Educational Workshop EW07: Viruses and the respiratory tract – current and future issues arranged with the European Respiratory Society (ERS) for the EU- funded Network of Excellence Genomics to Combat Resistance against Antibiotics in Community-acquired LRTI in Europe (GRACE) Convenors: Rogg(g,)er Finch (Nottingham, UK) Lia van der Hoek (Amsterdam, NL)
Faculty: Alberto Papi (Ferrara, IT) Marco Contoli (Ferrara, IT) Alison Bermingham (London, UK) Lia van der Hoek (Amsterdam, NL) Albert Osterhaus (Rotterdam, NL)
Papi - Viral interactions with respiratory tract in health and disease
Viral interactions with respiratory tract in health and diseasedisease
Marco Contoli, MD, PhD Prof. Alberto Papi, MD
Research Centre on Asthma and COPD Section of Respiratory Medicine Department of Clinical and Experimental Medicine University of Ferrara - ItalyItaly
Respiratory viruses with impact on the lung
Respiratory viruses
• rhinoviruses • enteroviruses
• influenza viruses A & B
• parainfluenza viruses • respiratory syncytial virus
• adenoviruses
3 Papi - Viral interactions with respiratory tract in health and disease
Detection methods
• Virus cultures • Serology • Antigen detection (immunofluorescence, hemagglutination…)
• PCR and related methodologies
Do viral infections in early life protec againstagainst asthmaasthma??
Respiratory Infections in Infancy May Protect Against Development of Asthma
•• ≥≥2 episodes of “common cold” beforebefore age 1 yr decrease risk of asthma b y age 7 by ~50%by ~50% • Other viral infections - eg, herpes, varicella, measles - also protective
(Illi S et al. BMJ. 2001)
4 Papi - Viral interactions with respiratory tract in health and disease
Do viral infections in early life induce asthma?asthma?
Effect of Day Care in Infancy and Number of Older Siblings on Asthma Risk
Children who had ≥≥2 older siblings or attended day care during first 6 mo of life had increased risk of wheeze early in life but decreased risk later.later.
2.72.7 P=0.01P=0.01 risk zingzing e P=0.03P=0.03 1.01.0 P=0.001P=0.001 P<0.001P<0.001 P<0.001P<0.001 0.40.4 Adjusted relativ Adjusted of frequent whee 0.10.1 112233445566778899101011 12 13131414 Age (yr)(yr)Age
(Ball TM et al. N EnglJ Med. 2000)
…..Resolving the paradox ...…
5 Papi - Viral interactions with respiratory tract in health and disease
Deficient type 1 immune response in RSV bronchiolitis
100000< 0.08
0.07 10000 0.06 g
-g 1000 0.05 0.04 4/IFN- 100 IL IL--4/IFN 0.03 in Gene Copies
0.02 10 F prote 0.01 1 0
URTI Bronchiolitis 0 Day 1-2 Day 5-7 Day 1-2 Day 5-7
URTI Bronchiolitis
Legg et al, AJRCCM 2003
Infections and asthma: the apparent paradox
Th2 Th1Th1 Th1 Th2Th2
Competent immune system RV Bronchiolitis developsdevelopsappropriate responses to infections in early life
Viral infections and asthma exacerbations
6 Papi - Viral interactions with respiratory tract in health and disease
Virus associated asthma exacerbations
ChildrenChildren AdultsAdults
Positive virus detection No virus detected
(Johnston et al. BMJ 1995) (Corne et al. Lancet 2002)
Two thirds of viruses detected are rhinoviruses
(Reviewed in Contoli, Johnston, Papi et al Clin Exp Allergy 2005 )
Mechanisms of virus induced asthma exacerbation: exacerbation: In vivo human models
1616 10
5 1212 0 88 rning PEF st score st o e -5 44
-10
Total ch 00
--44 -15
-20 % change m in Normals Asthmatics -4-3-2-101234567891011121314
Day post experimental RV16 infection
(Message SD, Mallia P, Contoli M, Papi A, Johnston SL et al. PNAS 2008)
Mechanisms of virus induced asthma exacerbation: exacerbation: In vivo human models
60 Asthmatic patients Normal subjects 40
tom score 20 tal chest p o T sym 0
4 6 8 10 Nasal lavage virus load (Log10 copies/mL)
(Message SD, Mallia P, Contoli M, Papi A, Johnston SL et al. PNAS 2008)
7 Papi - Viral interactions with respiratory tract in health and disease
Role of innate immune rersponse in virus induced asthma exacerbations
Respiratory virus induction of alphaalpha--, beta, beta-- and lambdalambda--interferonsinterferons in bronchial epithelial cells
Khaitov, Laza-Stanca, Edwards, Walton, Rohde, Contoli, Papi, Stanciu, Kotenko, S. L. Johnston Allergy 2009
Role of deficient type III interferoninterferon--λλ production in asthma exacerbations
RV16 infection in Bronchial IFNIFN λλ 1 in Bronchial Epithelial Cells Epithelial Cells
# 6 *** 2,500 **
2,000
µg RNA) 5 pression * / x
λ 1,500 IFN- copies
(pg/ml) 1,000 4 10 500 (log
0 e vRNA RV16 3 M RV16 M RV16 Normal Asthmatic
Normal Asthmatic
(Contoli, Message, Papi, Johnston et al. Nat Med 2006)
8 Papi - Viral interactions with respiratory tract in health and disease
Role of deficient type III interferoninterferon--λλ production in asthma exacerbations
5 40
0 1 30 -5
e frombaseline 20 alue) old score in FEV s v
-10 c Fall Fall -15 10 Total Total
(percent decrea -20 0 0 25 50 75 100 125 150 175 200 0 50 100 150 200 IFN-λ (pg/ml) IFN-λ (pg/mL)
(Contoli, Message, Papi, Johnston et al. Nat Med 2006)
Role of deficient type III interferoninterferon--λλ production in asthma exacerbations: ex vivo results
300 5
4 200 3 -epithelial cells) 8 level level in 8 BAL g/ml) in sputum - n p 2 ( 100
1 0 0 Interleukin Eosinophils 0 50 100 150 200 0 25 50 75 100 125 150 175 200 (percent of no IFNIFN--λλ (pg/ml)(pg/ml) IFNIFN--λλ(pg/ml)(pg/ml)
(Contoli, Message, Papi, Johnston et al. Nat Med 2006)
Antiviral Immune Response in Asthma
3.0 5 * * 2.5 4
2.0 3 e from baseline)
1.5 NA expression n from baseline n of apoptosis g o o R 2 m
1.0 β Inducti
(fold chan 1
0.5 IFN-in Fold inducti
0 0 N NA A N NA A
Medium RV16 Medium RV16
((WarkWark,, Johnston et al. J Exp Med 2005)
9 Papi - Viral interactions with respiratory tract in health and disease
Conclusions: state of the art
IFNIFN--ββ IFNsIFNs IFNIFN-- LPSLPS λλ ProPro--inflammatoyinflammatoy cytokines (RANTES, IPIP--10,10, ILIL--6,6, IL-IL-8)8)
Viral Viral Th1/Th2Th1/Th2 ReplicationReplication
ISGF3ISGF3
CytoplasmCytoplasm vRNAvRNA PKRPKR ApoptosisApoptosis IRFIRF--3/73/7 p53p53 NucleusNucleus PKRPKR MxMx Inhibition of NFNF--kBkB OASOAS Viral ADARADAR ReplicationReplication Epithelial cells Macrophages
Frequency, severity and duration of rhinovirus infections in asthmatic and nonand non--asthmaticasthmatic individuals: a longitudinal cohort study
Symtoms Partecipants with Healthy partecipants asthma (n=76) (n=76)(n=76) URTURT Severity (score) 2 (0-(0-12)12) 2 (0-(0-7)7) Duration (days) 3 (0-(0-11)11) 3 (0-(0-17)17) LRTLRT Severity (score) 1 (0-(0-17)17) 0 (0-(0-7)*7)* Duration (days) 2.5 (0-(0-35)35) 0 (0-(0-22)†22)† Data are median (range). *p=0.001; †p=0.005 (Corne et al. Lancet 2002)
• Respiratory infection in asthamatic patients are more severe. • They are more prone to clinically severe infections
Epidemiological relationship between the common cold and exacerbation frequency in COPD
** 80 70 2 60 50 40 30 1 lds ratio % lds ratio iated with exac/ 20 d frequency/yr o c 10 0 0 Total c Total colTotal col Total colTotal Colds asso Colds InfreqInfreq FreqFreq InfreqInfreq FreqFreq
Exacerbation frequency in chronic obstructive pulmonary disease is associated with an increased frequency of acquiring the common cold, rather than an increased propensity to exacerbation once a cold has been acquired.
(adapted from Hurst et al. ERJ 2005)
10 Papi - Viral interactions with respiratory tract in health and disease
Detection of rhinovirus in induced sputum at exacerbation of COPD
25 6
20 5
15 4 % tom score
10 p 3
5 Sym 2
0 1
((SeemungalSeemungal et al. ERJ 2000)
Respiratory viruses, viruses, symptom and inflammatory markers in acute exacerbation and stable COPD
4 15
3
ptom count ptom 10 m count recovery time recovery count m m o 2
5
Tatl daily sy daily Tatl 1 Median daily Median daily total sympt
((SeemungalSeemungal et al. AJRCCM 2001)
Viruses and bacteria in COPD exacerbations
No pathogen VirusesViruses 21%21% 24%24%
25%25% BacteriaBacteria 30%30%
Viruses &Viruses & BacteriaBacteria
Papi, Fabbri & Johnston et al. AJRCCM 2006
11 Papi - Viral interactions with respiratory tract in health and disease
Eosinophils increased only in virus related AE
** ** **
10 ** **** ****
8 phils g o u 6 /mg pl /mg 66 4 1010 Sputum Eosin Sputum
2
00 EEESSSEESSEESSE S
Virus Virus & Bacteria No pathogen Bacteria Papi, Fabbri & Johnston et al. AJRCCM 2006
Conclusions
• Viral infections are the most frequent cause of asthma and COPD exacerbations
•• Impaired antiviral response in asthma
• The role of viral infections is understimated in COPD
12 Contoli - Upper respiratory tract viral infections with impact on the lung
Upper Respiratory tract viral infections with impact on the lunglungthe
Marco Contoli, MD, PhD Prof. Alberto Papi, MD
Research Centre on Asthma and COPD Section of Respiratory Medicine Department of Clinical and Experimental Medicine University of Ferrara - ItalyItaly
Respiratory viruses
• rhinoviruses • enteroviruses
• influenza viruses A & B
• coronaviruses
• parainfluenza viruses • respiratory syncytial virus
• adenoviruses
Detection methods
• Virus cultures • Serology • Antigen detection (immunofluorescence, hemagglutination…)
• PCR and related methodologies
13 Contoli - Upper respiratory tract viral infections with impact on the lung
Virus associated asthma exacerbations
ChildrenChildren AdultsAdults
Positive virus detection No virus detected
(Johnston et al. BMJ 1995) (Corne et al. Lancet 2002)
Two thirds of viruses detected are rhinoviruses
(Reviewed in Contoli, Johnston, Papi et al Clin Exp Allergy 2005 )
Experimental Infection Localization of RVs in the bronchi
• Rhinovirus infects lower airwaysairways
(Papadopoulos, Papi, Johnston J Infect Dis 2000)
Experimental infection: B - Immunology Th2 cellsTh2 cells
Eosinophils Dendritic T cells Basophils cells
Th1 cellsTh1 cells Macrophages Neutrophils
•• In vitro:vitro – A defective type 1 response to rhinovirus in atopic asthma. (Papadopoulos, Papi, Johnston et al. Thorax 2002) •• In vivo:In vivo: – RV infection in atopic subjects is associated to low Th1/Th2 antiviral responses (Gern 2000 AJRCCM)AJRCCM)
14 Contoli - Upper respiratory tract viral infections with impact on the lung
Experimental infection: C - Inflammation Neutrophils IL-8, Groα activation chemotaxis
GM-CSF, Eotaxin, RANTES, MIP-1α Eosinophils survival, chemotaxis IL-1β, MIP-1α, MCP-1, TNFα Macrophage virus s
IFNα / β, MIP-1α NK cells activation
RANTES, IL-6 T lymphocytes activation, chemotaxis MHC I, ICAM-1, VCAM-1 IFNα / β
Experimental infection: C - Inflammation
•• In asthmatic subjects rhinovirus infection induces prolonged eosinophil infiltration of the bronchial m u co sa mucosa (Fraenkel et al. AJRCCM 1995)
• Increased levels of eosinophil cationic protein were found in the sputum of RV- infected subjects (Grunberg et al. AJRCCM 1997)
Inflammatory common pathway : Adhesion molecules
•• Epithelial ICAMICAM--11 expression is increased in asthmaasthmain
(Bentley et al. JACI 1993)
Surface epithelial ICAMICAM--11 increases after allergen challengechallenge (Vignola, Am Rev Respir Dis 1993)
15 Contoli - Upper respiratory tract viral infections with impact on the lung
Common pathwaypathway:: Adhesion molecules
•• ICAMICAM--11 is the cellular receptor for 90% RV •• Rhinovirus increases epithelial ICAMICAM--11
(Papi, Johnston J Biol Chem 1999) (Grunberg, et al Clin Exp Allergy 2000)2000)
Rhinovirus infection of the airway epithelial cells In vitro models
Inflammatory RhinovirusRhinovirus ResponseResponse ICAMICAM--11 ICAMICAM--11
II--kB degradation XX
NF-kB Oxidant formation
NF-kB
Reducing agents XX (Papi A, Johnstion SL. FASEB J 2002)
Papi A, Contoli M, Pinamonti S et al; J Biol Chem 2008
16 Contoli - Upper respiratory tract viral infections with impact on the lung
Causes of virusvirus--inducedinduced airway obstruction
PlasmaPlasma Mucus leakageleakage hypersecretion Inflammatory cell recruitment and activation
VirusVirus--infectedinfected epitheliumepithelium
Neural activation AirwayAirway Hyperresponsiveness (Contoli etal. Clin Exp Allergy 2005)
Effect of viral infection on Glucocorticoid Responsiveness
GCS Cell membrane
GR
mRNA
hsp90
nucleus
Steroid responsive +GRE -GRE target genes
Rhinovirus and childhood asthma ChildhoodChildhoodOriginOrigin of AsThma (COAST)
First 3 yearsof life
70
(%) OR=10OR=10 60 OR=8OR=8
ears (%) 50 y yy 40
at 6 30 OR=2.6OR=2.6 20 10 OR=1OR=1 Asthma at 6 0 Neither RSV only RV only RV & RSV
Wheezing illnesses
(Jackson et al. AJRCCM 2008)
17 Contoli - Upper respiratory tract viral infections with impact on the lung
Relative contribution of rhinovirus wheezing illnesses and aeroallergen sensitization to risk of asthma at 6 years
OR (95% CI)
RV wheezing Aeroallergen sensitization
First year of life 2.8 (1.4 – 5.6) 3.6 (1.7 – 7.7)
Third year of life 25.6 (8.2 – 3.4 (1.7 – 6.9) 79.6)
(Jackson et al. AJRCCM 2008)
Mechanisms by which viral infectioninfectioncancan lead to an increased Th2 response (mouse model)model)
Type I interferon receptorreceptor--dependentdependentexpression of high affinity IgE receptot VirusVirus SendaiSendai
CrossCross--linkinglinking FcεεRI
Dendritic cells
CCL28CCL28
IL-13 producing CD4+ T-cell
((GraysonGrayson et al. J Exp Med 2007)
ModulatoryModulatoryeffecteffectof viral infectioninfectiononon allergen induced Th2 inflammationinflammation,, following in vitro stimulation with japanese cedar pollen
250 2000
200 1500
150 pg/ml 1000
pg/ml 100 500 50
0 0
ILIL--44ILIL--55
((LiuLiu et al. J MedVirol 2007)
18 Contoli - Upper respiratory tract viral infections with impact on the lung
Conclusions
Viral infections impact on the lunglung:: • Inflammatory effects ••SteroidSteroid sensitivity • Immunological consequences
19 Contoli - Viral pneumonia – primary and co-pathogenic infections
Viral pneumonia – primary and co-pathogenic infections
Marco Contoli, MD, PhD
Research Centre on Asthma and COPD Section of Respiratory Medicine Department of Clinical and Experimental Medicine University of Ferrara - ItalyItaly
Pneumonia: definition
Acute infectious disease
Documentation of abnormalities at chest radiografy
Frequent association with aspecific resp ira tory symp toms
Possibly associated with systemic inflammatory symtoms
No correlations with aetiology, radiology and clinical severity
2008
20 Contoli - Viral pneumonia – primary and co-pathogenic infections
Epidemiological classification of pneumonia
Community- Hospital-acquired, acquired and ventilator- pneumonia associated (()CAP) pneumonia
Correlations with pathogens
Therapeutical implications
(ATS statement 2005 and 2001)
Epidemiology
CommunityCommunity--acquiredacquiredpneumonia: aetiology
Haemophilus.influenzae (4%) Mycoplasma pneumoniae (8%) Legionella spp (5%) Gram- ,enterobatteri (3%) Virus (8%) Chlamydia psittaci(2%) Coxiella burnetii (2%) Chlamydia pneumoniae (12%) Staphylococcus aureus (2%)
Streptococcus Unknown (24.5%) pneumoniae (28%)
21 Contoli - Viral pneumonia – primary and co-pathogenic infections
Improved Diagnosis of the Etiology of CommunityCommunity--AcquiredAcquired Pneumonia with RealReal--TimeTime Polymerase Chain ReactionReaction
20 18 16 14 12 Conv 10 PCR
No. Of patients 8 6 4 2 0
(Templeton et al. Clin Inf Dis 2005)
Incidence and characteristics of viral communitycommunity--acquiredacquired pneumonia in adults
Of 304 patients with CAP, a viral diagnosis was made in 88 (29%)
14 12 10 8 % 6 4 2 0
(Jennings et al. Thorax 2008)
Viral Infection in Adults Hospitalized With Community- Community-AcquiredAcquired Pneumonia : Prevalence,Prevalence, Pathogens,Pathogens, and Presentation
Of 193 patients with CAP, a viral diagnosis was made in 29 (15%)
8 7
ases 6 c 5 4 3
Number of 2 1 0
(Johnstone et al. Chest 2008)
22 Contoli - Viral pneumonia – primary and co-pathogenic infections
SARS: Severe Acute Respiratory Syndrome
SARS CoV
(Li et al Nature 2003) (Holmes et al. NEJM 2003)
Avian Influenza A (H5N1) Virus Infection in Humans
Influenza A H5N1
(NEJM 2008)
2009 H1N1 Influenza
Febrile respiratory illness in children from southern California caused by infection with a novel influenza A (H1N1) virus
H1N1 was isolated in 47 cases of rapidly ppgrogressive severe pneumonia that resulted in 12 known deaths
By June 11, 2009, nearly 30,000 cases of 2009 H1N1 virus had been confirmed across 74 countries = pandemic influenzainfluenza Influenza A H1N1 Sullivan S J et al. Mayo Clin Proc. 2010;85:64-76
23 Contoli - Viral pneumonia – primary and co-pathogenic infections
CDC Estimates of 2009 H1N1 Cases and Related Hospitalizations and DeathsDeaths from April 2009 - January 16, 2010, By Age Group
Clinical manifestationsmanifestations:: specifiifiific fffor ae tio ltillogy??
Managment of communitycommunity--acquiredacquired pneumoniapneumonia
Signs and symptoms cannot predict the atiologicatiologic agentagent
(Halm et al. NEJM 2003)
24 Contoli - Viral pneumonia – primary and co-pathogenic infections
Viral Infection in Adults Hospitalized With Community- Community-AcquiredAcquired Pneumonia : Prevalence,Prevalence, Pathogens,Pathogens, and Presentation
100
80
60 Viral % 40 Bacterial Mixed 20 Unknown
0 Cough Sputum Dyspnea
(Johnstone et al. Chest 2008)
Incidence and characteristics of viral communitycommunity--acquiredacquired pneumonia in adults
(Jennings et al. Thorax 2008)
Cytokines & Hormokines upon Infection
TNFa IL6 PCT CRP ug/ml Fatal Outcome 5
4
3
2
1
0 0 122436486072h
Endotoxin iv adapted from Meisner M, J Lab Med 1999 Dandona P, et al. J Clin Endocrinol Metab 1994 Harbarth S, AJRCCM 2001 Becker KL, J Clin Endocrinol Metab 2004
25 Contoli - Viral pneumonia – primary and co-pathogenic infections
Calcitonin in healthy people
Control Sepsis
Thyroid White Blood Cells „Healthy“ Perit. Macrophage Calcitonin Spleen Lung Liver Kidney Adrenal Brain Spine Pancreas Stomach Small Intestine Colon Heart Muscle Skin Visceral Fat Testes Müller B, et al. JCEM 2001
Calcitonin precursors (CTpr) in infected patients
Control Sepsis
Thyroid White Blood Cells Perit. Macrophage Spleen Lung Liver Kidney CTpr Infection Adrenal incl. PCT Brain Spine Pancreas Stomach Small Intestine Colon Heart Muscle Skin Visceral Fat Testes Müller B, et al. JCEM 2001
(Christ-Crain, Muller et al. Lancet 2004)
26 Contoli - Viral pneumonia – primary and co-pathogenic infections
Comorbidities and CAP etiology
70 60 50
40 Viral % 30 Bacterial 20 Mixed 10 Unknown 0 CD RD IFS
(Johnstone et al. Chest 2008)
Comorbidities and CAP severity
Vedi anche jama 2009
CAP severity and etiology
(Jennings et al. Thorax 2008) (Templeton et al. Clin Inf Dis 2005)
27 Contoli - Viral pneumonia – primary and co-pathogenic infections
CAP severity and etiology
Postmortem lung specimens of 77 fatal H1N1 cases documented coinfection in 22 cases (29%)
Streptococcus pneumoniae predominated (10 cases) (10 cases)
These findings highlight the importance of: • early detection and treatment of bacterial pneumonia in patients with 2009 H1N1 influenza • pneumococcal vaccine for those in whom it is indicated.
CDC CDC MMWR Morb MortalMortal Wkly Rep. 2009;58(38):10712009;58(38):1071--1074.1074.
Interaction between bacteria and virusand virus ilin lower resp itiratory trac tiftit infections
Rhinovirus infections in COPD Criterion for exacerbation: increase over baseline in LRT symptom score of >2 for 2 days
Upper & lower respiratory tract scores
(Mallia, Johnston et al. Respir Res 2006)
28 Contoli - Viral pneumonia – primary and co-pathogenic infections
A human experimental model of rhinovirus induced exacerbations of Chronic Obstructive Pulmonary Disease
Preliminary data indicates that in vivo experimental RV infection in COPD patients leads to increased bacterial load in thihe airways
(Mallia P, Message S, Gielen V, Contoli M, Papi A, Johnston SL, et al. Submitted)
Conclusions
Viral infection is an understimated cause of pneumoniapneumonia
Lack of specific pharmacological treatment
Role of prevention: vaccination
Interaction between virus and bacteria deserves further investigations
29 30 31 32 33 34 35 36 37 38 39 The best option for virus discovery, VIDISCA combined with high throughput sequencing
Michel de Vries1, Marta Canuti1, Nuno R. Faria1, Maarten F. Jebbink1, Angela C.M. Luyf 2,
Barbera DC van Schaik2, Marja Jakobs3, Richard Molenkamp4, Martin Deijs1, Frank Baas3,
Lia van der Hoek1
1 Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam. 2 Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Bioinformatics Laboratory, Academic Medical Center of the University of Amsterdam. 3 Department of Neurogenetics, Academic Medical Center of the University of Amsterdam, 4 Laboratory of Clinical Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam.
In approximately 20% of the adults with respiratory tract illness no known pathogen can be detected. A yet unknown virus could be the cause of the illness and a sequence independent amplifications method such as virus discovery cDNA-AFLP (VIDSICA), can be used to identify the virus. The
VIDISCA method uses restriction enzyme recognition sites to digest all nucleic acids in a sample, a ligation step to add adaptors, and a PCR with primers that anneal to the adaptors to amplify the input viral DNA or viral RNA1. The method was successfully used to identify human coronavirus NL63
(HCoV-NL63 1) and picornaviruses 2. These discoveries were all from cultured virus isolates and not from clinical samples, since the VIDISCA assay is not sensitive enough to detect viruses from uncultured material. In theory the latest high throughput sequencing techniques may enhance
VIDISCA sensitivity and enable virus identification in cases where conventional VIDISCA may not.
In conventional VIDISCA a virus specific amplification product has to be visible and recognizably different from the negative control PCR products. This means selection of virus specific amplification by eye, whereas VIDISCA combined with high throughput sequencing does not need recognizable visualization. With next generation sequencing large amounts of sequences are generated, thus it allows everything in a sample to be sequenced, including background (mainly rRNA amplification products) and potentially unknown viruses.
40 We evaluated this theoretical sensitivity improvement by sequencing VIDISCA amplified products with the Roche 454-FLX system (VIDISCA-454, see figure). As input twelve randomly chosen nasopharyngeal washings of patients all containing known respiratory viruses were used. The outcome of the standard VIDISCA was compared to the results with VIDISCA-454. All samples were analyzed double-blind to prevent biased analyzing of the sequences.
With Roche-454-FLX a total of 83135 sequences were generated of which 68 were viral
(HCoV-229E, HCoV-OC43, RSV, HRV, hMPV). Standard VIDISCA revealed the identity of only 1 virus
(HCoV-229E) in the 12 nasopharyngeal samples, whereas with the VIDISCA-454 the viruses in 6 of the 12 samples could be correctly identified.
In conclusion, high throughput sequencing and VIDISCA increases the sensitivity of the discovery method to a level that allows virus discovery directly from nasopharyngeal aspirates.
Reference List
1. van der Hoek L., Pyrc K, Jebbink MF et al. Identification of a new human coronavirus. Nat Med 2004;10(4):368-373.
2. de Vries M, Pyrc K, Berkhout R et al. Human parechovirus type 1, 3, 4, 5, and 6 detection in picornavirus cultures. J Clin Microbiol 2008;46(2):759-762.
41
Figure. Schematic overview of the steps in VIDISCA-454
42 Van der Hoek - Future diagnostic approaches to viral respiratory tract infections
Future diagnostic approaches to viral respiratory tract infections
Human bocavirus (HBoV)
Lia van der Hoek
Laboratory of Experimental Virology
We have so far… 1. Influenzavirus A (H1, H3, H5, H7) 2. Influenzavirus B 3. Enterovirus 4. Parechovirus 5. Adenovirus 6. RSV 7. Rhinovirus type A/B Detection nowadays: 8. PIV-1 • (Real-time) RT-PCRs 9. PIV-2 10. PIV-3 • Luminex approach 11. PIV-4 12. HCoV-229E 13. HCoV-OC43 Keep Ct into account with 14. hMPV identification of the pathogen 15. Bocavirus * 16. HCoV-NL63 17. HCoV-HKU1 * * No culture system 18. KIPyV * 19. WUPyV *
Laboratory of 20. Rhinovirus type C * Experimental Virology
What should we know
Koch’s postulates, causative role of disease: • The microorganism must be found in all organisms suffering from the disease, but not in healthy organisms. • The microorganism must be isolated from a diseased organism and grown in pure culture. • The cultured microorganism should cause disease when introduced into a healthy organism. • The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.
Problems - Only in organisms with the disease - Virus culture - Suitable animal model
Laboratory of Experimental Virology
43 Van der Hoek - Future diagnostic approaches to viral respiratory tract infections
Culture is needed in discovery
• Sequence independent amplifications • Identification • Genome characterisations • Worldwide spread • Association with disease
• Inhibition by antivirals ? • Replication kinetics ? • Pathogenicity ? • Koch’s postulates ?
Laboratory of Experimental Virology
Human bocavirus (HBoV-1)
• Identified in 2005 within pools of human nasopharyngeal aspirates (Allander et al PNAS 2005).
• The genomic DNA reference sequence is 5.299 nt in length. • Without flanking terminal hairpin structures.
• Infections have been found worldwide • Respiratory samples • Serum, fecal and urine samples
• Frequently found in children under the age of 2 years • High frequency of co-infections with other respiratory viruses • Acute wheezing
• Despite the current knowledge, no in vitro or in vivo model has been established that supports replication.
Laboratory of Experimental Virology
Phylogeny of Parvovirinae
Laboratory of Experimental Virology
44 Van der Hoek - Future diagnostic approaches to viral respiratory tract infections
Human airway epithelial culture
• Tracheal or bronchial epithelial cells from donors
• Dedifferentiation
• Grow until confluence
• Exposure to air on apical side
• Medium at basolateral side
• Differentiation (5 to 6 weeks)
Laboratory of Experimental Virology
Benefits of differentiated human airway epithelia (HAE)
– Morphologically and functionally resembles the human airways in vivo
– Susceptibility towards viruses coincides with the degree of differentiation
Propagation of HBoV on HAE ?
Laboratory of Experimental Virology
HBoV1 replication on HAE: Apical release of the virus / ml d Viral loa Viral
Hours post-inoculation
Laboratory of Experimental Virology Dijkman, Koekkoek, Molenkamp, Schildgen, van der Hoek J. Virology
45 Van der Hoek - Future diagnostic approaches to viral respiratory tract infections
Propagation? Yes
• Intracellular: spliced mRNA
• Release of virus particles only apical (so far…)
• Apical harvest can be passaged
• After first replication on HAE, replication in cell lines: ….. No (so far)
Laboratory of Experimental Virology
Transcript map of HBoV1
Laboratory of Dijkman, Koekkoek, Molenkamp, Schildgen, van der Hoek J. Virology 2009 Experimental Virology
And there is more to do…
• Type of cells infected • Receptor • Entry route • Release of the virus • Antivirals • Find animal model
• Other unculterables: HCoV-HKU1, Rhinovirus type C, Polyomaviruses
Laboratory of Experimental Virology
46 Van der Hoek - Future diagnostic approaches to viral respiratory tract infections
With special thanks to:
• AMC, Lab Experimental Virology Ronald Dijkman University of Amsterdam Krzysztof Pyrc Amsterdam, the Netherlands Maarten F. Jebbink Martin Deijs Michel de Vries
• Municipal Health Service Amsterdam, Wilma Vermeulen-Oost The Netherlands Ron Berkhout
• Bonn University, Germany Oliver Schildgen
Laboratory of Experimental Virology
47 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of Experimental Virology Identifying novel respiratory viruses
The technology and examples from the GRACE approach
Lia van der Hoek
www.pathogendiscovery.com
Laboratory of Experimental Virology Dep. Medical Microbiology AMC Amsterdam The Netherlands
Laboratory of experimental virology
Ronald Dijkman Michel de Vries Martin Deijs Maarten F. Jebbink Krzysztof Pyrc Marta Canuti Lia van der Hoek
Laboratory of In GRACE Experimental Virology
Lower respiratory tract infections in adults
All known pathogens are tested (bacterial, viral)
+/- 20% remains pathogen negative
Caused by an unknown virus?
48 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of Today Experimental Virology
I: Introduction VIDISCA
II: Discovery of HCoV-NL63
III: VIDISCA-454 the GRACE approach
VIDISCA (Virus Discovery cDNA-AFLP) Laboratory of Experimental Virology
Laboratory of VIDISCA: RNA and DNA viruses Experimental Virology
Parvo B19 plasma HIV-1 virus culture HBV plasma (ss-DNA) (ss-RNA) (partially ds-DNA) M M -+ -+M M -+
49 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of Experimental Virology Part II
Virus discovery example: HCoV-NL63
Laboratory of Patient NL63 Experimental Virology
7-month-old child with bronchiolitis, conjunctivitis and fever (Jan 2003)
Routine diagnostics negative for all known respiratory viruses RSV, Adenoviruses, Influenzavirus A and B, Parainfluenza virus types 1, 2 or 3, Rhinoviruses (PCR), Meta-pneumovirus (PCR), Enterovirus (PCR), Human coronaviruses HCoV-OC43 and HCoV-229E (PCR)
tMK cells: Cytopathic effect (CPE)
Laboratory of CPE on LLC-MK2 cells Experimental Virology
NL63
control
50 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of VIDISCA on NL63 Experimental Virology
1 of the 16 selective PCRs:
NL NL M P P 63 63 cC cC - - M
In total 13 fragments with similarity to coronaviruses
Laboratory of Phylogenetic analysis Experimental Virology
HCoV-NL63 HCoV-229E PEDV FIPV G1 CCoV PRCoV TGEV SARS-CoV BCoV HCoV-OC43 G2 MHV CoV-HKU1 TCoV IBV G3
0.2
Laboratory of Not a recombinant Experimental Virology
van der Hoek et al, Nature Medicine 2004
51 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of Experimental Virology
A previously unknown virus, what does it cause?
Further investigation of the virus
Treatment options
Laboratory of HCoV-NL63 worldwide Experimental Virology
Published The Netherlands (n=8) 1.4% van der Hoek et al 2004 The Netherlands (n=5) 2.9% Fouchier et al 2004 Australia (n=16) 2.1 % Arden et al 2005 Japan (n=3) 2.5 % Ebihara et al 2005 Canada (n=19) 3.6 % Bastien et al 2005 USA (n=79) 8.8 % Esper et al 2005 Belgium (n=7) 2.3 % Moes et al 2005 France (n= 28) 9.3 % Vabret et al 2005 Hong Kong (n= 15) 2.6 % Chiu et al 2005 Canada (n=26) 2.1 % Bastien et al 2005 Japan (n=5) 12%1.2 % Suzuki et al 2005 Switzerland (n=6) 7.3% Kaiser et al 2005 Germany (n=49) 5.2% van der Hoek et al 2005 Canada (n=12) 3.0% Boivin et al 2005 Korea (n=8) 1.6 % Choi et al 2006 Italy (n=9) 1.1% Gerna et al 2006 Hong Kong (n=53) 1.3% Lau et al 2006 Sweden (n=12) 5.7% Koetz et al 2006 Switzerland (n=6) 1.1% Garbino et al 2006 Australia (n=6) 1.9 % Arden et al 2006 Korea (n=14) 1.7% Han et al 2007 USA (n=11) 1.0% Kuypers et al 2007 Italy (n=1) 0.5% Pierangeli et al 2007 Greece (n=2) 1.0 % Papa et al 2007 Italy (n=13) 3.0 % Gerna et al 2007 Korea (n=3) 1.3% Chung et al 2007 USA (n=2) 0.7% Mahony et al 2007 USA (n=4) 4.8% Kistler et al 2007 Australia (n=8) 1.1% Lambert et al 2007
Laboratory of How common is 229E/NL63 infection? Experimental Virology
-----: HCoV-229E antibodies ___: HCoV-NL63 antibodies
Dijkman et al J. Clin Microb. 2008
52 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of Seroconversion 229E and NL63 Experimental Virology
HCoV-NL63 HCoV-229E
Dijkman et al J. Clin Microb. 2008
Laboratory of Experimental Virology
So every child becomes infected, but in how many cases does it require
1. Hospitalization?
2. Visit to the general practitioner/paediatrician?
The PRI.DE study
Laboratory of Experimental Virology (Parainfluenza- und Respiratory- Syncytial-Virus- Infektionen in Deutschland)
Population based study of children under the age 3 years with LRTI
Lower Respiratory Tract Infections (LRTI): Apnoea (under the age of 6 months) Laryngotracheitis (croup) Bronchitis Bronchiolitis Pneumonia
Visiting the hospital or peadiatrician* Peadiatrician: out patient clinica. In Germany >95% of children have a peadiatrician
Multiple hospitals and practices in cities in the north (Hamburg), east (Dresden), south (Freiburg) and west (Bochum) of Germany
53 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of M&Ms Experimental Virology
Samples: recruitment from November 1999 to October 2001. Total of 3654 respiratory samples
All tested for RSV, PIV-1, PIV-2, PIV-3, and influenza.
Randomized 1733 samples tested for HCoV-NL63 RNA by real- time RT-PCR
Laboratory of Experimental Virology Only high HCoV-NL63
Annual incidence of HCoV-NL63 in children (<3 yr) 7 per 1000 children (95% CI: 3 - 13 per 1000 children) visit the physician per year Absolute number: 16.929 children per year in Germany
Hospitalization rate: 22 per 100.000 children (95% CI: 7 – 49 per 100.000 children) have to be hospitalized per year Absolute number: 522 children per year in Germany
Rarely hospitalized, more often visit to the outpatient clinic
Van der Hoek et al J Clin Virol 2010
Croup associated with
Laboratory of Experimental Virology HCoV-NL63 and PIV
Van der Hoek et al PLoS Medicine 2005
Confirmed by: Choi et al Clin. Inf. Dis. 2006 Han et al J. Clin Virol 2007 Wu et al Eur. J. Pediatrics 2007
54 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of Receptor for HCoV-NL63 Experimental Virology
Group 1 HCoV-229E CD13 TGEV CD13 FIPV CD13 PrCoV CD13 HCoV-NL63 ?
Group 2 MHV CAECAM SARS-CoV angggiotensin converting enzyme 2 (ACE2) HCoV-OC43 Sialic acids
78 HCoV-NL63 100 HCoV-229E CD13 96 PEDV CD13 FIPV CD13 100 CCoV 99 TGEV CD13 100 PRCV SARS-CoV ACE2 MHV 67 CAECAM 100 EqCoV 100 HCoV-OC43 Sialic acids 95 BCoV 100 IBV 0.1
Laboratory of Overexpression of receptors Experimental Virology
Hofmann et al PNAS 2005
Laboratory of Conclusions Experimental Virology
• The VIDISCA method is suitable for identification of RNA and DNA viruses from Culture. Nature Med 2004
Nature Med 2004 • HCoV-NL63 is a previously unknown human coronavirus and it has Virology Journal 2004 Emerg. Inf Dis 2005 spread worldwide BioMed Central 2005 J.Mol Biol 2007
• It circulates in the winter months Nature Med 2004
J. Clin Microbiology • Every child becomes infected in youth 2008
• Rarely requires hospital uptake J Clin Virol 2010
PLoS Medicine • Infection is associated with croup in children 2005
• HCoV-NL63 is the only other coronavirus that uses the same receptor PNAS 2005 as SARS-CoV for entry
• HCoV-NL63 infections can be treated AAC 2006
55 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of BUT….. Experimental Virology
The VIDISCA method is suitable for identification of RNA and DNA viruses from virus culture.
We want to detect viruses without the need to culture (direct from patient material)
Laboratory of Experimental Virology Part III
VIDISCA-454 The GRACE approach
VIDISCA Laboratory of Experimental Virology 454
56 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of Next Generation Sequencing Experimental Virology
Clonal amplification by emulsion PCR: clonal on a bead
At the AMC: 454 sequencer of Roche.
Per run a maximum of 1.5 E6 beads can be used resulting in about 400.000 quality sequences.
Laboratory of monitor VIDISCA-454 Experimental Virology
Coxsackievirus B4 virus culture supernatant
12 Respiratory samples with known viruses
Laboratory of Virus culture VIDISCA-454 Experimental Virology
MID Nr. of sequences Viral sequences % of total
1 2805 2477 88.3
2 3890 3282 84.4
3 2369 2007 84.7
4 2782 2534 91.1
5 3056 2540 83.1
6 2271 2000 88.1
7 1429 1354 94.8
8 3830 2961 77.3
9 3018 2821 93.5
10 3040 2840 93.4
11 5216 4991 95.7
12 2891 2740 94.8
Total 36597 32547 88.9
57 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Laboratory of Clinical sample VIDISCA-454 Experimental Virology
Twelve clinical samples VIDISCA VIDISCA-454 containing known viruses
Virus indentified 16
HCoV-229E HCoV-OC43 HCoV-229E RSV (2X) Rhinovirus hMPV
Laboratory of Conclusion Experimental Virology
VIDISCA-454 in NPA is a very good virus discovery technique,
Ready to test GRACE samples for unknown viruses
Acknowledgments Laboratory of Experimental Virology Municipal Health Service Amsterdam, Wilma Vermeulen-Oost NL63 The Netherlands Ron Berkhout discovery Joke Spaargaren
University Hospital Freiburg, Germany Gabriela Ihorst St Joseph Hospital, Freiburg, Germany Johannes Forster NL63 Wyeth Pharma GmbH, Münster, Germany Gudula Peterson Croup Ruhr University Bochum, Germany Klaus Sure + Alexander Stang Burden of Klaus Überla disease University Hospital of Caen, France Astrid Vabret
University of Auckland, New Zealand Lea Deng NL63 Howard A. Ross evolutio n University of California, San Diego, USA Chisato Shimizu Sharon L. Reed Jane C. Burns NL63 Layola University, Maywoo, USA Susan C. Baker Kawasaki Northwestern University, Chicago, USA Francesca Garcia disease Anne H. Rowley Stanford T. Shulman Vanderbilt Univ. Med. Center, Nashville, USA Helen K.B.Talbot John V. Williams NL63 Serocon- Bonn University, Germany Marcel Müller version
58 Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach
Acknowledgments II Laboratory of Experimental Virology
AMC, Sequence Department of Neurogenetics Marja Jakobs facility AMC, Amsterdam, The Netherlands Frank Baas
Department of Clinical Virology, AMC, Amsterdam, the Netherlands Richard Molenkamp Clinical samples
Clinical Eppgyidemiology and biostatistics Barbara van Schaik IT- AMC, Amsterdam, the Netherlands Angela Luijf support
Laboratory of Experimental Virology Lia van der Hoek AMC, Amsterdam, the Netherlands Michel de Vries Krzysztof Pyrc VIDISCA Maarten F. Jebbink Ronald Dijkman Martin Deijs Nuno M. Faria Marta Canuti Loes Jachimowski
59 60 61 62 63 64 65 66 67 68 69 70 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
ESCMID 10-13 April 2010 Vienna, Austria
Vaccine prevention of non-influenza viral respiratory disease: current and future.
Ab Osterhaus
Human respiratory tract disease - Associated pathogens -
540 respiratory GP patients , Netherlands, 1996-1997, culture and PCR (NIVEL) Bacteria, 16%
Mycoplasma pneumoniae, 1% Chlamydia pneumoniae, 1% Parainfluenza virus, 1% Adenovirus, 1% Negative, 36% Coronavirus OC43, 2% Enterovirus, 4%
RSV, 5%
Influenza B virus, 9%
Rhinovirus, 22% Influenza A (H3N2) virus, 14%
Newly identified human respiratory viruses in last 15 years alone
- AIV`s * influenza virus 1997… - Hendra-/NipahV paramyxovirus 2000… - hMPV * paramyxovirus 2001 - SARS-CoV * coronavirus 2003 - HCoV-NL63 * coronavirus 2004 - HCoV-HKU1 coronavirus 2005 - HBoV parvovirus 2005 - KI/WU-PyV polyomavirus 2007 - MelV (KamV) orthoreovirus 2007 (2009) - H1N1v influenza virus 2009 -… - animal origin * ErasmusMC involvement
71 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future Order Mononegavirales, family Paramyxoviridae
v.d.Hoogen et al., Nat.Med. 2001
DNA Maximum likelihood, Polymerase ORF Morbillivirus PDVRPV TuV CDV Henipahvirus MV hPIV3 HeV bPIV3 Respirovirus Paramyxovirinae NiV SeV hPIV1 Pneumovirinae
hRSV NDV Avulavirus Pneumovirus bRSV LPMV Metapneumovirus MuV Rubulavirus APV hPIV2 SV5 SV41 0.1 hMPVhMPV
Newly discovered human paramyxovirus hMPV - Risk groups -
- (Young) children ~10 % of children with RTI
- Immunocompp()romised individuals (fatal cases!)
- Elderly
- Normal individuals ~5 % of RTI in community surveillance studies
v.d.Hoogen et al., Nat.Med. 2001 Osterhaus and Fouchier, Lancet 2003 v.d. Hoogen et al., JID 2003
Human metapneumovirus - F protein -
-HMPV F protein is major determinant of protection -Highly conserved 3 4 2 -Genetic lineages are highly 1 5 6 related antigenically A2 7 8 F gene A1
B1 16 15 B2 14 13 9 10 12 11 0.1
72 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Human metapneumovirus - Vaccine approaches -
Vaccine Animal model Authors
Soluble F protein Hamsters, cynomolgus macaques Herfst, 2007/2008
Soluble F protein and Cotton rats Cseke, 2007 F DNA vaccine De Swart, 2007 FI-HMPV Macaques, cotton rats Yim, 2007 HI-HMPV Mice Hamelin, 2007 Cpts HMPV Hamsters, cynomolgus macaques Herfst, 2008 Biacchesi, 2004/2005 HMPV deletion mutants Hamsters, AGM Buchholz, 2005 Chimeric HMPV / AMPV-C Hamsters, AGM Pham, 2005 B/HPIV3 expressing F Hamsters, AGM Tang, 2003/2005 Alphavirus replicon particles Mice, cotton rats Mok, 2008 expressing F T-lymphocyte vaccine Mice Herd, 2006
b/hPIV3 expressing hMPV F gene
AvrII 104 AvrII 1774
b/h PIV3 N P/C M hPIV3 F hPIV3 HN L
b/h hMPV F1 F N P/C M hPIV3 F hPIV3 HN L
b/h hMPV F2 N F P/C M hPIV3 F hPIV3 HN L
Tang R. S. et al., 2003, J Virol 77:10819-28 MedImmune, Inc
b/hPIV3 expressing hMPV F gene
Replication of bovine/human PIV3 expressing the hMPV F protein in position 1 or 2 of the PIV3 genome in hamsters.
Mean virus titer on day 4 post-infection b, c (log10 TCID50/g tissue + S.E.) Virus a Nasal turbinates Lungs
b/h PIV3 4.8 + 0.2 5.6 + 0.6 b/h hMPV F1 5.3 + 0.5 5.7 + 0.4 b/h hMPV F2 5.7 + 0.5 4.6 + 0.3 hMPV 5.3 + 0.1 3.6 + 0.3 a Groups of six hamster were inoculated intranasally with 1 x 106 pfu of indicated virus. b Standard error. c TCID50 assays were read for CPE on Day 10 post-infection. MedImmune, Inc
73 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future b/hPIV3 expressing hMPV F gene
Hamsters immunized with bovine/human PIV3 expressing hMPV F protein are protected from challenge with hMPV or hPIV3.
Challenge hPIV3 hMPV virus: Mean virus titer on day 4 post-challenge Mean virus titer on day 4 post-challenge b b (log10 TCID50/g tissue + S.E.) (log10 pfu/g tissue + S.E.) Immunizing Nasal Turbinates Lungs Nasal Turbinates Lungs Virusa b/h PIV3 <1.3 + 0.2 <1.1 + 0.1 ND ND b/h hMPV F1 <1.3 + 0.1 <1.1 + 0.1 3.5 + 0.8 <0.5 + 0.2 b/h hMPV F2 <1.2 + 0.1 <1.2 + 0.1 <0.9 + 0.4 <0.5 + 0.1 hMPV ND ND <0.8 + 0.3 <0.4 + 0.0 placebo 4.3 + 0.3 4.5 + 0.5 6.0 + 0.3 4.5 + 1.3
a Virus used to immunize groups of six hamsters on Day 0. b On Day 28, the hamsters were challenged with 106 pfu of hPIV3 or hMPV/NL/1/00. ND = not determined. MedImmune, Inc
F subunit vaccine - Immunization / challenge -
-Vaccines: * F1/00 (A1) Specol * F1/00 Iscom * F1/00 nonAdj * F1/99 (()pB1) Specol * F1/99 Iscom * F1/99 nonAdj *Specol * Iscom * PBS
-Hamsters were immunized twice, 3 week interval (10ug F) -Challenge with 106 TCID50 of recNL/1/00 (A1) -Lungs and nasal turbinates were collected 4 dpi
F subunit vaccine - Immunization / challenge -
9 8 -- 7 ------viral titers 6 -- -- /g NT -- in NT 0 5 4 3 2 Log-10 TCID5 1 0
74 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
F subunit vaccine - Immunization / challenge -
6
5 virus titers 4 /g NT in lungs 0
3 ------2
------Log-10 TCID5 1
0
Subunit vaccines - Immunization / challenge – PRVN -
PRVN-assay:
F1/99 F1/00 F1/99 F1/00 F1/99 F1/00 Spec Spec IM IM nonA nonA 1/00 (A) 230 3563 163 2363 953 1/99 (B) 4546 1044 4816 963 9 11 Ratio A-B 3.4 2.5 4.8 Ratio B-A 20 30 1
Mean PRVN titers (8 animals / group), homologous titers are underlined
Cold-passaged viruses - cpNL/1/99 (B1) -
-NL/1/99 (B1) was passaged at gradually decreasing temperatures (25 ºC), pass 35 was sequenced -Recombinant virus (HMPV11) was cloned and rescued
-Four ts RSV mutations were found that can be introduced in recombinant HMPV -Recombinant virus (NL/1/99 (B1) backbone) containing 3 mutations was cloned and rescued (HMPV/R3)
-Shut-off temperature ≥ 38 ºC in vitro -Viruses attenuated in hamsters
75 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Infection of macaques - Waning immunity -
Van den Hoogen et al., J. Gen Virol, 2007
Immunization of macaques - F subunit / cptsHMPV11 -
6 -t=0 first imm, 10µg Fsol (Iscom) / 10 TCID50 HMPV11 / PBS -t=28 second imm.
6.5 -t=84 heterologous challenge 10 TCID50 -1, 3, 5, 9 dpi collection of throat swabs / BAL samples
Immunization of macaques -PRVN -
PBS F/Iscom HMPV11
1/99 (B1) Homol.
1/00 (A1) Heterol.
76 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Immunization of macaques - T-cell proliferation -
Cold-passaged viruses - Results challenge infection -
9 6 6 Imm: 10 TCID50 7 Chall: 10 TCID50 6 4 /gram lung 0/gram NT 0/gram 0
3 2 Log10 TCID5 0 Log10 TCID5 0 t t S 1 3 S 1 3 Vaccine 1 Vaccine 1 B W R B W R V / V / P P P V P V M P M P H M H M H H
Immunization of macaques - Virus detection by Taqman -
Throat swabs
BAL samples
77 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Human metapneumovirus - Conclusion / discussion -
-Cross-protection with adj. F subunit and cpts vaccines in hamsters -HMPV-specific responses were induced in macaques, but animals were not protected against infection -AGMs probably more suitable model, limited availability of reagents to study immunological responses -Ideal experimental animal model is not available, development of such a model may be needed
-Only transient protective immunity is induced after wt infection in macaques, so an effective vaccine should ideally be more immunogenic and protective than natural HMPV infection -Waning immunity: longer time frame between immunization and challenge infection should be considered for vaccination experiments
Paramyxovirus in SARS patients
Hong Kong researchers announce fingings of Paramyxoviruses in SARS patients Similar findings communicated from Canada through SARS etiology network
Rota et al., Science 2003 HCoV HKU v.d. Hoek et al., Nature Med., 2004 - Phylogeny - Fouchier et al., PNAS 2004 Woo et al., J.Virol., 2005
78 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Cynomolgus macaques (Macaca fascicularis)
Simultaneous SCV-hMPV Infection-experiments:
•SCV •hMPV •SCV followed by hMPV
Fouchier et al.,Nature 2003 Kuiken et al., Lancet 2004
Gross pathology in aged macaques
3-5 yrs young 2331 ∧ 10199 ∧ 9081 ∧ 10029 ∧
10-20 yrs aged
324 ∨ 334 ∨ 223 ∨ 507 ∨
35 30 25 20 15
10 4A low4D4D4A1A 4B4C1A1B low low 5 de Lang et al., PLoS Path. 2007
Grosspathology score (%) 0 Smits et al., Plos Path. 2010 young-adult aged
Administration of pegylated IFN-alpha in aged macaques
4A low4D4D4A1A 4B4C1A1B low low
Haagmans et al., Nature Med. 2004 Smits et al., Plos Path. 2010
79 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Vaccine-induced enhancement of viral infections Huisman et al., Vaccine 2009
Possible problems with “a candidate SARS vaccine”
• Lack of efficacy -- problems with animal coronavirus vaccines!
• Safety concerns
-- antibody mediated enhancement (AME) example: FIP candidate vaccines!
-- predisposition for more serious disease examples: i.a. RSV- and measles vaccines!
Antibody mediated enhancement in FIP
• Hallmarks:
- faster onset of disease - more f ulminant co urse of disease
• Hypothesis:
- S-specific antibodies mediate infection of macrophages
80 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Inactivated paramyxovirus vaccines: lessons for SARS?
1960’s: development of inactivated RSV and MV vaccines: formaldehyde-inactivated whole virus preparations (FI-RSV / FI-MV) precipitated with aluminium phosphate / - hydroxide
Vaccination induced short-lived antibody and inbalanced T cell responses
Vaccination predisposed infants for enhanced disease following subsequent natural infection with the respective viruses
→Immunopathology !!!!!
Adverse effects paramyxovirus vaccines
• Formalin-inactivated paramyxovirus vaccines adjuvanted with alum can predispose to hypersensitivity responses
• Similar pathology seen for FI-RSV, FI-MV, and FI-hMPV
• Differences: • FI-RSV study: mild eosinophilic tracheobronchitis, but two fatal cases (with low eosinophil counts!) • FI-MV study: severe eosinophilic tracheobronchitis • FI-hMPV study: eosinophilic alveolitis
Histopathology: infiltration of eosinophils
day 5 day 13 Eosinophilic tracheo-bronchitis day 5 day 13 day 5 day 13 Eosinophilic bronchiolitis day 5 RL de Swart et al., day 13 J Virol 76: 11561 (2002) day 5 Diffuse eosinophilic alveolitis
day 13 Blue: control animals Red: FI-RSV primed animals
81 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Two FI-RSV animals died with hyperinflation
days 0-9 day 12
• Histopathology: no abnormalities • In vitro correlates: high IL-13 and IL-5 responders in the absence of detectable IFN-γ responses RL de Swart et al., J Virol 76: 11561 (2002)
Marshall and Enserink (Science 2004)
Antiviral effect of purified human SARS immunoglobulins in Macaques: protection and no indication for AME
Haagmans et al., in preparation
1400
1200 Human anti-SARS antibody ELISA titers in macaques 0 and 1 day after i.p. injection of different 1000 amounts of human SARS antiserum. 800
Ab titer Ab 600
400
200
0
1e+7 SARS CoV titres in the lungs of 1e+6 macaques treated 5 days earlier with different amounts of human SARS antiserum 1e+5 and infected 4 days earlier with SARS CoV
1e+4 SCV TCID50/ml SCV
1e+3
1e+2 control 0.5 ml 5 ml 50 ml
82 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Prophylactic treatment of ferrets with huMab C3104: SARS-CoV replication, shedding and pathology
AB 10e7 10e6 Pharynx
Lung 10e6 10e5
10e5
/mL 10e4 50 10e4 10e3 TCID 10e3 SARS-CoV / eq mL 10e2 10e2
10 p< 0.001 <10
day 4 0 2 4 0 2 4 days .
C D
10 Lung Pathology 8 * 6
percentage 4
2 *
0 p=0.013
day 4 ter Meulen et al, Lancet 2004
A DNA vaccine induces protective immunity in mice Yang et al., (Nature 2004) NIAID-NIH study
Mice were immunised with plasmids encoding different forms of the SARS CoV spike protein
SARS CoV spike protein expressed by attenuated Vaccinia virus protectively immunizes mice (Bisht et al., PNAS 2004) NIAID-NIH study
Intranasal or intramuscular immunisation of mice with MVA-S at 0 and 4 weeks
Two dayygs after intranasal challenge SARS coV titers were determined in Nasal turbinates and in the lungs
83 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Immunization of ferrets with modified vaccinia virus Ankara based recombinant vaccine against SARS CoV: challenge protection (Weingartl et al., J.V. 2004) Winnipeg study
Immunization of ferrets with modified vaccinia virus Ankara based recombinant vaccine against SARS CoV: enhanced inflammation in the liver (Weingartl et al., J.Virol. 2004)
MVA
rMVA-S Boosted VN response associated “periportal mononuclear panlobular hepatitis” NB: ADV?
PBS
Naïve
Winnipeg study
Protective efficacy of BHPIV3 recombinants expressing SARS-CoV proteins in hamsters (Buchholz et al.,PNAS 2004) NIAID-NIH study
84 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Vaccination of ferrets with inactivated SARS-CoV
Haagmans et al
- BPL inactivated SARS CoV - in the presence or absence of aluminiumhydroxide (alum) or MF59 4 - SARS CoV challenge with 10 TCID50 SARS CoV
immunization Challenge or Challenge (MF59 group) (alum group)
week 0 4 7 22
Necropsies were taken 5 days after challenge
Vaccination of ferrets with inactivated SARS-CoV in alum Haagmans et al., in preparation
b a P<0.05 6 500 5 r VN-Abs 400 Pharyngeal 4 300 swabs /g tissue) /g 3 10 N Ab tite 200 g ARS CoV Eq. o 2 V S (l 100 1 0 0 0 5 10 15 20 25 control 104 105 106 weeks after vaccination c d P<0.05 P<0.05 Lungs 7 7 Lungs 6 6 (RT-PCR) (titration) 5 5 4 /g tissue) /g 4 /g tissue) 10 10 3 3 (log SARSEq. CoV (log 2 SARS CoV TCID50 CoV SARS 2 1 1 0 NB 106 ~ 1 μg control 104 105 106 control 104 105 106
Vaccination of macaques with inactivated SARS CoV (w/wo alum)
- BPL inactivated SARS CoV - in the absence or presence of aluminium hydroxide 7 - SARS CoV challenge with 10 TCID50 SARS CoV
immunization Challenge
week 0 4 19
Necropsies were carried out 5 days after challenge
Haagmans et al., in preparation
85 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Vaccination of macaques with inactivated SARS CoV (w/wo alum)
Haagmans et al., in preparation
a b no adjuvant adjuvant
1250 LST 1500 400 VN-Abs producticytokine IFN-γ 1000 IL-5 300 (pg/ml) 750 1000 200 500
VN Ab titer 500 100 o 250 n
0 0 0 0 5 10 15 20 ctrl...... 106 107 ctrl 106 107 weeks after vaccination c d Lungs no adjuvant adjuvant no adjuvant adjuvant Lungs P<0.05 P<0.05 P<0.05 P<0.05 (titration) 6 6 6 6 (RT-PCR) (log SARS CoV Eq. CoV SARS 50 5 5 5 5
4 4 10 4 4 tissue) /g
/g tissue) /g 3 3
10 3 3 2 2 (log SARS CoV TCID CoV SARS 2 2 1 1
1 1 0 0 ctrl 106 107 ctrl 106 107 ctrl 106 107 ctrl 106 107
Vaccination of macaques with inactivated SARS CoV: Virus neutralizing antibody titers after 2 vaccinations Rotterdam studies
256
128 N titer V 64
32 D.L.
SARS CoV CoV SARS 16
8 Vaccine - + ++ - + ++ Adjuvant - - - + + +
Dose and adjuvant dependent VN antibody induction
Vaccination of macaques with inactivated SARS CoV: SARS CoV PCR titers in the lungs 5 days after challenge Rotterdam studies
10 6
10 5
0 /g 4
5 10
10 3
10 2 * SCV TCID * 10 1 D.L.
10 0 Vaccine - + ++ - + ++ Adjuvant - - - + + +
Dose and adjuvant dependent protection!
86 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Diffuse alveolar damage in virus controls
Peribronchiolar and perivascular lymphoid cuffing in macaques vaccinated with inactivated SARSSARS--CoVCoV with alum
Eosinophil counts in the bronchus of macaques vaccinated with inactivated SARSSARS--CoVCoV with alum
50 45 40 35 ils per field 30
25 20 15 10
Number of eosinoph of Number 5 0 AI042 AF437 AG865 AH246 AH444 AH494 AH547 AH635 AH639 AH647 AG958 AH451 AH531 AH799 AH081 AH260 AH638 AG927 AG940 AH439 AH514 AH201 AH622 AH625 buffer adjuvant 10*5.9 virus 10*5.9 virus + 10*6.9 virus 10*6,9 virus + adjuvant adjuvant
87 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Presence of IL-IL-1313 mRNA in SARSSARS--CoVCoV challenged vaccinated macaques
IL-4 IFN-Y IL-13 400 120 t 200 t p 300 150 80 200 100 num ber num ber 40 num ber 100
relative transcri relative 50 relative transcrip relative relative transcript relative
0 0 PBS SARS CoV vaccine + SARS CoV 0 PBS SARS CoV vaccine + SARS CoV PBS SARS CoV vaccine + SARS CoV
cowpox Since eradication smallpox: more animal poxvirus infections in humans?
Wolfs et al. E. I. D. 2002
monkeypox cowpox
ProMED-mail 9 Jan 2003
cowpox
Pelkonen et al. E.I.D. 2003; 9:1458-1461
Preventive smallpox vaccination with Elstree-RIVM against lethal MPXV infection
Plasma viral load (log10)
C 7 † C: Control XX
6 E: Elstree-RIVM E*: Elstree-BN 5 M/M M/E: MVA + Elstree-RIVM E*
E M/M: MVA + MVA 4
M/E 3
0 5 10 15 20 25 30
Time after infection (days) Stittelaar et al 2005 JVI
88 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Survival of monkeys subjected to different post- exposure treatments after lethal MPXV infection
100
VI: HPMPO-DAPy [6 doses] (n= III: Cidofovir [5 doses] (n=6) 75 IV: Cidofovir [6 doses] (n=6)
V: HPMPO-DAPy [5 doses] (n= 50 % Survival
25 II: Elstree (n=6)
I: Controls (n=17)* 0
013571013 1821 28 V V V VVV
V V V VVV VI: V
V V VVV V: V Time (days after exposure)
V V VVV IV: V III: V Stittelaar et al., Nature, 2006 II:
Order Mononegavirales, family Paramyxoviridae
Morbilliviruses: a continuing story!!!
DNA Maximum likelihood, Polymerase ORF Morbillivirus PDVRPV TuV CDV MV Osterhaus et al., Nature 1988 Henipahvirus Jensen et al.,Science 2002 hPIV3 HeVHeV bPIV3 Respirovirus Paramyxovirinae NiVNiV SeV hPIV1 Pneumovirinae
hRSV NDV Avulavirus Pneumovirus bRSV LPMV MuV Rubulavirus Metapneumovirus APV hPIV2 SV5 SV41 0.1 hMPVhMPV
Morbilliviruses crossing species barriers
Antarctic 1955: CDV in CbCrabeat er seal s CDV in Baikal seals CDV in Caspian seals Nature 1988 EID 2000
CDV in Serengeti lions DMV in Med. monk seals CDV in Jap. macaques Vaccine 1994 Nature 1997 Vet. Microbiol 1989
89 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Morbilliviruses crossing species barriers
Antarctic 1955: CDV in CbCrabeat er seal s CDV in Baikal seals CDV in Caspian seals Nature 1988 EID 2000
CDV in Serengeti lions DMV in Med. monk seals CDV in Jap. Macaques Vaccine 1994 Nature 1997 Vet. Microbiol 1989 should we continue measles vaccination for ever?
Evaluation of new generation MV vaccines
Evaluation of alternative vaccination routes
90 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Evaluation of alternative vaccination routes
CONCLUSIONS:
In the last decade alone a dozen new respiratory viruses have been identified in humans Several are the result of inter-species transmission The relative clinical impact of most is not yet known Candidate vaccines are being developed with classical and novel technologies Novel administration routes (intranasal, dry powder) are explored Safety and efficacy of most of candidate vaccines is not yet clear Rapid response is crucial to contain newly emerging viruses
Acknowledgements: hMPV
Virology Animal experiments Bernadette van den Hoogen Geert van Amerongen MedImmune, Inc Miranda de Graaf Robert Dias-d’Ullois Roderick Tang Theo Bestebroer Jeanne Schickli Monique Spronken Pathology Fiona Fernandes Leo Sppgrong Thijs Kuiken Leenas Bicha Chantal Verheyen Aurelia Haller Onno Schaap Electron Microscopy Richard Spaete Rob van Lavieren Bé Niemeyer Nancy Ulbrandt Edwin Fries Arnita Barnes Martin Schutten Clinical Kannaki Senthil Bert Niesters Gerard van Doornum Jeanne Schickli Jan de Jong Richard Spaete Eefje Schrauwen Jantijn Fockens Karim Hussain Ronald de Groot Theo Harmsen James Simon Statistics Rik de Swart Roel van Eijk Walter Beyer Ron Fouchier Jeroen Maertzdorf
91 Osterhaus - Vaccine prevention of non-influenza viral respiratory disease: current and future
Acknowledgements: Rotterdam SARS studies
Dept. Virology, WHO & Members of the WHO Erasmus MC SARS aetiology team Theo Bestebroer Centers for Disease Control & Prevention, Atlanta, USA Bé Niemeyer Central Public Health Laboratory, London, UK Georgina Aron Public Health Laboratory Centre, Hongkong, SAR China Robert Dias-d’Ullois PiPrince of fWl Wales H ospit itlal, SARChiSAR China Queen Mary Hospital, SAR China Gerard van Doornum Singapore General Hospital, Singapore Martin Schutten Federal Laboratories for Health Canada, Winnipeg, Canada Bert Niesters Health Canada, Ottawa, Canada Geert van Amerongen Bernhard-Nocht Institute, Hamburg, Germany Bart Haagmans Institut Pasteur, Paris, France Byron Martina National Institute of Infectious Disease, Tokyo, Japan Thijs Kuiken Guus Rimmelzwaan Several industrial partners Ron Fouchier James Simon ViroNovative BV Ab Osterhaus
Department of Virology
Emerging virus infections
Email: [email protected]
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