Vaccine Safety:

Evaluating the Science

January 3-7, 2011,

Tryall Club,Jamaica

Aluminum as a neurotoxin: the

evidence from cell culture, in vivo, and

human studies

Christopher A. Shaw

Depts. of Ophthalmology and Visual

Sciences, Experimental Medicine, and

Program in Neuroscience

Topics to be addressed

• Conflict of interest

• Neurological disease clusters

• ALS epidemiology

• Following ALS-PDC

• Gulf War Syndrome and aluminum

• Aluminum neurotoxicity in various

preparations

• Mimicking motor neuron disease with

aluminum adjuvants

• Conclusions and future directions

Evaluating critical vs. non-critical

events in neurological disease

• Causal events start disease process

-essential to know

• Coincident events

- not directly relevant, can be ignored?

• Compensatory events

- something to increase?

• Prevention = causality

• Early treatment = timeline of critical stages

• Cure = ??? (no consensus on what this means)

~30

0 Symptoms of

disease No clinical symptoms, but

disease process ongoing

Healthy neurons

Central

Nervous

System

“Health”

100

Time

Conventional

Diagnosis

Neurological disease

progression:AD, PD, ALS

Prevention

Intervention

Palliative Care

?? Cure

Causal Event(s)

Neuro-

degenerative

Cascade

Causality: environment, genetics, and age?

Age

Young

Old

Genetics

Low High Detoxification

Low High [Toxin]

Environment

•Cycad toxicity

•Rotenone/Pesticides

•MPTP

•Lead/

Heavy metals

•High cholesterol/

Diet

•Viral infections

Susceptibility Genes

•ApoE

•Glutathione Transferase

•CYP genes

•ND3 (complex 1 enzyme)

High Low

Abnormal Toxicity Disease

Phenotype Causative Genes

•mAPP/tau

•PS1/PS2

•mSOD1

•alsin

•α-synuclein

•Parkin

•UCHL-1

•LKKR2

Alzheimer‟s

Disease

Parkinson‟s

Disease

ALS

• Xenobiotic metabolism

•Toxic accumulation

•Previous CNS trauma

• Cerebral Perfusion

Clues from neurological disease

„clusters‟

• Defn: a large number of cases of the disease with time and geography constraints

• The Centers for Disease Control and Prevention (CDC) define a cluster investigation as, "a review of an unusual number, real or perceived, of health events (for example, reports of cancer) grouped together in time and location." Cluster investigations seek to confirm cases of the disease; establish whether the reported cases represent an unusually high occurrence of the disease; and explore potential causes when possible.

Ex: Eastern Canada: domoic acid in mussels (example of bio-magnification)

ALS:

• ALS-parkinsonism dementia complex (ALS-PDC) of the Western Pacific

• Gulf War Syndrome ALS?

Changing ASD prevalence

0

50000

100000

150000

200000

250000

300000

350000

400000

3-22 ys

6-22 ys

• ALS („lytico‟); parkinsonism with

dementia („bodig‟); combined form

possible.

• Lytico, bodig once 100-400x greater

than ALS in N. America; leading

cause of death.

• Familial clustering, but no clear

genetic inheritance; genetic

susceptibility (e.g., apo E), not

causality. Importance of gene-

environment interactions.

• Males > Females: 2-3:1

• Strange features of ALS-PDC:

olfactory disturbance; early exposure

linked to delayed onset?

• Strong link to dietary neurotoxin

contained in cycad seeds (Cycas

micronesica).

Guam

ALS-PDC of Guam

ALS-PDC • Earliest recorded case of ALS-

PDC phenotype in Chamorro population in early 1800s.

• Peak in 1950s/60s: decline in ALS-PDC coincides with change in dietary habits.

• ALS has declined to approx. N. American levels; PDC still higher.

• Saipan vs. Guam: why different?

• Single major ALS, PD, AD

disease „cluster‟.

• Water soluble BOAA, BMAA,

cycasin/MAM not involved (ie.,

no bats involved).

• Neurological “Rosetta Stone”.

What are the toxins in cycad?

Evidence for:

• cycasin/MAM: genotoxic effects, do not give

ALS-PDC-like outcomes

• BMAA: weak NMDA agonist; AMPA agonist; no

ALS-PDC outcomes (…and not from bats)

• BOAA: lathyrism not really ALS-PDC

• Other “toxins”? Role for sterol glucosides.

• Other environmental factors: high aluminum ??

A simple experiment: Cycad (w/out BMAA, BOAA, cycasin/MAM)

+ Mouse = ALS-PDC

4 Dimensions

•Behavioural: motor,

cognitive and olfactory

•Cellular

• Biochemical

Time

Behaviour: Motor tests

Wire Hang –Muscle Strength

Paw Print –Gait Length Measure Rotarod –Motor Coordination and Strength

Leg Extension –Motor

Neuron Integrity

Leg Extension

0 10 20 30 40 500

1

2

Control

Cycad#

[* p<0.05

+ p<0.001

# p<0.0001

+++ +++++ +++

Day

# o

f h

ind

leg

s e

xte

nd

ed

Magnetic resonance microscopy

Volume measurements: MRM

D C

B A

MRM: some examples

f

a b c

d e

Ventral Horn

Control Cycad-fed0.0

0.2

0.4

0.6

0.8

1.0-20%

GroupN

orm

ali

zed

Vo

lum

e

Cycad feeding: measurable ALS

phenotype

Leg Extension

0 10 20 30 40 500

1

2

Control

Cycad#

[* p<0.05

+ p<0.001

# p<0.0001

+++ +++++ +++

Day

# o

f h

ind

leg

s e

xte

nd

ed

Motor Neuron Count

Control Cycad-fed0

5

10

15

20

*

Group

MN

s c

ou

nte

d

GFAP

Labelled

Ventral Horn Volume

Control Cycad Fed0.0

0.2

0.4

0.6

0.8

1.0

Group

No

rmali

zed

Vo

lum

e

Gray Matter

Control Cycad-fed0.0

0.2

0.4

0.6

0.8

1.0

1.2

- 21%**

Group

No

rmali

zed

Vo

lum

e

MRI Volume Analysis

SC

Cycad Fed

SC

Control

GLT-1B

Labelling

ALS-PDC vs cycad-fed mice

Table 1. Comparison of ALS-PDC symptoms and cycad-induced outcomes in mice and rats

ALS-PDC

Cycad-fed

mice

Progressive motor weakness

+

+

Gait impairment

+

+

Cognitive deficits

+

+

Olfactory dysfunction

+

+

Tau

+

?/+ culture

Amyloid and -synuclein deposits

+new cases/20%

-/? (rats +)

Motor neuron loss

+

+

Hippocampal/cortical neuron loss

+

+

SNpc DA neuron loss

+

+

Dopamine D2r up; DAT down

Not determined

+

Reactive astrocytes/microglia

+

+/+ (GFAP:

mice and rats)

Oxidative stress

Not determined

protein/lipid

ALS-PDC and GWS ALS:

similarities?

• ALS-PDC:

-cycad sterol glucosides

-aluminum (Gadjusek)

• GWS ALS:

-cholesterol glucoside in H. pylori and

Mycoplasma fermentans

-aluminum: AVA and others

• Synergy of SGs and Al?

Down the neurological disease

rabbit hole

H1N1

• PRODUCT INFORMATION LEAFLET

• Arepanrix™ H1N1

• AS03-Adjuvanted H1N1 Pandemic Influenza

Vaccine

• Version 1 approved October 21, 2009

• Health Canada has authorized the sale of

Arepanrix™ H1N1 based on limited clinical

testing in humans under the provision of an

Interim Order (IO) issued on October 13, 2009.

This is it? Non-clinical data reveal no special hazard for humans based on

conventional studies of safety pharmacology, acute and repeated dose

toxicity, local tolerance, female fertility, embryo-fetal and postnatal

toxicity up to the end of the lactation period.

Two reproductive studies were conducted with AS03-adjuvanted H5N1

antigen and evaluated the effect on embryo-fetal and peri-and post-natal

development in rats, following intramuscular administration. Although

no definite conclusion could be reached, regarding a possible relation to

treatment with the H5N1 vaccine and/or the adjuvant AS03, and other

findings were considered normal, the following observations deserve to

be mentioned: In the first study, there was an increased incidence of

fetal malformations with markedly medially thickened/kinked ribs

and bent scapula as well as an increased incidence of dilated ureter

and delayed neurobehavioral maturation. In the second study, there

was an increased incidence of post-implantation loss, and the fetal

variation of dilated ureter. Not all findings were observed in both

studies, and hence the toxicological significance is uncertain.

Aluminium

adjuvant

Vaccine Trade name Manufacturer Amount (μg) per

dose

Al hydroxide

DTaP

Infanrix

GlaxoSmithKline

625 18

HepA Havrix GlaxoSmithKline 250 18

HepB EngerixB GlaxoSmithKline 250 18

Hib PedVax Hib Merck and Co 225 18

Anthrax Biothrax Bioport Corp 600 43

Al phosphate PCV Prevnar Wyeth 125 18

MenC Meningitec Wyeth 125 42

Al sulphate HepB Recombivax Merck and Co 500 18

Table 1. Aluminium adjuvant content in licensed vaccines.

DTaP=Diphtheria, Tetanus, and acellular Persussis; HepA=Hepatitis A; HepB=Hepatitis B;

Hib=Haemophilus influenza type b; PCV=Pneumococcal;

MenC=Meningococcal group C

In this protection experiment, the ferrets (six ferrets/group) were

immunized intramuscularly with vaccine candidate containing three

different doses of H5N1 antigen (7.5, 3.8 and 1.9 μg of HA antigen)

adjuvanted with the standard dose or half dose of AS03. Control groups

included ferrets immunized with adjuvant alone and non-adjuvanted

vaccine (7.5 μg HA). Ferrets immunized with the non adjuvanted

H5N1 influenza vaccine were not protected from death and showed

similar reduced lung viral loads and degree of viral shedding in the

upper respiratory tract as those exhibited by ferrets immunized with the

adjuvant alone. Conversely the combination of a range of doses of

H5N1 antigen with AS03 adjuvant was able to protect against mortality

and to reduce lung virus loads and viral shedding after intra-tracheal

challenge with a homologous wild type H5N1 virus. Serological testing

indicated a direct correlation In this protection experiment, the ferrets

(six ferrets/group) were immunized intramuscularly with vaccine

candidate containing three different doses of H5N1 antigen (7.5, 3.8

and 1.9 μg of HA antigen) adjuvanted with the standard dose or half

dose of AS03. Control groups included ferrets immunized with

adjuvant alone and non-adjuvanted vaccine (7.5 μg HA). Ferrets

immunized with the non adjuvanted H5N1 influenza vaccine were not

protected from death and showed similar reduced lung viral loads and

degree of viral shedding in the upper respiratory tract as those

exhibited by ferrets immunized with the adjuvant alone. Conversely the

combination of a range of doses of H5N1 antigen with AS03 adjuvant

was able to protect against mortality and to reduce lung virus loads and

viral shedding after intra-tracheal challenge with a homologous wild

type H5N1 virus. Serological testing indicated a direct correlation

“Pervasive uncertainty”? Just how

pervasive? Curiouser and curiouser...

Glenny, A.T., Pope, C.G., Waddington, H., and U.

Wallace The antigenic value of toxoid precipitated

by potassium alum.

J Pathol Bacteriol 29 (1926): 38-45.

Vaccine 20 (2002) S1–S4

Conference report

Workshop summary

Aluminum in vaccines

•Aluminum hydroxide induce cell death in NSC34 cells for 3 days (One way ANOVA, *p<0.05; #p<0.001)

0.0 1.0 5.0 10.0 25.0 50.0 100.00

10

20

30

Al(OH)3 Concentration (M)

Perc

en

tag

e o

f C

ell

Death

* *

#

#

##

IMR-32 cells with aluminum

chloride treatment

ChAT labelled MNs

Aluminum Toxicity: NeuN and Activated Caspase-3

E

A B

C D

F

Control Aluminum

Motor neuron from lumbar SC

from aluminum treated mouse

NeuN and Caspase-3 LabelingLumbar SC

NeuN Caspase-3 Double0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5Control

Aluminum

Squalene

Aluminum+Squalene

*#

*

**#

Marker

No

rmali

zed

nu

mb

er

of

po

sit

ive l

ab

ele

d c

ell

s p

er

sam

ple

are

a

A

NeuN and Caspase-3 Labelingin Red Nucleus

NeuN Caspase-3 Double0.0

0.5

1.0

1.5

2.0Control

Aluminum

Squalene

Aluminum+Squalene

Marker

No

rmali

zed

nu

mb

er

of

po

sit

ive l

ab

ele

d c

ell

s i

nsam

ple

are

a

C NeuN and Caspase-3 Labelingin Substantia Nigra

NeuN Caspase-3 Double0.0

0.5

1.0

1.5Control

Aluminum

Squalene

Aluminum+Squalene

Marker

No

rmali

zed

nu

mb

er

of

po

sit

ive l

ab

ele

d c

ell

s p

er

sam

ple

are

a

D

NeuN and Caspase-3 Labelingin Primary Motor Cortex

NeuN Caspase-3 Double0.0

0.5

1.0

1.5

2.0

2.5Control

Aluminum

Squalene

Aluminum+Squalene

*#

#

*

*

*

Marker

No

rmali

zed

nu

mb

er

of

po

sit

ive l

ab

ele

d c

ell

s p

er

sam

ple

are

a

B

NeuN and Caspase-3 Labelingin DG of Hippocampus

NeuN Caspase-3 Double0

1

2Control

Aluminum

Squalene

Aluminum+Squalene

**

Marker

No

rmali

zed

nu

mb

er

of

po

sit

ive l

ab

ele

d c

ell

s p

er

sam

ple

are

a

E

NeuN and activated caspase-3 cell counts

A B

GFAP

CON ALUM SQE A+S0

1

2

3

4

5Control

Aluminum

Squalene

Aluminum+Squalene

***

Group

No

rmali

zed

nu

mb

er

of

po

sit

ive l

ab

ele

d c

ell

per

sam

ple

are

a

C

A B

C D

E

GFAP Morin Aluminum: GFAP and Morin

D

Control Control Aluminum Aluminum

Morin

CON ALUM SQE A+S0

1

2

3

*

*Control

Aluminum

Squalene

Aluminum+Squalene

*P=0.025

Group

No

rmali

zed

nu

mb

er

of

po

sit

ve l

ab

ele

d c

ell

s p

er

sam

ple

d a

rea

Iba-1 labeling

Tau labelling

Motor behavioural data 2 (Shaw

and Petrik, 2009)

More...

Still more

Water maze test as an evaluation of learning and memory.

Mice injected 6× with aluminum hydroxide on average took

significantly longer to complete the maze compared to saline

injected mice (two-way ANOVA. *p = 0.0389).

Behavioural outcomes: 40mg/L dietary

AlCl•6H2O

male CD1 mice aged ~7-12 months

Baseline T2 T3 T4 T5 T6 T7

Dis

tance

(cm

)

0

6

7

8

9

10

11

12

Control (N =9-11)

Aluminum only (N=9-10)

Mean Distance to Zone Borderin Open Field Test (5min)

Baseline T2 T3 T4 T5 T6 T7 T8

Late

ncy (

se

cond

s)

0

25

50

75

100

125

150

Control (N =9-11)

Aluminum only (N=9-10)

Latency to Enter Light Area in the Light-Dark Box (5min)

Baseline T2 T3 T4 T5

Dura

tion (

seconds)

0

20

40

60

80Control (N =10-11)

Aluminum only (N=9-10)

Time in Open Areain Elevated Plus-Maze Test (5min)

Baseline T2 T3 T4 T5 T6 T7 T8

Dura

tion (

seconds)

0

20

40

60

80

100Control (N =9-11)

Aluminum only (N=9-10)

Time in Illuminated Area in the Light-Dark Box (5min)

Baseline T2 T3 T4 T5 T6 T7 T8

Fre

qu

en

cy

0

2

4

6

8

10

12 Control (N =9-11)

Aluminum only (N=9-10)

Entries into Centre of Illuminated Area in the Light-Dark Box (5min)

Baseline T2 T3 T4 T5 T6 T7

Du

ration

(seco

nd

s)

0

100

150

200

250

300

350

400

Control (N =9-11)

Aluminum only (N=9-10)

Time to Find all Reward Pelletsin Radial Arm Maze (max 10min)

Those pesky ASD data again

0

50000

100000

150000

200000

250000

300000

350000

400000

3-22 ys

6-22 ys

Aluminum adjuvants and ASD

Ye

arl

y %

in

cre

ase

in

# o

f A

SD

ca

ses

an

d v

acc

ine

Al-

bu

rde

n

0

20

40

60

80

100

120

140

160

180

200

% increase in # of ASDcases

% increase in vaccine-administered Al

R² = 0.85

0

50,000

100,000

150,000

200,000

250,000

300,000

350,000

4500 5000 5500 6000 6500

Cumulative Al burden (μg)

Conclusions/future directions • What we‟ve shown:

• Abundant evidence for aluminum in some

neurological disease states;

• Motor neuron loss with neuroinflamatory

markers;

• Behavioural deficits of motor and cognitive

function;

• What we need to do:

• Do a lot more of same with emphasis on

compound, route of administration, dose,

duration, age, gender, gene-toxin

interactions; key biomarkers, component

quantification...

• Translational steps leading to therapy.

Thanks to: Lab Collaborators

Daryl Bannon

Dr. Jason Wilson

Grace Lee

Michael Petrik

Rena Tabata

Dominica Kwok

Pierre Zwickers

Lucija Tomljenovic

• Granting Agencies

NSERC

Scottish Rite Charitable Foundation

US Army Medical Research and Materiel Command

NIH (NINDS)

ALSA/ALS Canada

Dr. Steve Blackband

Dr. Bob Brown

Dr. Ulla Craig

Dr. L. T. Kurland†

Dr. Tom Marler

More thanks:

Dwoskin Foundation

Kaitlin Fox Foundation

Thank you and questions?