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PRIONS –an update

Dr Nitin Gupta

Department of Microbiology

AIIMS, New Delhi

Contents

• History

• Introduction

• PrPC and PrPSc

• Replication in Prions

• Pathogenesis

• Animal Prion Diseases

• Human Prion Diseases

• Disinfection & Sterilization

• Therapeutics

• Prevention

Daniel Carleton Gajdusek

• Kuru by ritualistic consumption

• successfully transmitted to primates

• Suggested viral causation

• got Nobel prize for his work in 1976

Gajdusek, D. Carleton et al. Science155(3759): 212–214.

Stanley B. Prusiner

• purified the hypothetical infectious protein

• named it Prion (Proteinaceous Infectious particles)

won the Nobel Prize in Physiology or Medicine in 1997

Prions

• proteinaceous infectious particle

• resistant to inactivation by procedures that modify nucleic acids – UV &ionizing radiation

– Dry heat

– Formaldehyde & glutaraldehyde

– DNAse & RNAse

– Digestion with proteinase K

• prion protein designated as - PrP

Annu. Rev. Microbiol. 2013. 67:543–64

Misfolded Proteins

Annu. Rev. Microbiol. 2013. 67:543–64

PrPC

• PrPC (for Common or Cellular) or PrPsen (protease sensitive)

• normal monomeric properly folded protein

• mainly alpha-helical structure

• found on outer surface of neurons

• attached by a glycosylphosphatidyl-inositol (GPI) anchor to

CM(differentiates from other misfolded proteins)

Málaga-Trillo E et al. March 2009. PLoS Biology 7 (3): e55.

Abbott A (2010). Nature

cell-cell adhesion

and intracellular

signaling

cell-cell communication

myelination in

Schwann cells

maintenance of long-term

memory

Proposed

functions of

PrPc

PrPSc

• PrPSc (for Scrapie) or PrPres (protease resistant)

• abnormal and oligomeric form

• amino acid sequences are identical to that of PrPC

• it has a higher proportion of β-sheet structure

Annu. Rev. Microbiol. 2013. 67:543–64

Os pub health res persp 2013 4(1), 57-66

Replication in Prions

• Prion induces PrPC into PrPSc

• PrPSc acts as template to guide the misfolding

conformational alteration is not immunogenic

• There are two models for Prion replication

Aguzzi A (2008).PNAS, 105(1): 11–2

Heterodimer model of replication

Fibril model of prion replication

Human Transmission

Nature reviews, Dec 2013, vol13

Pathogenesis

Archives of Medical Research 36 (2005) 622–627

Infection and peripheral replication

Neuroinvasion Neurodegeneration

Nature reviews, Dec 2013, vol13

Nature reviews, Dec 2013, vol13

Animal Prion

Diseases

Scrapie Bovine Spongiform

Encephalopathy (

BSE)

Chronic Wasting

Disease (CWD)

Transmissible

mink

encephalopathy

Feline

spongiform

encephalopathy

Ungulate

spongiform

encephalopathy

compulsively scrape off their fleeces due to intense itching

Transmission occurs through birth fluid or faeces

Not infectious to humans

animals become ataxic and wasted

Lichens reduce no. of prions in soil

Massive outbreaks in 1980s and 90s

Due to contaminated cattle feed

transmitted to man as vCJD in 1996

Bovine Spongiform Encephalopathy

Lancet.2005.Volume 366, Issue 9488

Lancet.2005.Volume 366, Issue 9488

Chronic Wasting Disease

Human prion

diseases

Creutzfeldt-Jakob

Disease (CJD)

Variant Creutzfeldt-

Jakob Disease(vCJD)

Gerstmann-Straussler-

Scheinker Syndrome Kuru

Proteinase sensitive

neuronopathy(new

entity described in

2008)

Fatal Familial

Insomnia

Genetics in Prion disease

Gene coding normal protein in humans (”PRNP”)

located on the short arm of chromosome 20.

• Patients with the D178N mutation and

homozygous for valine at codon129 develop CJD,

homozygous for methionine at 129 have FFI

Kuru

• Literal meaning ‘Shivering’ or

‘Trembling

• Mainly affected women and young

children

• Presents with Ataxia, Unsteady gait,

Cerebellar disorders

• Associated with ritual

cannibalism(stopped in 1950s)

Fatal familial insomnia

• rare fatal disorder identified in Italian families.

• MC gene mutation detected at D178N.

• progressive insomnia

• Autonomic disturbance & endocrine disturbances

• Genetic studies-the diagnostic procedure of choice

Gerstmann-Straussler-Scheinker syndrome

• AD, Complete penetrance

• Middle age adults

• Cerebellar ataxia, nystagmus and gait abnormalities

• Demonstartion of PrP mutation( mc-P102L)-best way to

diagnose

CRUETZFELD JACOB DISEASE

Inherited mutation

Mc-missense mutn in codon200

PrPSc accumulation in brain

Familial CJD(15%)

No mutation

Exogenous

Human PrPSc •human pituitary

hormones(MC,13

0)

•dural graft

transplants(110)

•corneal

transplant(3)

•liver transplants

•contaminated

neurosurgical

instruments (6)

Iatrogenic CJD(1%)

Spontaneous mutation

May be age related

Spradic CJD(85%)

Conversion of

PrPc into PrPSc

• Incidence of CJD in world- 1 case/million population

• 1968-1997: 69 cases of CJD from different parts of

India(NIMHANS)

• 1990-1998:10 cases of CJD, GB pant, New Delhi

• 2010-2013:10 cases of CJD from Bangur Institute of

Neurosciences,Kolkata

Variant Creutzfeldt-Jakob disease

• First reported in 1996

• acquired by ingestion of

infected meat products(BSE)

• Transmitted by blood

• No vertical transmission

Os pub health res persp 2013 4(1), 57-66

Classical CJD vs Variant CJD

Characteristic Classic CJD Variant CJD

Median age at death 68 years 28 years

Median duration of illness 4-5 months 13-14 months

Periodic sharp waves on

electroencephalogram

Often present Often absent

"Pulvinar sign" on MRI Not reported Present in >75% of

cases

Presence of "florid plaques" on

neuropathology

Rare or absent Present in large

numbers

Presence of agent in lymphoid

tissue

Not readily

detected

Readily detected

(tonsilar biopsy tissue)

Diagnosis

of CJD

CSF

MRI

EEG Brain

biopsy

Genetic studies

• no cells

• normal glucose

• elevated protein

• CSF 14-3-3 protein

(sCJD)

•most useful

•Increased T2 signal- striatum

(sCJD)

•Increased T2 signal in post

thalamus-Pulvinar sign(vCJD)

•periodic sharp wave

complexes (PSWC)

•high specificity but a low

sensitivity

gold standard

HPE

a) Spongiform changes

b) Neuronal loss

c) Astrocytosis

d) Amyloid plaque

Brain Biopsy

Immunoassays

SANDWICH

ELISA

FORMAT

Clin Dev Immuno,Vol 2013, Article ID 360604,

Os pub health res persp 2013 4(1), 57-66

Distribution of infectivity

High infectivity

(BSL-3)

Brain

• Spinal cord

• Eye

Low infectivity

(BSL-2)

• CSF

• Kidney

• Liver

• Lung

• Lymph nodes/spleen

• Placenta

No infectivity

• Blood

• Tears

• Nasal mucous

• Saliva

• Sweat

• Serous exudate

• Milk

• Semen

• Urine

• Faeces

Sterilization & Disinfection

• infectivity strongly stabilized by drying or fixation with

alcohol, formalin or glutaraldehyde

• Surgical instruments contact high infectivity tissues, single

use recommended

• instruments contaminated by CSF-also single use

• Incineration for all disposable instruments, materials, and

wastes

Sterilization & Disinfection contd..

With Pre treament

Instruments immersed in NaOH or Sodium Hypochlorite

Subjected to Autoclave at 121°C for 30 min to 1hr

Rinsed in water and subjected to routine sterilisation

Without pre treatment

Autoclave at 134°C for 18 minutes

WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies

THERAPEUTICS

• Universally fatal and incurable

• Pentosan polysulfate by intraventricular infusion slows

disease progression

• Amantadine transiently improves symptoms

• Flupirtine slow cognitive decline

• Astemizole - anti-prion activity

Potential immunotherapy targets

Nature reviews, Dec 2013, vol13

• Innate immunity repeated TLR9 stimulation protective

• Active immunization difficult because tolerance to PrP

Synthetic PrP peptides can be used

• Passive immunisation 1) Abs generated by

immunizing Prnp neg mice with PrP peptides

recombinant PrP

native PrP purified from tissues.

2) PrP-specific monoclonal Ab inhibited prion accumulation in the spleen

Nature reviews, Dec 2013, vol13

THANK

YOU