Ebv & Multiple Sclerosis

The Charcot ProjectThe Charcot Project

The viral hypothesis of MSThe viral hypothesis of MS

Gavin Giovannoni

Neuroscience & Trauma Centre, Blizard InstituteBarts and The London School of Medicine and Dentistry

We work on the hypothesis that MS is an autoimmune disease

The autoimmune hypothesis

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Relapsing and Remitting Multiple Sclerosis: Pathology of the Newly Forming LesionRelapsing and Remitting Multiple Sclerosis: Pathology of the Newly Forming Lesion

Barnett & Prineas. Ann Neurol 2004;55:458–468

Magnetization Transfer Changes in the Normal Appearing White Matter Precede Magnetization Transfer Changes in the Normal Appearing White Matter Precede the Appearance of Enhancing Lesions in Patients with Multiple Sclerosis the Appearance of Enhancing Lesions in Patients with Multiple Sclerosis

Filippi et al. Ann Neurol 1998;43:809-814

Bermel et al. Ann Neuol 2012.

Predictors of long-term outcome in Predictors of long-term outcome in MSers treated with interferon beta-1aMSers treated with interferon beta-1a

Highly-effective DMTs

Daclizumab?

Daclizumab High-Yield Process (DAC HYP): First in Class IL-2 Immunomodulator

IL2Rβ(CD122) γcommon

(CD132)

αIL-2

βγ

IL-2

βγ

High AffinityIL-2 ReceptorIL2R

(CD25)

CD, cluster of differentiation; IL, interleukin; NK, natural killer.1. Depper JM et al. J Immunol. 1983;131:690-696; 2. McDyer JF et al. J Immunol. 2002;169:2736-2746; 3. Wuest SC et al. Nat Med. 2011;17:604-610; 4. Bielekova B. Proc Natl Acad Sci. 2006;103:5941-5946; 5. Martin JF et al. J Immunol. 2010;185:1311-1320; 6. Perry JSA et al. Sci Transl Med. 2012;4:145ra106.

Intermediate AffinityIL-2 Receptor

•DAC HYP selectively blocks the high-affinity IL2R by binding the subunit (CD25)

•Promotes a shift of IL-2 signaling towards the intermediate-affinity IL-2R

Biological impact of DAC HYP

•Inhibition of activated T cell responses1−3

•Expansion of CD56bright NK cells4,5

•Normalization of lymphoid tissue inducer cell numbers6

8

DECIDE: daclizumab HYP vs. interferon β-1a in RRMS Annualized Relapse Rate (ARR)

(n=922) (n=919)

Estimated from a negative binomial regression model adjusted for baseline relapse rate, history of prior IFN beta use, baseline EDSS (≤2.5 vs > 2.5) and baseline age (≤35 vs >35). Patients were censored at the earliest of the following events: 1) start of alternative MS medication, 2) 180 days post treatment discontinuation or 3) end of treatment period. CI, confidence interval.

Kappos et al. AECTRIMS 2014

DECIDE: daclizumab HYP vs. interferon β-1a in RRMS DECIDE: daclizumab HYP vs. interferon β-1a in RRMS Proportion Relapse FreeProportion Relapse Free

Percentage relapse free:Week 24: 88% vs. 83%Week 48: 81% vs. 71%Week 96: 73% vs. 59%Week 144: 67% vs 51%

Risk reduction: 41%; p<0.0001*

BL 12 24 36 48 60 72 84 96 108 120 132 144Time on study (weeks)

Risk reduction estimated from a Cox proportional hazards model adjusted for baseline relapse rate, history of prior IFN beta use, baseline EDSS and baseline age. Analysis includes INEC confirmed relapses. * Not statistically significant per the sequential closed testing procedure.


• Reductions in lesions (T2, Gd+, and T1) were observed as early as 24 weeks for DAC HYP vs IFN beta-1a (p<0.001 for all comparisons)

DECIDE: daclizumab HYP vs. interferon β-1a in RRMS DECIDE: daclizumab HYP vs. interferon β-1a in RRMS Effect on MRI-defined lesions at Week 96Effect on MRI-defined lesions at Week 96

*Adjusted mean for T2 and T1 lesions. †The number of new/newly enlarging T2 lesions at 96 weeks was a secondary endpoint. Data were adjusted for baseline lesion volume, history of prior IFN beta-1a use and baseline age (≤35 v >35). Missing data were not imputed and patients with no post baseline MRI were excluded from analysis of new/newly enlarging T2 lesions at 96 weeks. Missing data were imputed for all other analyses.

New T1 Hypointense Lesions‘black holes’New Gd+ Lesions

IFN beta-1a 30 mcg DAC HYP 150 mg

n=908n=841 n=864 n=909 n=900 n=899

New/Newly Enlarging T2 Lesions†

n=841


DECIDE: daclizumab HYP vs. interferon β-1a in RRMS DECIDE: daclizumab HYP vs. interferon β-1a in RRMS 3-Month and 6-Month Confirmed Disability Progression3-Month and 6-Month Confirmed Disability Progression

Risk reduction: 16%; p=0.16

Proportion with progressionWeek 48: 6% vs. 8% Week 96: 12% vs. 14% Week 144: 16% vs. 20%



Risk reduction: 27%; p=0.0332

Proportion with progressionWeek 48: 4% vs. 7% Week 96: 9% vs. 12% Week 144: 13% vs. 18%

3-month* 6-month†

*3-month confirmed: Patients censored after tentative progression (n=67) analyzed per primary method in the statistical analysis plan: all imputed as non-progressors; †6-month confirmed: Patients censored after tentative progression (n=108) imputed per observed rate in trial; tertiary endpoint. Estimated proportions are the average over imputed datasets. For both endpoints risk reductions based on Cox proportional hazards model adjusted for baseline EDSS, history of prior IFN use, and age.


Effect of Anti-CD25 Antibody DaclizumabEffect of Anti-CD25 Antibody Daclizumabin the Inhibition of Inflammation and in the Inhibition of Inflammation and Stabilization of Disease Progression in Stabilization of Disease Progression in

Multiple SclerosisMultiple Sclerosis

Bielikova et al. Arch Neurol. 2009;66(4):483-489

Monoclonal antibody blockade of IL-2 receptor during lymphopenia Monoclonal antibody blockade of IL-2 receptor during lymphopenia selectively depletes regulatory T cells in mice and humansselectively depletes regulatory T cells in mice and humans

Kappos et al. Blood 2011;118(11):3003-3012.

AAN 2014

AAN 2014

.

Epidemics or clusters of MS

• No documented cases of MS on the Faroe Islands until after World War II

• 55 cases since 1940• Occupied during World

War II• Authors concluded that

this was evidence of an MS epidemic caused by an agent introduced by the troops

• However a number of concerns remain

The annual incidence of MS (per 100 000 inhabitants) in the Faroe Islands since 1940

Kurtze et al 1993

0

2

4

6

8

10

12

1940 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990

Is the MS dogma wrong?

immune activationinnate and adaptive responses

focal inflammation

BBB breakdown

oligodendrocyte toxicity & demyelination

Acute axonal transection and loss

“autoimmune endophenotype”

axonal plasticity & remyelination

delayed neuroaxonal loss and gliosis

Gd-enhancementGd-enhancement

T2 & T1 lesionsT2 & T1 lesions

brain & spinal cord atrophybrain & spinal cord atrophy

release of soluble markersrelease of soluble markers

Clinical AttackClinical Attack

Disease ProgressionDisease Progression

Clinical RecoveryClinical Recovery

- biology

- clinical outcomes

- biomarkers

Is the MS dogma wrong?

immune activationinnate and adaptive responses

focal inflammation

BBB breakdown

oligodendrocyte toxicity & demyelination

Acute axonal transection and loss

“autoimmune endophenotype”

axonal plasticity & remyelination

delayed neuroaxonal loss and gliosis

Gd-enhancementGd-enhancement

T2 & T1 lesionsT2 & T1 lesions

brain & spinal cord atrophybrain & spinal cord atrophy

release of soluble markersrelease of soluble markers

Clinical AttackClinical Attack

Disease ProgressionDisease Progression

Clinical RecoveryClinical Recovery

- biology

- clinical outcomes

- biomarkers

VIRUS(EBV, HERVs)

Infectious agents in MS

. Ramagopalan et al 2009

. Handel et al, 2010

19390 MS patients and 16007 controls, p < 1019390 MS patients and 16007 controls, p < 10-54-54

Infectious Mononucleosis and MSInfectious Mononucleosis and MS

MS IM

Ramagopalan et al, 2011

Pearson R= 0.70, p=0.000025Pearson R= 0.70, p=0.000025

Causation theoryCausation theory

CausationCausation

http://www.campusi.com/bookFind/asp/bookFindPriceLoad.asp?prodId=0029266718

early adopters

late adopters

Causation - Koch’s PostulatesCausation - Koch’s Postulates

1.1. The specific organism should be shown The specific organism should be shown to be present in all cases of animals to be present in all cases of animals suffering from a specific disease but suffering from a specific disease but should not be found in healthy animals. should not be found in healthy animals.

2.2. The specific microorganism should be The specific microorganism should be isolated from the diseased animal and isolated from the diseased animal and grown in pure culture on artificial grown in pure culture on artificial laboratory media. laboratory media.

3.3. This freshly isolated microorganism, This freshly isolated microorganism, when inoculated into a healthy when inoculated into a healthy laboratory animal, should cause the laboratory animal, should cause the same disease seen in the original same disease seen in the original animal. animal.

4.4. The microorganism should be The microorganism should be reisolated in pure culture from the reisolated in pure culture from the experimental infection. experimental infection.

1843-1910

http://www.nmsi.ac.uk/piclib/subjectdetail.asp?sub=PER

Sir Bradford-Hill: Criteria for CausationSir Bradford-Hill: Criteria for Causation

Bradford-Hill A. The environment and disease: association or causation? Proc Royal Soc Med 1966;

58:295.

1.1. CONSISTENCY AND UNBIASEDNESS OF FINDINGS CONSISTENCY AND UNBIASEDNESS OF FINDINGS

2.2. STRENGTH OF ASSOCIATIONSTRENGTH OF ASSOCIATION

3.3. TEMPORAL SEQUENCETEMPORAL SEQUENCE

4.4. BIOLOGICAL GRADIENT (DOSE-RESPONSE RELATIONSHIP)BIOLOGICAL GRADIENT (DOSE-RESPONSE RELATIONSHIP)

5.5. SPECIFICITYSPECIFICITY

6.6. COHERENCE WITH BIOLOGICAL BACKGROUND AND PREVIOUS COHERENCE WITH BIOLOGICAL BACKGROUND AND PREVIOUS

KNOWLEDGEKNOWLEDGE

7.7. BIOLOGICAL PLAUSABILITYBIOLOGICAL PLAUSABILITY

8.8. REASONING BY ANALOGYREASONING BY ANALOGY

9.9. EXPERIMENTAL EVIDENCEEXPERIMENTAL EVIDENCE

Causation and Disease: A Chronological Journey

Alfred S. Evans Hardcover / Plenum Pub Corp

March 1993 / 0306442833

““Evidence” or “lack of evidence”Evidence” or “lack of evidence”

Infectious mononucleosisInfectious mononucleosis

Handel et al. 2010.

Small OR

Odds ratio of MS in subjects seronegative for EBVOdds ratio of MS in subjects seronegative for EBV

Ascherio et al, 2007

EBV Seropositivity titresEBV Seropositivity titres

99.2%99.2%

.

90.2%90.2%

Ascherio et al, 2000

EBV antibody titresEBV antibody titres

.Levin et al, 2005

Distribution of MS in EnglandDistribution of MS in England

Ramagopalan et al., JNNP 2011.Michael Goldacre; Unit of Health-Care Epidemiology, Department of Public Health, University of Oxford, Oxford

Distribution of IM in EnglandDistribution of IM in England

Ramagopalan et al., JNNP 2011.Michael Goldacre; Unit of Health-Care Epidemiology, Department of Public Health, University of Oxford, Oxford

Correlation Between Distribution of IM and MS in EnglandCorrelation Between Distribution of IM and MS in England

Females- Pearson R= 0.70, p=0.000025

Males- Pearson R= 0.55, p=0.003

Correlations don’t imply causationRamagopalan et al., JNNP 2011.

Primary infection with the EBV and risk of MSPrimary infection with the EBV and risk of MS

• Nested case-control study including from over 8 million military personnel with Nested case-control study including from over 8 million military personnel with serum stored in the Department of Defense Serum Repository. serum stored in the Department of Defense Serum Repository.

Levin et al. Ann Neurol 2010.

MSMSMSMS

ControlsControlsControlsControls N = 610

N = 30510/305 (3.3%) EBV –ve

32/610 (5.2%) EBV –ve 10/28 (35.7%) EBV –ve

10/10 (100%) EBV –ve

• MS risk is extremely low among individuals not infected with EBV, but it MS risk is extremely low among individuals not infected with EBV, but it increases sharply in the same individuals following EBV infection.increases sharply in the same individuals following EBV infection.

• Small study ; yet to be reproduced.Small study ; yet to be reproduced.

.

Levin et al, 2010

The ugly factThe ugly fact

““The great tragedy of Science; the slaying of a beautiful The great tragedy of Science; the slaying of a beautiful hypothesis by an ugly fact.” hypothesis by an ugly fact.”

Thomas Henry HuxleyThomas Henry Huxley

Viral serologies in children with MSViral serologies in children with MS

Banwell et al. Lancet Neurology, Sept. 2007

?

CongruencyCongruency

Compston & Coles, Lancet 2008.

Epidemiology Epidemiology worldwide distribution & migration studiesworldwide distribution & migration studies

Genetics of MS: the rate of MS in females is increasingGenetics of MS: the rate of MS in females is increasing

1Orton SM et al. Lancet Neurol 2006;5:932–936.

Smoking is a risk factor for multiple sclerosisSmoking is a risk factor for multiple sclerosis

Handel et al. PLoS One. 2011 Jan 13;6(1):e16149.

Ramagopalan et al. Arch Neurol 2011; 68:469-72.

Biological plausabilityBiological plausability

EBV & Disease ActivityEBV & Disease Activity

Farrell et al. Neurology 2009

EBNA-1 EBNA-1

NOT NOT VCA or EAVCA or EA

Dysregulated EBV infection in the MS brainDysregulated EBV infection in the MS brain

Serafini et al. J Exp Med. 2007 Nov 26;204(12):2899-912.

Negative follow-up studiesNegative follow-up studies

1.1. Torkildsen O, et al. Upregulation of Immunoglobulin-related Genes in Torkildsen O, et al. Upregulation of Immunoglobulin-related Genes in

Cortical Sections from Multiple Sclerosis Patients. Brain Pathol. 2009 Oct 16. Cortical Sections from Multiple Sclerosis Patients. Brain Pathol. 2009 Oct 16.

[Epub ahead of print] [Epub ahead of print]

2.2. Willis SN, et al. Epstein-Barr virus infection is not a characteristic feature of Willis SN, et al. Epstein-Barr virus infection is not a characteristic feature of

multiple sclerosis brain. Brain. 2009 Dec;132(Pt 12):3318-28. multiple sclerosis brain. Brain. 2009 Dec;132(Pt 12):3318-28.

3.3. Peferoen LA, et al. Epstein Barr virus is not a characteristic feature in the Peferoen LA, et al. Epstein Barr virus is not a characteristic feature in the

central nervous system in established multiple sclerosis. Brain. 2010 central nervous system in established multiple sclerosis. Brain. 2010

May;133(Pt 5):e137. Epub 2009 Nov 16. May;133(Pt 5):e137. Epub 2009 Nov 16.

4.4. Hans Lassman et al, personal communication. Hans Lassman et al, personal communication.

Innate immune activation is a hallmark of the active MS lesions

Tzartos et al., Neurology, in print

Intrathecal oligoclonal IgG bands (OCBs)Intrathecal oligoclonal IgG bands (OCBs)

1. Rand KH, et al. (1998) Molecular approach to find target(s) for oligoclonal bands in multiple sclerosis. J Neurol Neurosurg Psychiatry 65:48-55.

2. Bray PF, et al. (1992) Antibodies against Epstein-Barr nuclear antigen (EBNA) in multiple sclerosis CSF, and two pentapeptide sequence identities between EBNA and myelin basic protein. Neurology 42:1798-804.

3. Cepok S, et al. (2005) Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis. J Clin Invest 115:1352-60.

4. Rand KH, et al. (2000) Epstein-Barr virus nuclear antigen-1 (EBNA-1) associated oligoclonal bands in patients with multiple sclerosis. J Neurol Sci 173:32-9.

C+ / S-

AnalogyAnalogy

Axthelm et al. Ann Neurol 2010

Japanese Macaque Encephalomyelitis: Japanese Macaque Encephalomyelitis: A Spontaneous Multiple Sclerosis–like Disease in a Nonhuman PrimateA Spontaneous Multiple Sclerosis–like Disease in a Nonhuman Primate

HTLV-1 myelopathyHTLV-1 myelopathy

Experimental evidenceExperimental evidence

N Engl J Med 2008;358:676-88.

Highly-effective DMTs

Daclizumab?

Summimg-upSummimg-up

Sir Bradford-Hill: Criteria for Sir Bradford-Hill: Criteria for CausationCausation

Bradford-Hill A. The environment and disease: association or causation? Proc Royal Soc Med 1966;

58:295.

1.1. CONSISTENCY AND UNBIASEDNESS OF FINDINGS - CONSISTENCY AND UNBIASEDNESS OF FINDINGS - Yes Yes (not 100%)(not 100%)

2.2. STRENGTH OF ASSOCIATION – STRENGTH OF ASSOCIATION – ? ? // Yes Yes (RR ~ 2 to 3)(RR ~ 2 to 3)

3.3. TEMPORAL SEQUENCE - TEMPORAL SEQUENCE - YesYes

4.4. BIOLOGICAL GRADIENT (DOSE-RESPONSE RELATIONSHIP) - BIOLOGICAL GRADIENT (DOSE-RESPONSE RELATIONSHIP) - ? ? (not relevant to (not relevant to

infections)infections)

5.5. SPECIFICITYSPECIFICITY – – NoNo (not 100% other putative autoimmune diseases also associated with (not 100% other putative autoimmune diseases also associated with

EBV)EBV)

6.6. COHERENCE WITH BIOLOGICAL BACKGROUND AND PREVIOUS KNOWLEDGE - COHERENCE WITH BIOLOGICAL BACKGROUND AND PREVIOUS KNOWLEDGE -

YesYes

7.7. BIOLOGICAL PLAUSABILITY - BIOLOGICAL PLAUSABILITY - YesYes

8.8. REASONING BY ANALOGY - REASONING BY ANALOGY - YesYes

9.9. EXPERIMENTAL EVIDENCE - EXPERIMENTAL EVIDENCE - ??

Is EBV the cause of Is EBV the cause of MS?MS?

Is there a “Black Swan” flying in?

Charcot Project: viral aetiology

HIV and lower risk of MS: beginning to unravel a mystery using a record-linked database study

Nexø et al. Epidemiology 2013; 24:331-2

Treatment of HIV and Risk of Multiple Sclerosis

Gold et al. JNNP August 4, 2014 as 10.1136/jnnp-2014-307932.

Raltegravir → RRMS (INSPIRE STUDY)ClinicalTrials.gov ID: NCT01767701

Endogenous retroelements and autoimmune disease Endogenous retroelements and autoimmune disease

Volkman & Stetson. Nat Immunol 2014

EBV

? mutations

EBV: where to next?

Early infection

Late infection

Asymptomatic seroconversio

n

Infectious mononucleosus

At risk MS

vD/Sunlight Smoking

Genetics1. Epidemiologists2. Virologists3. Genomics4. Bioinformatics5. Immunologists6. Neurologists7. Pharmaceuticals

Vaccine

IM Rx

MS Rx

AcknowledgementsAcknowledgements• Ute MeierUte Meier

• Monica MartaMonica Marta

• Sreeram RamagopalanSreeram Ramagopalan

• Ruth DobsonRuth Dobson

• George EbersGeorge Ebers

• Jo ToppingJo Topping

• Georgina BurrowGeorgina Burrow

• Julian GoldJulian Gold

• Rachel FarrellRachel Farrell

• Cosimo MaggioreCosimo Maggiore

• Jaap MiddeldorpJaap Middeldorp

• Sandra AmorSandra Amor

• Dorothy CrawfordDorothy Crawford

• Karen McCauleyKaren McCauley

• Adam HandelAdam Handel

• Giulio DeSantoGiulio DeSanto

• Hadi Amir-MaghziHadi Amir-Maghzi

• Chris HawkesChris Hawkes

• Klaus SchmiererKlaus Schmierer

• David BakerDavid Baker

Health & Medicine

Ebv & Multiple Sclerosis