and a better understanding of the mechanisms of these abnormal immune

responses has the potential to lead to the development of novel therapeutic targets for

virus-induced exacerbations. The aim of this article is to review the current knowledge

regarding the role of viruses and immune modulation in acute exacerbations of chronic

pulmonary diseases and discuss exciting areas for future research and novel treatments.

Keywords

Asthma, chronic obstructive pulmonary disease, respiratory viruses, rhinovirus, interferon

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Introduction

Chronic airways diseases are a significant source of morbidity and mortality worldwide.

COPD is predicted to be the fourth leading cause of mortality by 2030[1] and in excess of

250 million people worldwide suffer from asthma. COPD and asthma are believed to be

caused by exposure to environmental agents (mainly cigarette smoke and aeroallergens,

respectively) in patients with a susceptible genetic background.

Despite differences in the pathophysiology, COPD and asthma have a similar presentation

and disease course: chronic respiratory symptoms, such as wheeze and breathlessness,

punctuated by periods of increased symptomatology, known as ‘acute exacerbations’ [2].

Acute exacerbations are significant events in the course of chronic pulmonary diseases as

they accelerate disease progression, impair quality of life, and are the predominant cause of

mortality. Additionally they often necessitate unscheduled healthcare visits, treatment costs

and hospitalizations[3].

Preventing exacerbations is a major unmet therapeutic goal. A crucial step towards this goal

is the recognition that acute exacerbations are most commonly due to respiratory virus

infection. It follows that the host immune response to virus infection may be impaired, and

that a better understanding of how the immune response differs in asthma and COPD has

the potential to lead to the development of new therapies. This article discusses the current

knowledge of the role of viruses and host immune responses in asthma and COPD.

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Virus infections as aetiological causes for acute asthma exacerbations

The Global Initiative for Asthma (GINA) defines asthma as ”a chronic inflammatory disorder

of the airways in which many cells and cellular elements play a role” [4]. Asthma is

characterized by airway hyperresponsiveness with subsequent diffuse reversible airways

obstruction, manifesting as wheeze, chest tightness, breathlessness and cough. Recent

discoveries have led to the re-classification of asthma as a heterogeneous condition

encompassing several endotypes. The classic pattern is that of T helper 2 lymphocyte (Th2)-

predominant inflammation, with high levels of airway eosinophils, IgE, mast cells and

exhaled nitric oxide (FeNO)[5]. Four additional asthma endotypes are recognised: adult

onset asthma with eosinophilia but an absence of allergic disease [6]; exercise-induced

asthma, which is likely to be mediated via activated mast cells; obesity-related asthma, who

have minimal Th2 inflammation; and a final group who also have little Th2 inflammation but

notably have a sputum neutrophilia and a Th type 17 response. Work is ongoing to precisely

define these subpopulations and offer better targeted therapy.

It has long been recognized that viral respiratory tract infections trigger exacerbations of

asthma in both adults and children but early studies reported low detection rates of viruses

in asthma exacerbations, casting doubt on this association[7, 8]. The development of

modern highly sensitive and specific molecular diagnostic techniques using reverse

transcriptase polymerase chain reaction (PCR) technology demonstrated the presence of

viruses in 80-85% of asthma exacerbations in school-aged children and ~80% of

exacerbations in adults [9-12], leading to a re-evaluation of the role of virus infections in

asthma.

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Viruses that are most commonly identified during asthma exacerbations include

rhinoviruses, influenza viruses, respiratory syncytial virus (RSV), coronaviruses, human

metapneumoviruses, parainfluenza viruses and adenoviruses. Influenza infection is common

during the winter, frequently emerging as a local or national epidemic. The 2009 H1N1

influenza A pandemic brought about a number of studies highlighting asthma as an

important comorbidity in those infected [13]. Moreover asthma was associated with

increased disease severity, with a higher risk of hospitalization, need for intensive care and

overall mortality [14-16].

A study of co-habiting partners, one of whom had asthma, found that although there was no

difference in frequency of rhinovirus infections, the asthmatics exhibited greater lower

respiratory symptoms and change in airway physiology [17]. It was subsequently shown that

experimental rhinovirus infection in asthmatics precipitates acute exacerbation, providing

direct evidence for a causative role of rhinoviruses [18, 19]. Additionally, the inflammatory

and symptomatic consequences of a rhinovirus infection in asthmatics are more severe than

in non-asthmatics. The demonstration that, following nasal inoculation, rhinovirus can infect

bronchial epithelium provided a mechanism by which rhinovirus might trigger lower

respiratory tract symptoms [20, 21]. These studies also highlight the differences in the

inflammatory and symptomatic response to virus infection in asthmatics and non-

asthmatics. Greater understanding of the mechanisms underlying virus-induced asthma

exacerbations is crucial in order to develop new treatment strategies.

Immune response to rhinovirus infection

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The majority of current in vitro and in vivo work focuses on rhinoviruses, since this is the

virus type most commonly detected in asthma exacerbations. Epidemiological studies

confirm that human rhinoviruses are the predominant virus type detected in asthma

exacerbations [22]. Rhinoviruses are small (30 nanometer), non-enveloped, single-stranded

RNA viruses from the Picornaviridae family [23]. 101 serotypes have been identified,

classified into major and minor subgroups [24] . The major group (over 90 serotypes) bind to

the human intercellular adhesion molecule-1 (ICAM-1) receptor [25]; the minor group bind

to low density lipoprotein receptors (LDLR)[26]. Alternatively they are grouped as

rhinovirus-A and -B based on genetic sequencing similarity. Most of these 101 viruses, plus

an additional ~60 C subtype strains (that are not easy to grow and therefore have not been

serotyped), are believed to be in circulation currently. Despite difficulties characterising the

rhinovirus C subtype, recent work suggests that human cadherin-related family member

3 (CDHR3) is a functional receptor for RV-C [27]. A & C subtypes are thought to be more

associated with wheezing illnesses than B subtypes.

Rhinoviruses primarily enter and replicate in epithelial cells, triggering a cascade of immune,

inflammatory responses and cytotoxicity [21, 28]. Human bronchial epithelial cells (HBEC)

are of great interest during viral respiratory infections because they serve as the major host

cell for viral replication [29]. Other cells, including airway smooth muscle cells [30],

fibroblasts [31] and alveolar macrophages [18], may also be important sites of infection,

inducing inflammatory mediators [18, 32-34], but significant viral replication seems to be

limited to epithelial cells [35].

Rhinovirus replication leads to synthesis of viral RNA, which is recognised by innate immune

receptors. These pattern recognition receptors include the cytosolic RNA helicases, retinoic

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acid inducible gene I (RIG-I), melanoma differentiation-associated protein-5 (MDA-5),

dsRNA/protein kinase receptor (PKR) and the toll-like receptors (TLR)-3 , -7 and -8 [36-39].

Following ligand and receptor binding there is a signalling cascade which leads to the

activation of transcription factors including interferon regulatory factors (IRF)-3 and-7,

nuclear factor-κB (NF-κB), activating transcription factor 2 (ATF2) and c-Jun [35, 40, 41]. The

activated transcription factors then translocate to the nucleus of bronchial epithelial cells to

induce transcription of type I and type III interferons (IFN-α/-β and IFN-λ1, 2, 3 respectively)

and many pro-inflammatory cytokines and chemokines including Interleukin(IL)-6, IL-8 (also

known as C-X-C motif ligand 8, CXCL8), IL-16, epithelial-derived neutrophil-activating peptide

78 (ENA-78, or CXCL5), C-C motif ligand 5 (CCL-5, also known as Regulated on Activation,

Normal T cell Expressed and Secreted, RANTES), and IFN-γ-induced protein 10 kDa (IP-10, or

CXCL10)[21, 42-49].

IFNs are of central importance in the innate immune response to viral infection[50]. Their

antiviral effects occur directly through inhibition of viral replication in cells, and indirectly

through stimulation of innate and adaptive immune responses. The direct antiviral activity

of type I IFNs is mediated by a number of mechanisms including blocking viral entry into

cells, control of viral transcription, cleavage of RNA, blocking translation, and induction of

apoptosis [51]. These protective effects are brought about through the up-regulation of IFN-

stimulated genes (ISGs) and the production of antiviral proteins[50]. The indirect effect of

IFNs is mediated through induction of cytokines and chemokines leading to recruitment of

natural killer cells and CD4 and CD8 T cells [52], up-regulation of the expression of MHC-I on

cells and up-regulation of antigen-presenting cell co-stimulatory molecules. Therefore, a

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robust IFN response is central to effective antiviral responses and resolution of virus

infection.

Abnormalities of immune responses associated with asthma

Deficient innate anti-viral immunity

The first report that indicated that the lungs’ innate immune response to virus infection may

be impaired in asthma emerged in 2005 when Wark et al investigated primary human

bronchial epithelial cells (HBECs) taken from normal and asthmatic subjects at bronchoscopy

[53]. They showed that rhinovirus replication was increased in the cells from asthmatics

compared with those from non-asthmatic subjects. Interestingly, they found induction of

IFN-β was both delayed and deficient in asthmatics and administration of exogenous IFN-β

resulted in induced apoptosis and reduced virus replication, demonstrating a causal link

between deficient IFN-β and increased virus replication. The observation of a restored

antiviral response with administration of type I IFN was validated in a subsequent study [54],

and our group has shown that IFN-α and IFN-β production by alveolar macrophages is also

impaired in asthmatics [55]. Contoli and colleagues[47] similarly showed deficient IFN-λ

production by HBECs and alveolar macrophages from a group of atopic asthmatics infected

ex vivo with rhinovirus-16. This study utilised a human rhinovirus induced asthma

exacerbation model to demonstrate that exacerbation severity was inversely proportional

to IFN-λ generation.

Not all studies have found deficient IFN responses in asthmatics [56-59]. It must also be

noted that the in vitro studies discussed are small, with different experimental conditions,

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such as cell culture techniques and virus dose (see table 1). Thus although IFN deficiency is

an interesting and biologically plausible possible mechanism underlying the observed

increased severity of virus infection in asthma, it has not been universally demonstrated.

Several studies have reported relationships between markers of asthma/allergy severity and

IFN responses[55, 60-62], and it may be that IFN delay/deficiency is only detected in more

severe/less well-controlled disease. Inhaled IFN-β recently proceeded to a phase two

randomized, placebo-controlled study in persistent asthma (British Thoracic Society (BTS)

guidelines steps 2-5) at the onset of a viral infection [63]. The primary outcome, change in

ACQ-6 score from baseline to Day 8, was not significantly different between the treatment

and placebo arms. However, in pre-specified subgroup analyses, IFN-β effectively prevented

virus-induced worsenings of asthma symptoms in patients with more severe disease (BTS

Steps 4–5), leading authors to conclude that future studies of IFN-β should target patients

with moderate or severe asthma. It is also possible that the exact nature of IFN deficiency is

specific to certain asthmatic phenotypes. Further work is indicated to understand these

mechanisms, with focus on greater subject numbers, careful patient selection and

characterization.

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Table 1. Summary of published studies assessing the role of type I and III IFNs in asthma

Studies supporting type I and/or III IFN deficiency in asthma Studies not supporting type I and/or III IFN deficiency in asthma

Study Key finding Study Key finding

Wark et al, 2005 [53] Delayed and deficient IFN-β,

impairment of apoptosis and

increased rhinovirus replication

in atopic asthmatic HBECs.

Lopez-Souza et al, 2009 [57] No significant difference

between asthmatic and

healthy subjects in rhinovirus

induced IFN-β expression in

nasal epithelial cells and

HBECs.

Contoli et al, 2006 [47] Atopic asthmatics have

deficient rhinovirus induced

IFN-λ production by HBECs and

alveolar macrophages.

Significant relationship between

degree of IFN deficiency and

rhinovirus induced asthma

exacerbation severity.

Bochkov et al, 2010 [56] Using microarrays and qPCR,

no difference in rhinovirus

induced type I or III IFN

expression between non

atopic-non asthmatics and

atopic asthmatics.

Gehlhar et al, 2006 [64] Respiratory syncytial virus (RSV)

and Newcastle disease virus

(NDV) induced IFN-alpha2

release from peripheral blood

cells of allergic asthmatic

patients was significantly

reduced compared with healthy

controls.

Sykes et al, 2014[58] No difference in rhinovirus

induced IFN-λ and, to a lesser

degree, IFN-β in the HBECs

from mild, well controlled

asthmatic subjects.

Bufe et al, 2002[65] Significantly lower levels of

virus induced IFN-α in blood

cultures of children with atopic

asthma than in non-atopic

asthmatics and healthy

children.

Patel et al, 2014 [66] This study examined ex vivo

influenza A and RSV infection

of HBECs. Whilst there was

impaired induction IFN- λ in

response to RSV and an

increase in influenza virus load

in asthmatic groups other

results were broadly similar.

Gill et al, 2010 [67] Influenza A induces significantly

less IFN-α in plasmacytoid

dendritic cells of asthmatics.

Relationship between degree of

IFN deficiency and serum IgE.

Cross-linking IgE on dendritic

cells impairs IFN induction.

Sykes et al, 2014 [59] In ex vivo BECs and PBMCs

no difference was observed in

TLR induced interferon

production between well

controlled asthmatic and non-

asthmatic subjects.

Uller et al, 2010 [68] Double stranded RNA induces

disproportionate expression of

thymic stromal lymphoprotein

(TSLP) versus IFN-β in

asthmatics ex vivo.

Spann et al, 2014 [69] In response to stimulation

from RSV or hMPV, no intrinsic

defect in IFN-β or -λ

production seen in tracheal

epithelial cells of children with

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wheeze and/or atopy.

likura et al, 2011 [70] IFN-α production was

significantly lower in rhinovirus

stimulated peripheral blood

mononuclear cells obtained

from asthmatic youths

compared to healthy controls.

Herbert et al, 2014 [71] Human airway epithelial cells,

co-cultured with/without IL-4

and IL-13 were stimulated with

poly I:C. There was no

reduction in anti-viral response

genes and a demonstrable

increase in mRNA for type III

interferon.

Forbes et al, 2012 [72] Peripheral blood mononuclear

cells taken from pregnant

asthmatics, asthmatics and

healthy controls were

stimultated with rhinovirus or a

TLR7 agonist. Significantly

reduced IFN-α and -λ, during

asthma exacerbations was

observed. Pregnant asthmatics

had significantly reduced IFN-λ

responses compared with

healthy non-pregnant women.

Edwards et al, 2012 [60] HBECs obtained from severe

asthmatic children have

profoundly impaired rhinovirus

-induced IFN-β and IFN-λ mRNA

and protein production.

Sykes et al, 2012 [55] Rhinovirus induction of type I

IFNs in BAL cells is delayed and

deficient in asthmatics and is

associated with greater airway

hyperresponsiveness and skin

prick test positivity.

Baraldo et al, 2012 [61] In HBECs from children,

deficient rhinovirus induced

IFN-β and IFN-λ present in

asthmatics irrespective of their

atopic status and in atopic

patients without asthma.

Relationships between IFN

deficiency and bronchial biopsy

eosinophilia, IL-4 positivity and

epithelial damage and with

total serum IgE.

Durrani et al, 2012 [73] Following IgE receptor cross-

linking in peripheral blood

mononuclear cells, rhinovirus

induction of IFN-α and IFN-λ

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were significantly lower in

allergic asthmatic children than

in allergic non-asthmatic

children and non-allergic non-

asthmatic children. IgE receptor

expression on plasmacytoid

dendritic cells was inversely

associated with IFN production.

Zhu et al, 2014 [74] The effect of Alternaria

alternata, commonly found in

the airways of asthmatics, was

investigated in primary tracheo-

bronchial epithelial cells

exposed to rhinovirus or dsRNA.

The effect of both fungus

together with virus/synthetic

viral mimicker was a marked

decrease in type I and type III

IFN gene expression.

Parsons et al, 2014 [75] Asthmatic pBECs had

significantly reduced release of

IFN-λ in response to rhinovirus

1B infection compared with

healthy cultures.

Spann et al, 2014 [69] Nasal epithelial cells from

children with wheeze and/or

atopy produced less IFN-β, but

not IFN-λ, in response to RSV

infection.

Wagener et al, 2014 [76] dsRNA-induced changes in gene

expression profiles of primary

nasal and bronchial epithelial

cells from patients with asthma,

rhinitis and controls.

Induction of several interferon-

related genes by dsRNA was

impaired in asthmatic primary

nasal and bronchial epithelial

cells compared to rhinitis and

healthy controls.

The mechanisms underpinning impaired induction of IFN in response to virus infection in

asthmatics are unclear. Suppressor of cytokine signalling (SOCS) 1 and SOCS3 are proteins

known to function as negative regulators of cytokines. In mice, SOCS1 and 2 negatively

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feedback on Th2 immunity, [77-80], whilst in human studies a genetic polymorphism

enhancing SOCS1 is associated with asthma [81] and T cell SOCS3 mRNA levels are increased

in asthmatic patients [77]. Edwards et al reported that rhinovirus infection and Th2 and pro-

inflammatory cytokines increase levels of SOCS1 and SOCS3 mRNA in HBECs, while SOCS1,

but not SOCS3, was increased in bronchial biopsies in asthmatic patients [82]. Increased

SOCS1 mRNA expression was associated with impaired IFN induction and increased virus

replication in HBECs from children with severe asthma. As SOCS1 inhibits both virus-

stimulated IFN induction, antagonism of SOCS1 is an attractive therapeutic target (see figure

1).

Transforming growth factor (TGF)-β is a multifunctional cytokine of interest because it

mediates suppression of both IFN-λ and IFN-β in primary bronchial epithelial cells from

healthy subjects exposed to rhinovirus [83], and IFN-α and IFN-β in rhinovirus-infected

fibroblasts [84]. Work by Mathur and colleagues further investigated the role of TGF-β

mediated IFN suppression by co-culturing rhinovirus infected BEAS-2B monolayers with

eosinophils to demonstrate enhanced suppression of IFN induction [85]. This fits with the

observation that airway eosinophilia is associated with an increased risk of exacerbation in

asthma.

An alternative mechanism of IFN suppression is through the prominent Th2 cytokine, IL-13,

which induces IL-1 receptor associated kinase M (IRAK-M). IRAK-M over-expression is

present in the asthmatic airway, where it appears to promote lung epithelial rhinovirus

replication and autophagy but crucially inhibits rhinovirus-induced IFN-β and IFN-λ1

expression [86]. In vitro, recombinant IL-13 suppressed dsRNA-induced expression of IFN-λs

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in airway epithelial cells whilst a Janus kinase inhibitor prevented the IL-13 mediated

suppression [87].

To investigate effects of asthmatic treatment on innate anti-viral immunity, peripheral blood

mononuclear cells were infected with rhinovirus and pre-treated with budesonide with and

without formoterol. There was reduced type I IFN induction in budesonide treated cells

from both healthy and asthmatic donors [88]. An influenza A murine infection model

supported this finding when it demonstrated more severe disease in the steroid treated

group and interestingly went on to show adjuvant IFN treatment markedly reduced GCS-

amplified infections in human airway cells and in mouse lung [89].

Virus induction of type 2 immunity

Mechanisms by which virus infections (which induce a type 1 immune response), worsen

the type 2 inflammatory pattern predominantly seen in asthma, are debated. Our group has

recently used novel methods to sample airway lining fluid from the nose (nasosorption) and

the bronchus (bronchosorption) combined with low volume protein detection methods to

demonstrate for the first time that the type 2 cytokines IL-4, IL-5, and IL-13 are all induced in

vivo in asthmatic, but not in normal subjects in a rhinovirus-induced exacerbation model

[19]. Furthermore, levels of IL-5 and IL-13 during infection correlated significantly with

exacerbation severity, suggesting that the induction of type 2 cytokines might be

functionally important.

The importance of epithelial-derived mediators that are capable of promoting the type 2

immune response is emerging from animal studies and molecules implicated include thymic

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stromal lymphopoietin (TSLP), IL-25, and IL-33. Their importance in human asthma

exacerbations was, until very recently, unknown. Our group investigated the role of IL-25 in

this context and found that rhinovirus-infected HBECs from asthmatic donors had greater IL-

25 induction, which correlated with donor atopic status. Human IL-25 levels were induced

by experimental rhinovirus infection in vivo and expression was greater in asthmatics at

baseline and during infection. In mice, rhinovirus infection also induced IL-25 and

augmented allergen-induced IL-25 and blockade of the IL-25 receptor markedly suppressed

many rhinovirus-induced exacerbation-specific responses, including type 2 cytokines and

chemokines, mucus production, and recruitment of eosinophils, lymphocytes and

neutrophils, IL-4+ basophils and Th2 cells, as well as ILC2s. Therefore, asthmatic epithelial

cells have an increased intrinsic capacity for IL-25 expression in response to a viral infection,

and IL-25 is a key mediator of rhinovirus induced exacerbations of pulmonary inflammation

[90].

We also recently investigated the role of IL-33 as an inducer of type 2 inflammation. IL-33

was induced by rhinovirus in the asthmatic airway in vivo and IL-33 levels in asthmatic

subjects were related to rhinovirus-induced asthma exacerbation severity. Further,

induction of IL-33 correlated with viral load and levels of IL-5 and IL-13 induced by infection.

Rhinovirus infection of human primary BECs in vitro strongly induced IL-33, and culture of

human T cells and ILC2s with supernatants of rhinovirus-infected BECs (but not with

supernatants of mock-infected BECs) strongly induced type 2 cytokines (IL-4, IL-5 and IL-13

in T cells, and IL-5 and IL-13 in ILC2s). These inductions were entirely dependent on IL-33, as

blocking the IL-33 receptor in these co-cultures completely suppressed the inductions

observed in the cultures with supernatants of rhinovirus infected BECs. Thus, virus-induced

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IL-33 released from BECs and IL-33-responsive T cells and ILC2s are key mechanistic links

between viral infection and exacerbation of asthma [19].

In summary, there is evidence that anti-viral immune responses in asthmatics are impaired

(delayed and deficient) and this likely underlies increased disease severity following virus

infection in asthma. Further work is required to determine whether these impairments are

common to all asthmatics or whether they are unique to a phenotypic cluster. A novel virus

infection/epithelial cell-derived cytokine/T cell/ILC2/type 2 cytokine pathway has been

identified, in which IL-25 and IL-33 and their receptors appear exciting new targets for

development of novel therapies for asthma exacerbations.

Virus infections as aetiological causes for acute COPD exacerbations

As in asthma, viruses and in particular rhinoviruses are increasingly thought to be a major

cause of COPD exacerbations. It has long been known that exacerbation frequency doubles

in winter months [91, 92], with many exacerbations preceded by coryzal symptoms [93-95].

With the advent of PCR, viruses have been detected in 22-64% of COPD exacerbations [94,

96-118], with rhinoviruses the most prevalent. Positive viral PCR during acute exacerbations

is unlikely to represent chronic infection or seasonal carriage, as viral PCR is rarely positive

when patients are sampled prior to [103, 104, 118] or after an exacerbation [94, 112, 113],

or in time-matched controls [97, 101, 106, 115].

Similarly to asthma, causal evidence that viruses can induce exacerbations comes from a

human model of experimental rhinovirus infection in COPD [119-123]. Inoculation of stable

COPD patients with rhinovirus induces lower respiratory tract symptoms, airflow

16

obstruction, and systemic and airway inflammation mimicking an exacerbation. Indeed in

experimental infection studies, exacerbations, using the same definition as studies

investigating naturally occurring exacerbations, occurred in over 90% of rhinovirus infected

COPD subjects, suggesting that the virus detection frequencies reported in naturally

occurring exacerbations (22-64%) are actually a gross underestimate of the true frequency

of viruses as precipitants of exacerbations.

Abnormalities of immune responses associated with COPD

Deficient innate anti-viral immunity

COPD is associated with profound changes in innate immunity that are likely to be relevant

in the pathogenesis of exacerbations. Smoking damages mucociliary clearance [124], and

the rhinovirus binding receptor ICAM-1 is upregulated on bronchial epithelial cells in COPD

[125]. Alveolar macrophages, which police the lungs to form a first line of defence, are

defective in COPD, with impairments in their ability to phagocytose bacteria [126, 127] and

efferocytose dead and dying cells [128] compared to alveolar macrophages from healthy

smoking and non-smoking controls.

Several studies have identified a ‘frequent exacerbator’ phenotype [129] who are likely

more susceptible to virus and/or bacterial infection [118, 130]. Alveolar macrophages taken

from such patients (defined as having had an exacerbation during a one year period) and

exposed to bacteria or TLR ligands ex vivo demonstrated impaired induction of CXCL8/IL-8

and TNF-α, compared to macrophages from patients who were exacerbation free for a year

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[130]. More studies are needed to elucidate differences in the immune responses of

frequent exacerbators that might predispose them to infections.

Unlike asthma, it is unclear whether innate IFN responses are defective in COPD. Varying

responses have been demonstrated in mouse models of COPD. One study reported that

mice exposed to four weekly doses of elastase and LPS followed by rhinovirus infection had

an impaired IFN-α/β response with increased virus loads compared to mice receiving

rhinovirus alone[131]. A separate study which attempted to replicate this model showed

impaired IFN responses but lower rather than higher virus loads, and reported an

alternative model of single dose elastase challenge combined with rhinovirus infection in

which IFN-λ was reduced compared to mice treated with rhinovirus alone, but IFN-β and

rhinovirus loads were unaffected [132]. By contrast, IFN responses have been shown to be

increased in cigarette smoke- and influenza-exposed mice [131, 133, 134].

In human studies, rhinovirus-exposed epithelial cells from COPD subjects showed increased

rhinovirus replication ex vivo, but surprisingly increased rather than decreased induction of

IFN-λ, with undetectable levels of IFN-α/β [135] (for IFN-α this is not surprising as epithelial

cells do not produce IFN-α [136]). Another study also found increased IFN-λ mRNA and

protein and increased IFN-β mRNA in COPD epithelial cells compared to cells from healthy

subjects, but did not find any differences in rhinovirus replication [137]. In contrast, BAL

cells from COPD patients infected ex vivo with rhinovirus demonstrated significantly

impaired IFN-β production, with non-significant trends for reductions in IFN-α and IFN-λ

[119]. Further studies of IFN induction in response to viral infection in epithelial and BAL

cells in COPD are clearly needed.

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Airway inflammation in stable COPD

Even during clinical stability (i.e. in the absence of viral infection), COPD results in a

persistent pro-inflammatory state with increased numbers of immune cells, inflammatory

mediators, and proteinases [138, 139]. Chemokines that recruit neutrophils, macrophages

and T cells are found at elevated levels [139] – and are in turn secreted by the newly

recruited immune cells. Specifically, CCL2/MCP-1 and CXCL1 (also known as growth-

regulated oncogene alpha, GRO-α) attract circulating monocytes, the precursors of

macrophages [140]. E-selectin on endothelial cells, to which neutrophils bind, is upregulated

in COPD [125]. Their chemotaxis is then mediated by increased levels of leukotriene (LT) B4,

CXCL1/GRO-α, CXCL5/ENA-78, and CXCL8/IL-8 [140]. CXCL9 (or Monokine Induced by

Gamma interferon, MIG), CXCL10/IP-10 and CXCL11 (or Interferon-inducible T-cell Alpha

Chemoattractant, I-TAC) act on the CXCR3 receptor on T cells, guiding them to the lungs

[141].

Neutrophils, macrophages and T cells are not only present in greater numbers, but are

functionally different from those of non-smoker and non-obstructed smoker controls.

Neutrophils from COPD patients are activated, as indicated by the presence of secreted

granule proteins [142], and migrate at a faster rate but with less accuracy than controls

[143]. Similarly, alveolar macrophages from COPD patients secrete more inflammatory

mediators and elastolytic enzymes compared to controls [144], as well as demonstrating the

impairments in phagocytosis and efferocytosis previously described.

19

This abundance of immune cells secrete pro-inflammatory cytokines, particularly TNF-α and

IL-6 [145]. Two more factors add to this inflammatory milieu: microbial colonisation of the

damaged lungs of COPD patients [146], and oxidants, both exogenous (e.g. cigarette smoke

[147]) and endogenous (e.g. reactive oxygen species produced by neutrophils). These

activate NF-κB, in turn inducing various inflammatory mediators [145]. Biopsies have

confirmed increased expression of NF-κB in COPD [148].

Virus induction of airway inflammation

One difficulty in studying the response to viral infection in COPD is identifying changes that

are over-and-above what would be seen in either virus infection alone or COPD alone.

Surprisingly few in vitro studies compare the responses of cells from COPD patients to viral

infection with those from healthy controls [119, 135, 137]. Similarly, few studies of naturally

occurring exacerbations have examined the inflammatory responses specific to viral

infection; most group viral, bacterial and non-infectious aetiologies together, potentially

masking important differences. Even studies that try to separate these will inadvertently

misclassify as ‘virus negative’ exacerbations that were initiated by a virus that escaped

detection; this is likely to be a large proportion, as alluded to previously.

A challenge in animal research has been to produce a model of COPD that accurately mimics

the pathology observed in man. Short-term cigarette smoke exposure (<4 weeks) is

insufficient to cause emphysema or airway remodelling, whilst lipopolysaccharide (LPS) and

elastase replicate the anatomical features but with a different inflammatory infiltrate [149].

Moreover many use influenza, which may be less relevant in human COPD where there is

widespread uptake of the influenza vaccine.

20

Experimental rhinovirus infection in humans with COPD offers the greatest prospect of

elucidating the sequence of events culminating in an exacerbation, but such studies are

logistically challenging and restricted to patients with mild/moderate disease. Nonetheless,

this model uniquely provides the opportunity to sample repeatedly as the exacerbation

evolves, allowing accurate dissection of the timings of responses and their relationship to

not only viral load, but also to secondary bacterial infections.

Studies that look specifically at viral infection in COPD compared to controls suggest that

there is an exaggerated inflammatory response (see Table 2). In vitro and human studies

almost uniformly show an increase in chemokines. The principal conflicting study, showing

deficient induction of CXCL10/IP-10 after experimental rhinovirus infection, used BAL cells

cultured ex vivo that were predominantly (>95%) macrophages [119]; it may be that

macrophages are not the principle source of CXCL10/IP-10 in this setting. Indeed monocytes

from the blood of volunteer donors secrete little CXCL10/IP-10 in response to rhinovirus, but

when co-cultured with epithelial cells there is a synergistic augmentation of CXCL10/IP-10

levels [150]. This is predominantly from epithelial cells, as it persists if those cells are

cultured with medium from rhinovirus-exposed monocytes, but not vice versa. It may be

significant that epithelial cells can produce pro-inflammatory cytokines under the influence

of macrophages, as paradoxically few epithelial cells are infected after inoculation with

rhinovirus [151].

As expected with the increase in chemokines, greater numbers of neutrophils,

macrophages, and lymphocytes are found following viral infection in COPD [119, 132-134,

152]. Sputum eosinophilia has also been seen in virus-associated naturally occurring

exacerbations [112], although a separate study identified a cluster of eosinophil-

21

predominant exacerbations which were largely virus negative [103]. In a recently described

elastase-induced mouse model of COPD , enhanced cellular airways inflammation (increased

neutrophils, lymphocytes and macrophages in BAL) was observed in mice treated with

combined elastase and rhinovirus compared to rhinovirus alone [132].

The picture in terms of pro-inflammatory cytokines is less clear. In vitro, in mouse models,

and in naturally occurring infection studies, viral infection induces more IL-6 in COPD than

controls [134, 135, 137, 152-155], although this appears to be localised to the airways [101,

156]. Divergent findings have been reported regarding TNF-α after viral infection in COPD.

Some studies have found levels of TNF-α to be unchanged [101, 102, 119, 135], raised no

more than in controls [137], or even reduced in some animal models [132, 152]. Conversely,

TNF-α was increased in the BAL of cigarette smoke-exposed mice infected with influenza

compared to controls [134], although a more recent study failed to replicate this finding

[152]. This may be specific to influenza; the conflicting results came from in vitro studies

using rhinovirus [135, 137], rhinovirus-infected mice [132], human experimental rhinovirus

infection [119], and naturally occurring exacerbation studies, only a minority of which were

influenza PCR positive [101, 102]. But a more recent study of experimentally-infected COPD

patients also found raised levels of TNF-α, this time in sputum [155].

Rhinovirus exposure of epithelial cells from COPD patients also failed to induce IL-1β and IL-

18 in vitro [135], and although IL-18 was seen in a mouse model [133], this may be an

influenza-specific effect as influenza activates the NLRP3 inflammasome, leading to

increased IL-1β/IL-18 [157]. However increased IL-1β was found in the sputum of

22

experimentally-infected COPD patients [155]; it is unclear how to reconcile these

contradictory results.

23

Table 2: Inflammatory changes in viral infections in COPD

Mediator In vitro1 Animal studies2 Naturally occurring infection3

Experimental infection in

humansChemokines

CXCL10/IP-10

↑ [135, 137]↓ (ex vivo, >95%

macrophages) [119]

↔ (elastase/LPS) ↑(elastase only)

[132]↓ [152]

↑ serum [101, 103, 156] +

sputum [103]↑ (BAL) [155]

CXCL8/IL-8

↑ [135]↑ (ex vivo, >95%

macrophages) [119]

↔ serum [101, 156] + sputum

[102, 153]

↑ (sputum)↔ (BAL) [119,

155]↑ (nasal lavage)

[120]

CCL5/RANTES ↑ [137]↔ (elastase/LPS) ↑(elastase only)

[132]

↑ (sputum) [103]↔ serum [101]

CXCL1/GRO-α ↑ [135]

CCL2/MCP1 ↔ [132] ↑ (sputum) [103]↔ serum [101]

CXCL11 ↑ (serum + sputum) [103]

Inflammatory cells

Neutrophils n/a↑ [131, 133, 134]

↔ [132]↓ [152]

↔ sputum [102] ↑ (BAL, sputum, blood) [119, 155]

Macrophages n/a↑ [131, 133, 152]↑ (mononuclear)

[134]

Lymphocytes n/a ↑ [131-133, 152] ↑ (BAL) [119, 123]

Eosinophils n/a ↑ (sputum) [112]Cytokines

IL-6 ↑ [135, 137]↔ (ex vivo) [119]

↑ [134]↑ [152]↓ [132]

↑ sputum [153, 154]

↔ serum [101, 156]

↑ (BAL) ↔ (sputum)

[119]↑ (nasal lavage)

[120]

TNF-α ↔ [135, 137] also ex vivo [119]

↑ [131, 134]↓ [132, 152]

↔ serum [101] or sputum [102]

↔ (BAL, sputum) [119]

↑ (sputum)[155]IL-1β ↔ [135] ↔ serum [101] ↑ (sputum) [155]IL-18 ↔ [135] ↑ [133]IL-10 ↓ [152] ↑ (serum) [101]IL-13 ↑ [131, 132] ↔ serum [101]IL-12/IL-23 p40 ↑ [133]Type I IFN (α/β) ↓ (ex vivo) [119]

undetectable [135]

↑ [134]↑ (to Poly I:C)

[133]

24

↓ β [152] ↓ [131, 132]↓ β [152]

Type II IFN (γ) ↔ [135]↑ [131, 133]

↔ [134]↓ [152]

↑ (serum) [103]↔ (serum) [101]

Type III IFN (λ1/IL29, λ2/IL28)

↑ λ1/2 [135] λ1 [137]

↓ λ1 [152]↔ ex vivo [119]

↓ λ3 [152], (mRNA) [132]

Selected others

Neutrophil elastase

↑ (sputum)↔ (BAL) [119,

121, 155]MMP-9 ↑[158] ↑ (sputum)[155]Antimictrobial peptides (secretory leukoprotease inhibitor, elafin)

↓ (sputum) [121]

Markers of oxidative stress (8-hydroxy-2’-deoxyguanosine, 3-nitrotyrosine)

↑ (sputum)[155]

1 Shows response of cells from COPD patients to viral infection relative to the response of cells from healthy controls to the same viral challenge (other in vitro studies excluded).2 Limited to animal models using long-term (>2 weeks) cigarette smoke exposure or elastase/LPS combined with viral infection. Results shown for changes post-virus exposure in the animal model of COPD vs in healthy mice.3 Only includes findings from exacerbations with confirmed viral infection.

25

Future therapeutic approaches in viral exacerbations of airway disease

Bronchodilators and inhaled corticosteroids (ICS) are the mainstay of treatment [159, 160].

However in asthma, ICS reduce exacerbations by 25-60% [161-164], and they fail to

significantly reduce symptoms in neutrophilic asthma [165, 166] and during experimental

rhinovirus infection [167-169]. In COPD, treatment with long acting muscarinic antagonists

(LAMA), long acting beta agonists (LABA) or ICS-LABA combinations only brings about a 14-

27% relative risk reduction in exacerbations [170]. Combining all three agents (LAMA, ICS

and LABA ‘triple therapy’) brings only a modest additional reduction in exacerbation rates

over LAMA or ICS-LABA alone, with over 30% still experiencing one exacerbation a year

[171]. Thus there remains an unmet need for treatments for acute exacerbations.

The advances in our understanding of the immunopathogenesis of exacerbations have

identified promising new therapeutic approaches, some of which are beginning to become

available (see Table 3). Thus reconstitution of deficient IFN responses in asthma with inhaled

IFN-β is under investigation [63]. Anti-inflammatory approaches have been more successful

in asthma, with monoclonal antibodies against IgE already available for selected patients,

and anti-IL-4, anti-IL-5 and anti-IL-13 in development. Future targets are likely to include IL-

25 and IL-33. In COPD, inhibiting phosphodiesterase 4 (PDE4)-mediated hydrolyzation of

cAMP, increasing cAMP concentrations and dampening the inflammatory response, reduces

exacerbation frequency [172, 173].

Therapies targeting pro-inflammatory cytokines, chemokines and their receptors are also

under investigation, although as these changes are less specific to airways diseases, many

candidates are being trialled in other conditions such as rheumatoid arthritis [174-176].

There are also efforts to disrupt inflammatory pathways further downstream (e.g. p38

26

MAPK, Phosphoinositide 3-Kinase (PI3K)), and to reinforce the host’s anti-oxidant defences

(e.g. with N-acetylcysteine (NAC) and carbocisteine [177-179]. However manipulating the

inflammatory response risks impairing the immune system, thus research must proceed

cautiously. Indeed trials of an anti-TNF agent in COPD showed a non-significant trend

towards increased pneumonia and malignancy in the treated subjects [180], while in severe

asthma a similar study was stopped early due to increased numbers of serious infections

and the one death and all eight malignancies occurring in the treated subjects[181].

An alternative strategy is to target the virus. Influenza vaccines are well established, but

development of vaccines against other respiratory viruses, particularly rhinoviruses, is

complicated by the antigenic diversity and high mutation and recombination rates.

Advances in bioinformatics are enabling identification of conserved regions across serotypes

that could form suitable vaccine targets [182]. Compounds targeting blockade of viral

attachment, internalization and/or replication are in development.

27

Table 3: Emerging and potential approaches for treating for viral exacerbations

Therapeutic approach Specific treatment StatusRestoring the immune responseInterferon treatment Inhaled IFN-β In development, benefit in

moderate asthma in early trial [63].

Reducing sensitivity to inhaled allergensAttenuating IgE-mediated allergic responses

Anti-IgE (omalizumab) Reduce exacerbations and steroid use [183, 184].

Reducing inflammationInhibiting type 2 cytokines in asthma

Anti-IL-5 (mepolizumab, reslizumab)

In refractory eosinophilic asthma, reduces exacerbations [185-187].

Anti-IL-13 (lebrikizumab, tralokinumab)

In clinical trials [188].

Anti -IL-4 /-13 receptor (dupilumab)

In clinical trials [189].

Inhibiting pro-inflammatory cytokines in COPD

Anti-IL-6 (e.g. siltuximab) and anti-IL-6R (e.g. tocilizumab)

Being investigated in other neutrophilic conditions e.g. rheumatoid arthritis [176].

Disrupting chemokine signalling and chemotaxis/adhesion

Antibodies and antagonists of CXCL10/IP-10 and its receptor CXCR3

Being investigated in other neutrophilic conditions e.g. rheumatoid arthritis [174, 175].

Antibodies and antagonists of CXCL8/IL-8 and its receptors CXCR1/2

Trial of anti-IL-8 in COPD found less dyspnoea but no change in other outcomes [190].Trials of CXCR2 antagonists in COPD have had mixed results [191, 192].

Selectin antagonist (bimosiamose)

Trial in COPD found reduced inflammatory markers but not powered to detect changes in clinical parameters [193].

Other anti-inflammatory strategies

Blocking Pattern Recognition Receptors, e.g. TLR4 antagonist (eritoran)

Eritoran shown to prevent mortality from influenza in mice [194].

Inhibiting components of the inflammatory pathway, e.g. PDE4 inhibitors, p38 MAPK inhibitors, PI3K inhibitors

The PDE4 inhibitor roflumilast resulted in a 17% absolute risk reduction in exacerbations and an improvement in FEV1 in severe COPD (FEV1 <50% predicted) [172]. Early trials with p38 MAPK inhibitors have yielded mixed results in terms of sputum neutrophilia and lung function[195, 196]. Inhibition of PI3K has been shown to restore steroid responsiveness in vitro in COPD

28

[197] and reduces the inflammatory response to rhinovirus in vitro and in mice [198].

Anti-oxidants, e.g. NAC, Nuclear erythroid-2- Related Factor 2 (Nrf2) activator

Increasing evidence that NAC reduces COPD exacerbations [177-179]. In vitro activation of Nrf2 restores steroid sensitivity in macrophages from COPD patients; trials are ongoing [199].

Macrolides (and non-antibiotic macrolides)

Reduce exacerbation frequency in COPD [200], but unclear if antimicrobial or anti-inflammatory effect. Non-antibiotic macrolides in development given concerns regarding resistance [201].

Targeting the virusVaccination Influenza vaccine Reduces exacerbations in COPD

[202]Rhinovirus vaccines In a mouse model,

immunisation with a capsid protein that is highly conserved across serotypes resulted in more effective viral clearance after challenge with heterotypic serotypes [182]

Preventing viral entry Blocking ICAM-1 (tremacamra, anti-ICAM-1)

Tremacamra reduced symptoms, virus titres and CXCL8 levels in nasal lavage, but expensive with onerous dosing schedule [203].Anti-ICAM-1 prevents RV-14 and RV-16 infection in mice [204].

Antiviral Targeting rhinovirus capsid (pleconaril, vapendavir)

Pleconaril produced symptomatic relief in colds [205] but rejected by US FDA on safety grounds. Risk-benefit ratio may be different in chronic lung disease, hence ongoing trials in asthma (ClinicalTrials.gov identifiers NCT00394914 (pleconaril) and NCT02367313 (vapendavir)).

Inhibiting rhinovirus protease 3C (rupintrivir)

Development halted [206].

Influenza neuraminidase inhibitors (oseltamivir, zanamivir)

Benefit confirmed in a recent Lancet paper [207].

29

Anti-RSV F protein (palivizumab)

Licensed for use in high risk infants [208].

Conclusion

In the past three decades, substantial data has implicated respiratory viruses as a major

cause of acute exacerbations in asthma and COPD. In both of these important chronic

conditions defective host immune responses to virus infection is evident. However, whether

this occurs through a common mechanism, or whether the processes differ between

diseases, is unclear. Further research is required to clarify the exact mechanisms of

increased susceptibility to virus infection in pulmonary diseases, the interactions between

viruses and bacteria, and how these impact on host immune responses. A better

understanding of these mechanisms has the potential to lead to the development of novel

treatment targets in different chronic pulmonary diseases to reduce the impact of virus

induced acute exacerbations. Targeting a novel virus infection/epithelial cell-derived

cytokine/T cell/ILC2/type 2 cytokine pathway is highly promising for development of novel

therapies for asthma exacerbations.

Figure 1 legend

Epithelial and immune cell responses to rhinovirus infection including the influence of

SOCS1. Following infection; pro-inflammatory cytokines, chemokines, interferons and

growth factors are induced. This leads to airway inflammation, as well as mucus

hypersecretion and likely airway remodelling. SOCS1, which appears to be increased in

asthmatics, downregulates the antiviral response.

30

Despite difficulties characterising the rhinovirus C subtype, recent work suggests that

human cadherin-related family member 3 (CDHR3) is a functional receptor for RV-C [27].

Abbreviations

ATF2 = activating transcription factor 2

BEC = bronchial epithelial cells

CCL = Chemokine (C-C motif) ligand

CCR = CC chemokine receptor

CXCL = Chemokine (C-X-C motif) ligand

CXCR = CXC chemokine receptor

DALYs = Disability Adjusted Life Years

dsRNA = double stranded

EGFR = epidermal growth factor receptor

ENA = epithelial-derived neutrophil-activating peptide

ENA-78 = Epithelial cell-derived Neutrophil Attractant 78 (also known as CXCL5)

FEV1 = forced expiratory volume in one-second

FeNO = exhaled nitric oxide

GINA = Global Initiative for Asthma

GRO-α = Growth-regulated oncogene alpha (also known as CXCL1)

HBEC = human bronchial epithelial cells

hMPV = human metapneumovirus

31

HRV = human rhinovirus

ICAM-1 = intercellular adhesion molecule-1

ICS = Inhaled corticosteroids

IFN = Interferon

IL = Interleukin

ILD = interstitial lung disease

IP-10 = IFN-γ-Inducible Protein 10 (also known as CXCL10)

IFN = Interferon

IRF = interferon regulatory factor

ISGs = interferon stimulated genes

I-TAC = Interferon-inducible T-cell Alpha Chemoattractant (also known as CXCL11)

LDLR = low density lipoprotein receptors

LTB4 = Leukotriene B4

MCP-1 = monocyte chemotactic protein 1

MDA-5 = melanoma differentiation-associated protein-5 MCP1

MHC-I = major histocompatibility complex class I

Mig = Monokine induced by gamma interferon (also known as CXCL9)

mRNA = messenger ribonucleic acid

NDV = Newcastle disease virus

NF-κB = nuclear factor-κB

NO2 = Nitric dioxide

NRF2 = Nuclear erythroid-2- Related Factor 2

PAMP = Pathogen-Associated Molecule Patterns

pBEC = primary bronchial epithelial cells

32

PBMC = Peripheral blood mononuclear cells

PCR = polymerase chain reaction

PI3K = Phosphoinositide 3-Kinase

PKR = protein kinase receptor

PRR = Pattern Recognition Receptors

qPCR = quantitative polymerase chain reaction

RANTES = Regulated on Activation, Normal T cell Expressed and Secreted (also known as

CCL5)

RIG-I = retinoic acid inducible gene I

RV = Rhinovirus

RSV =

Th2 = T helper 2 lymphocytes

TLR = Toll Like Receptor

TNF = tumour necrosis factor

TSLP = Thymic stromal lymphoprotein

WHO = World health organisation

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