Infection and Immunity in Autism Spectrum DisordersNew Tools for Pathogen Discovery

18-19 April 2007Institute of Medicine

Washington, DC

W. Ian LipkinColumbia University and the Northeast Biodefense Center

NIAID, NINDS, NHLBI, NEI, DOD, CAN

PANDORA’S BOX PROJECT

MECHANISMS OF MICROBIAL PATHOGENESISPart I

Direct effects at infection sitereplication causes cell damagePoliovirusMotor neurons are killed, causing paralysis

toxin alters local physiologyVibrio choleracholera toxin alters ion transport in intestine, causing diarrhea

Effects distal to infection sitemicrobe produces a toxin with distant effectClostridium botulinumBotulinum toxin interferes with neurotransmitter function at nerve-muscle junction

Host responses influence outcomes

host response to microbe causes or augments damage at infection siteHepatitis BImmune response kills infected liver cells

microbe causes immunosuppression, increasing susceptibility to other agentsMeasles virus, HIVExacerbation of tuberculosis; opportunistic infections

Effects depend on maturational status of host organ systems at time of infection

microbe may be teratogenic depending on gestational periodRubella virusFirst trimester congenital infection results in a spectrum of birth defects

MECHANISMS OF MICROBIAL PATHOGENESISPart II

Effects depend on complex pathogen:host interactionsInterference with differentiated cell functionLymphocytic choriomeningitis viruspersistent viral infection alters neurotransmitter or hormone production without killing cells (dementia, diabetes, hypothyroidism)

Molecular mimicryStreptococcusantibodies to bacteria also bind to host, causing cardiac or CNS damage (autoimmunity)

Longterm effects of infection?Multiple sclerosislatitude where one lives before puberty determines risk for a disease that begins in early adulthood

Psychiatric disordersGestational exposure increases risk for schizophrenia, a disorder that first manifests in adolescence or early adulthood

MECHANISMS OF MICROBIAL PATHOGENESISPart III

Emerging Microbial Concepts:Infectious Causes of Chronic Disease

DiseaseCervical CAChronic Hepatitis/HepatocarcinomaLyme ArthritisPeptic Ulcer DiseaseWhipple DiseaseBladder CAPremature BirthCerebral Palsy

CauseHuman papilloma virusHepatitis B and C virusesBorrelia burgdorferiHelicobacter pyloriTropheryma whippeliiSchistosoma haematobiumVariousVarious

Compelling evidence

Emerging Microbial Concepts:Chronic Diseases Potentially Linked to Infection

DiseaseCardio/Cerebrovascular DiseaseDiabetes MellitusObsessive Compulsive Disorder(PANDAS)SchizophreniaInflammatory Bowel DiseaseIdiopathic Pulmonary FibrosisAutismAcute Macular Degeneration

CauseDental/sinus infectionsEnteroviruses, other virusesGroup A streptococcus

Influenza virus????

INTERPLAY OF GENETIC CONTEXT AND TIMING OF ENVIRONMENTAL INSULTS ON PATHOGENESIS

Fetus(a la da Vinci)

Stage 1Week 1

FERTILIZEDOOCYTE

Stage 10Week 4

NEURALFOLD

FUSES

ENVIRONMENTALFACTORS

GENETICFACTORS

DevelopmentalDisorders(Autism)

child

earlyadult

lateadult

Psychiatric & DemyelinatingDisorders (MS)

Degenerativedisorders

Toxins: thalidomide, valproic acid, HgMaternal infection: rubella, influenza (polyIC) Hornig, 2000

Signs/Symptoms of Infection Are NotUnique to Specific Agents

Growth of the Viral Database by Viral Family

A Staged Strategy for Pathogen Discovery

UncharacterizedSample

MassTag PCR• Respiratory disease • Hemorrhagic fevers • Meningoencephalitides• Poxviruses $15/assay, 96 samples in 6 Hours

QuantitativeReal-Time PCR

Consensus PCRCloning & Sequencing

GreeneChips• Viral• Pan-pathogen • Respiratory$75/assay, 8 samples in 15 hrs

Shotgun Sequencing$5,000/assay – 1 week

A STAGED STRATEGY FOR PATHOGEN DETECTION

MassTag PCR

Clinical specimenClinical specimen

Short candidate list<30 agents

New technology for sensitive, highly multiplexed, rapid differential diagnosis of:

Respiratory Infections Meningitides/EncephalitidesHemorrhagic Fevers Gastroenteritides/Diarrheas

Rhinovirus NY (Human rhinovirus C vs Human Enterovirus E)Discovery of a novel virus associated with influenza like illness

HRV NY041

HRV A039

H. influenzae

S. pneumoniae

HPIV-1037

HRV034

M. pneumoniae030

HRV NY028

HRV A026

M. pneumoniae025

HMPV017

S. pneumoniae

HMPV016

HCoV-OC43009

S. pneumoniae004

HRV NY003

AgentSample

HRV B077

HRV NY074

M. pneumoniae072

HRSV-B071

HCoV-OC43070

S. pneumoniae

HRV NY063

M. pneumoniae061

S. pneumoniae

HRV NY060

H. influenzae056

HEV052

S. pneumoniae050

H. influenzae

S. pneumoniae

HRV B045

HRV NY042

S. pneumoniae

FLUBV

FLUAV1140

N. meningiditis

FLUAV1135

HRV

FLUAV1126

S. pneumoniae

FLUBV1119

HRV

FLUAV1101

HSV

HRV NY1085

HRV B

FLUBV1083

S. pneumoniae

HEV

FLUBV1081

Pathogens in Flu-Negative SpecimensAdditional Pathogens inFlu-Positive Specimens

Lamson (2006) J Infectious Dis 194: 1398-1402

Global Distribution of a “New” VirusHuman Enterovirus E vs Human Rhinovirus C

HRV group A

HRV group BEV

New Clade

A STAGED STRATEGY FOR PATHOGEN DETECTION

Clinical specimenClinical specimen

Long candidate list

GreeneChipsHighly multiplexed, differential diagnosis of infectious disease

Virus surveillance Respiratory pathogens; influenza serotyping GreeneChipVr1.5 GreeneChipResp

Virus discoveryGreeneChipVr2.0

Greene Pathogen DatabaseComprehensive, curated database of pathogen sequences

WHO Outbreak Surveillance Differential diagnosis of hemorrhagic fever

Clinical Specimen DNA

Random primed PCR Secondary PCR to incorporateshort DNA tag

Hybridize unlabeled templatein multi-well slides

Secondary hybridization to incorporate 3DNA flourophore

GreeneChip Protocols

Agilent arrays• In situ synthesis• High density/multiplex format• Rapid modification

NCBI Sequence data (Nat. Center for Biotechnology Information)

ICTVdB (Int.Comittee on Taxonomy of Viruses)

Pfam database(Protein FamiIies)

Viral sequence database382,512

Greene Pathogen Databases

RDP (Ribosomal Database Project)

+NCBI

Pan-microbial sequence database382,512 viruses

41,790 bacteria 4,109 fungus2,626 parasites

ExperimentDatabase

PI, affiliation, clinical,geography, results, comments

H5N1

Web-based Analysis of GreeneChip DataAccounts: Australian Biosecurity CRC (Boyle); CDC (Nichol & Towner); Stanford

(Hirschberg & Davis); Institut Pasteur Shanghai (Deubel); Univ of Colorado(Holmes & Dominguez); Wadsworth Center,NYSDOH (St. George & Dean)

Enteroviruses in Feces of Healthy Norwegian Children Aged 3-28 Months (145 of 1,255 samples)

2.43HEVUntyped

1.62CAV19HEV-C

1.62HEV-BaHEV-B

0.81EV30HEV-B

0.81EV25HEV-B

8.911EV18HEV-B

1.62EV13HEV-B

0.81EV11HEV-B

2.43EV9HEV-B

0.81EV5HEV-B

1.62EV3HEV-B

3.24CBV5HEV-B

2.43CBV4HEV-B

7.39CBV3HEV-B

4.05CBV1HEV-B

3.24CAV9HEV-B

3.24HEV-AaHEV-A

14.518HEV71HEV-A

0.81CAV16HEV-A

4.86CAV14HEV-A

5.67CAV10HEV-A

10.513CAV6HEV-A

5.67CAV5HEV-A

8.911CAV4HEV-A

2.43CAV2HEV-A

Percent (n=124)EpisodesSerotypeSpecies

5’ UTR mutationsmay inhibit EVfitness

A STAGED STRATEGY FOR PATHOGEN DETECTION

Hi Throughput Sequencing

Disclosure454 Life Sciences Scientific Advisory Board 2003-present

Novel high throughput pyrosequencing slide-based platformAlgorithm for Reductive Analysis of Sequence DataProgram for automated identification of pathogen sequences

Outbreak of Transplant-Associated Encephalitis Identification of a novel arenavirus

Colony Collapse Disorder Profile of an emerging threat to agriculture and economic welfare

Primer

Repeat

Trimming FilteringClustering Assembly

Raw Reads

host

ContigsTrimmed Reads Unique Reads Non-host ReadsReductive Analysis of Highthroughput Sequencing Data

Contigs and SingletsContigs and Singlets

Candidates withNucleotide Homology

Candidates withNucleotide Homology

BLASTN

Candidates withProtein HomologyCandidates with

Protein Homology

BLASTX

viruses

bacteria

fungi

metazoans

parasites

Sequence ð TaxonomySequence ð Taxonomy

Candidate organism

High Throughput Sequencing in Pathogen Surveillance and Discovery

Encephalitis MassTag PCR

140,000 sequences obtained

14 (0.01%) Old World Arenavirus sequences

S-segmentGreeneChipVr1.5 array

High throughput sequencing of pooledrandom PCR products

2007: 3 cases of transplant associated disease Organ Donor: 57 y/o M (thalamic bleed)

Recipients: 63 y/o F (kidney), 44 y/o F (kidney), 64 y/o F (liver)

• S-segment, 12 sequences• L-segment, 2 sequences

Identical sequence in donor and recipients

50,000 RNA copies in liver and kidney<100 RNA copies in brain and serum

To Bee or Not to Bee…Colony Collapse Disorder

Global pollination of > 90 fruit and vegetable crops$14.6B/year in US alone

Family apoidea

AlfalfaAlmondAppleApricot (some varieties)ArtichokeAvocadoBlackberryBlack locustBlueberryBoysenberryBroadbeansBroccoliBrussels sproutsBuckwheatCabbageCarawayCarrotsCatalpaCauliflowerCeleryCherryChestnutChicoryChinese cabbage

Clover (some varieties)CoconutCoffeeCollardsCorianderCottonCrabappleCranberriesCrownvetchCucumbersCurrantsDewberryDillEggplantEndiveFennelFigsFlaxGarlicGooseberriesGrapes (muscadine)GrapefruitGuarGuava

Holly KaleKenafKiwiKohlrabiLeekLespedeza (bush)Lima beansLoquatsMacadamia nutMaple (red)Mandarin orangeMangoMuskmelonsMustardNectarinesOkra OnionsOrange PapayaParsleyParsnipsPeachesPear

Pepper Persimmon (native)Plums and PrunePumpkinsRadishRapeRaspberryRutabagaSafflower SquashStrawberrySunflowerSweetcloverTangeloTangerineTendergreensTrefoilTurnipsVetchWatermelonsYucca

viruses

bacteria

fungi

metazoans

parasites

Varroa destructor mite

Kynetoplastide parasite species

Nosema sp.

Neisseria species

Gamma proteobacteria

Viruses

ssRNA (+)

Dicistroviridae

Cripavirus

Black Queen Virus

Israel acute paralysis virus

Kashmir bee virus

Iflavirus

Sacbrood virus

Deformed wing virus

Chronic bee paralysis virus

Large unselected cohort (100,000)

Prospective collections of clinical data and biologic specimens begins early in gestationBiological and clinical phenotypes Longitudinal trajectory of diseaseFunctional genomics, proteomics, toxicology, as well as genetics

Resources establishedClinical databases (questionnaires, videography, clinical exams, outcomes)Biobanks of unique clinical materials

Maternal blood during pregnancy and at termPaternal bloodCord blood

Autism Birth CohortGENE X ENVIRONMENT X TIMING INTERACTIONS IN DISEASE

UO1-NS047537

www.abc.columbia.edu

Summary

Microbial pathogenesis is complex• Susceptibility is a function of genes, age, other factors• Mechanisms can be direct, indirect• Expression of disease may be delayed

The microbiome is largely uncharted.

The advent of new tools for microbial surveillance canchange the landscape of chronic as well as acute diseases.

Realization of the promise of these tools will require aninvestment not dissimilar to that allocated for genetics.

In the period that Einstein was active as a professor one of his students came to him and said: “The questions of this year’s exams are the same as last year!”

“True, “ Einstein said, “but this year all the answersare different.”

GENE-ENVIRONMENT-TIMING INTERACTIONS