Enterovirus Infections and Type 1

Diabetes
- Can a Vaccine Test Causality?

Malin Flodstrm Tullberg, Ph.D.

Karolinska Institutet, Stockholm, Sweden

No conflict of interest
Malin Flodstrm Tullberg

September 5, 2014

Type 1 Diabetes -

A Disease with a Multifactorial Origin

EPIGENETICS
?
GENES
Candidate genes (IDDM1-19):
HLA II (IDDM1)
VNTR in insulin gene (IDDM2)
CTLA-4 (IDDM12)
MDA5/IFIH1 (IDDM19)
etc
Malin Flodstrm Tullberg

ENVIRONMENT
Possible factors:
Hygiene
Hormones
Stress
Dietary Factors
Microbial infections,
enteroviruses
September 5, 2014

Environmental factors

Enteroviruses

Small single stranded RNA viruses within the picornavirus

family. Cause lytic or persistent infections.

Infects via the fecal-oral route. The gastrointestinal mucosa is

the primary replication site.

Asymptomatic infections, acute respiratory disease,

myocarditis, meningitis, and acute pancreatitis.

Chronic dilated cardiomyopathy, Type 1 Diabetes (primarily

Coxsackieviruses (CVBs)).

Malin Flodstrm Tullberg

September 5, 2014

Environmental factors

Enteroviruses

Temporal association between enterovirus infections and

induction of beta-cell autoimmunity.

Autoantibody positive individuals progress more quickly to T1D if

infected with enterovirus (DAISY).

Enteroviruses have been isolated at the onset of T1D.

Virus RNA and/or viral proteins found in pancreata of T1D

patients.

Infect and damage/destroy beta cells in vitro.

Malin Flodstrm Tullberg

September 5, 2014

The Question:
Is there a causal relationship between
enterovirus infections and the
development of type 1 diabetes?

Vaccine

Malin Flodstrm Tullberg

September 5, 2014

Challenges for Vaccine

Development:
Coverage?
> 100 genotypes within the enterovirus genus
Safety?
Potential risk for induction of autoimmunity
Efficacy?
Low efficacy of vaccines in young infants

Malin Flodstrm Tullberg

September 5, 2014

Challenges for Vaccine

Development:
Coverage?
> 100 genotypes within the enterovirus genus
Safety?
Potential risk for induction of autoimmunity
Efficacy?
Low efficacy of vaccines in young infants

Malin Flodstrm Tullberg

September 5, 2014

Challenges for Vaccine Development:

> 100 Serotypes within the

Enterovirus Genus

-> It may be possible to narrow down the number of

enterovirus types suspected to trigger T1D

Malin Flodstrm Tullberg

September 5, 2014

Challenges for Vaccine

Development:
Coverage?
> 100 genotypes within the enterovirus genus
Safety?
Potential risk for induction of autoimmunity
Efficacy?
Low efficacy of vaccines in young infants

Malin Flodstrm Tullberg

September 5, 2014

Challenges for Vaccine Development:

Risk for Induction of

Autoimmunity

Temporal association between enterovirus infections and induction of

beta-cell autoimmunity.

Autoantibody positive individuals progress more quickly to T1D if

infected with enterovirus (DAISY).

CVB3 and CVB4 accelerates diabetes development in pre-diabetic

NOD mice.

Malin Flodstrm Tullberg

September 5, 2014

Aim

To address whether a CVB1 infection accelerates

disease (diabetes) onset in NOD mice

Malin Flodstrm Tullberg

September 5, 2014

Safety testing

A CVB1 Infection Accelerates Diabetes

Development in Pre-diabetic NOD mouse
Untreated

T1D development

Buffer treated

CVB1 infected

Age at diabetes
development, weeks

Virus
infection

30

p=0.01

20

10

nt
r

B
ea
uf
te
fe
d
rtr
ea
te
d
In
fe
ct
ed

* P < 0.05

Larsson et al. In revision.

Malin Flodstrm Tullberg

September 5, 2014

Challenges for Vaccine Development:

Risk for Induction of

Autoimmunity

Temporal association between enterovirus infections and induction of

beta-cell autoimmunity.

Autoantibody positive individuals progress more quickly to T1D if

infected with enterovirus (DAISY).

CVB3, CVB4 and CVB1 accelerates diabetes development in NOD

mice.

-> Initial safety testing can be done in the NOD mouse

Malin Flodstrm Tullberg

September 5, 2014

Aim

To test the efficacy and safety of a prototype vaccine to

CVB1

Malin Flodstrm Tullberg

September 5, 2014

Vaccine Approach & Efficacy Testing

Non-adjuvanted formalin inactivated CVB1 virus
Balb/c mice
Challenge w CVB1

Immunizations

Blood sampling
Pancreas sampling

D0 D2

D21

D35

D42

D49

D51

D56

Efficacy testing:

FIGURE 2A Virus titers BALB/c

Vaccination Protects Balb/c mice

from CVB1 Infection

Untreated, n=10

Malin Flodstrm Tullberg

y
da
ed

Va

cc

in

at
re
nt
U

,d
ed

at

ed

7
ay

7
at
in
cc
Va

nt
U

Va

cc

in

re

at

at

ed

ed

,d

,d

ay

2
ay

2
ay
,d
ed
at
re
nt

Vaccinated, n=10

***

od

,d
ay
lo
od

**

**

ea
s,

Pancreatic
viral titers
Pancreas
***

,d
ay

Pa
nc
r

***

Virus titer, log10 Eq TCID50/ml

Blood virus
titers BALB/c
Viremia

lo titer, log10
2
Virus
Eq TCID50/ml

Log10 CCID50/ml or g

***

Larsson et al. In revision.

September 5, 2014

Safety Testing

Female NOD mice

Immunizations
Blood sampling

7 months
of age
11

14

16

18

Age in weeks

Blood Glucose Measurements

Malin Flodstrm Tullberg

September 5, 2014

Safety testing

Vaccination Induces Neutralizing

Antibodies in NOD mice
S5 Pilot exp, vacciantions
Neutralization titer, log2

15

10

Buffer, n=6
Vaccinated, n=5

17-8
3
Larsson et al. In revision.

ay

35

42

16
2

(e
nd
)

io
n)
D
ay

(3
r

in
nd

je
ct

in

io
n)

14
1

je
ct

11
0

(2

21
ay

Malin Flodstrm Tullberg

ay

(1
st

in

je
ct

Age (weeks)
# of inj.

io
n)

September 5, 2014

Safety testing

No Accelerated Diabetes Onset in

Vaccinated NOD Mice
Untreated

T1D development

Buffer treated
Vaccinated

Age at diabetes
development, weeks

Vaccination (x3)

30

n.s.

20

10

U
nt
re
B
uf
at
fe
ed
rtr
ea
te
Va
d
cc
in
at
ed

Larsson et al. In revision.

Malin Flodstrm Tullberg

September 5, 2014

Challenges for Vaccine

Development:
Coverage?
> 100 genotypes within the enterovirus genus
Safety?
Potential risk for induction of autoimmunity (e.g. via molecular
mimicry)
Efficacy?
Low efficacy of vaccines in young infants

Malin Flodstrm Tullberg

September 5, 2014

Challenges for Vaccine Development:

Low Efficacy of Vaccines

in Infants
Infections with enterovirus are common even in young children

Autoantibodies may appear early in life

Low efficacy of vaccines in very young children
Passive transfer of antibodies from mother to child
-> Possibility to vaccinate women before child birth alternatively
during pregnancy?

Malin Flodstrm Tullberg

September 5, 2014

Aim

To address whether immunity to enteroviruses is

passively transferred from mother to child

Malin Flodstrm Tullberg

September 5, 2014

Passive Immunization:

Can Maternal Antibodies Protect from

Infection and Diabetes Development?

4-6 weeks
Neutralizing
Antibodies?

Malin Flodstrm Tullberg

September 5, 2014

Passive Immunization:

Offspring From Previously Infected Females

have Neutralizing Antibodies to Enterovirus
***

***

***

*** p < 0.01

Larsson et al. Diabetologia, 2013

Malin Flodstrm Tullberg

September 5, 2014

Passive Immunization:

Passive Immunity Protects Offspring

from Enterovirus Infection
Viremia, day 3 p.i.

***

Pancreas virus
titers day 3 p.i.

**

** P < 0.01; *** P < 0.001

Larsson et al. Diabetologia, 2013
Malin Flodstrm Tullberg

September 5, 2014

Passive Immunization:

Are Maternal Antibodies Protecting

Offspring from Infection and Type 1
Diabetes?

Malin Flodstrm Tullberg

September 5, 2014

The suppressor of cytokine signaling-1

(SOCS-1) transgenic mouse

SOCS-1-Tg NOD mice express SOCS-1

under the control of the insulin promoter
SOCS-1-Tg NOD mice harbor beta cells
that cannot respond to interferons (IFNs)

Malin Flodstrm Tullberg

September 5, 2014

SOCS-1-tg Mice Develop Diabetes

Following Infection with Enterovirus
Non-Tg

SOCS-1-Tg
Blood Glucose mg/dl

Blood Glucose mg/dl

500
400
300

200
100
0
5

10

Days p.i.

600
500
400
300
200
100
0
5

10 Days p.i.

Flodstrm, et al Nat. Immunol., 2002; Flodstrm, et al Diabetes, 2003

Malin Flodstrm Tullberg

September 5, 2014

SOCS-1 Transgenic Mice Loose

their beta cells after CVB Infection
NOD
Insulin

SOCS-1-TG
Glucagon

Insulin

Glucagon

Control,
Uninfected,

CVB.

CVB3,
d.28 p.i. from
infected mother
Malin
Pr
Larsson
Flodstrm Tullberg

N/A

N/A

Flodstrm, et al. Nat Immunol, 2002;

Flodstrm, et al. Diabetes, 2003;
Larsson et al. Diabetologia, 2013
September
5 september
5, 2014

30

Experimental set-up

Malin Flodstrm Tullberg

September 5, 2014

Passive Immunity Protects Offspring

from Enterovirus-Induced Diabetes

***

*** P < 0.001

Larsson et al. Diabetologia, 2013

Malin Flodstrm Tullberg

September 5, 2014

Passive Immunity Protects Offspring from

Tissue Damage During Enterovirus Infection
SOCS-1-tg
SOCS-1-tg
islet, d.28 p.i.
islet, d.7 p.i.
(nave mother) (infected mother)

Insulin

Glucagon
Larsson et al. Diabetologia, 2013
Malin Flodstrm Tullberg

September 5, 2014

Conclusions
CVB1 accelerates diabetes development in NOD mice
The prototype CVB1 vaccine appears to be safe and confers
protection from infection without accelerating beta cell
autoimmunity
Maternal antibodies protect neonates from virus-induced
damage and type 1 diabetes

Malin Flodstrm Tullberg

September 5, 2014

Future Research

Additional studies to identify potentially diabetogenic

enteroviruses
Development and testing of a prototype multi-valent vaccine

Malin Flodstrm Tullberg

September 5, 2014

Acknowledgements
Flodstrm Tullberg group, Karolinska Institutet
Katharina Lind
Pr Larsson
Emma Svedin
Renata Utorova
Olli Laitinen
Virginia Stone
Erna Domsgen
Sebastian Kapell
Alvin Brodin
Stella Jacobson
Kanth Tadepally

University of Tampere, Finland

Heikki Hyty and associates

University of Helsinki, FInland

Mikael Knip and associates
Vactech Oy, Finland
Minni Koivonen
Olli Laitinen
Sanofi Pasteur, France
Valerie Lecouturier
Nicolas Devard
Pascal Chaux
Jeffrey Almond

The Swedish Child Diabetes Foundation, The Swedish Diabetes Association Research Foundation, EFSD/Novo
Nordic, EU (Marie Curie, and FP7 PEVNET), Karolinska Institutet including the strategic research program in
Diabetes, Swedens innovation agency (VINNOVA).

Malin Flodstrm Tullberg

September 5, 2014