Hookworm InfectionHookworm Infection

PrevalencePrevalence In 2002, WHO estimated 1.3 billion infected.In 2002, WHO estimated 1.3 billion infected. 65,000 deaths from its associated anemia.65,000 deaths from its associated anemia. Predominates in tropic and subtropic regionsPredominates in tropic and subtropic regions Disease of developing and under-developed Disease of developing and under-developed

world, disease of the poorest of the poorworld, disease of the poorest of the poor Most vulnerable: Most vulnerable:

ChildrenChildren Pregnant womenPregnant women Persons without shoes or adequate protective clothingPersons without shoes or adequate protective clothing AgriculturalistsAgriculturalists

Worldwide InfectionWorldwide Infection

PrevalencePrevalence US infection rates are very lowUS infection rates are very low

very low prevalence in Southeastern USvery low prevalence in Southeastern US Zoonotic transmission of hookworm species or acquired

tropical dermatitis more common Ancylostoma caninum – Abdominal pain, eosinophilic Ancylostoma caninum – Abdominal pain, eosinophilic

enteritisenteritis Ancylostoma brazilinese – Cutaneous larva migransAncylostoma brazilinese – Cutaneous larva migrans

Was widespread in Southeastern US in early 20th C. Was widespread in Southeastern US in early 20th C. The Rockefeller Sanitary Commission (1905-1919) was The Rockefeller Sanitary Commission (1905-1919) was

founded in response to eradicate hookworm.founded in response to eradicate hookworm.

Hookworm infection is not major a public Hookworm infection is not major a public health concern, research support limitedhealth concern, research support limited

Offending PathogensOffending Pathogens

Majority of hookworm infections in Majority of hookworm infections in humans caused byhumans caused by

1)1) Ancylostoma duodenale Ancylostoma duodenale (an-cy-CLO-sto-ma doe-AH-den-al)(an-cy-CLO-sto-ma doe-AH-den-al)

2)2) Necator americanus Necator americanus (ne-KAY-tor am-er-i-CON-us)(ne-KAY-tor am-er-i-CON-us)

Global DistributionGlobal Distribution

A. duodenaleA. duodenale and and N. N. americanusamericanus

Soil-transmitted helminthic nematodesSoil-transmitted helminthic nematodes infection through skin contact with soil infection through skin contact with soil

laden with hookworm larvae.laden with hookworm larvae. helminth a.k.a. wormhelminth a.k.a. worm nematode a.k.a. roundworm --> non-nematode a.k.a. roundworm --> non-

segmented, having cylindrical bodies segmented, having cylindrical bodies that narrow at each end, with a simple that narrow at each end, with a simple gut tube. gut tube.

InfectionInfection

Hookworm Egg and Hookworm Egg and LarvaeLarvae

                      \

N. americanus N. americanus andand A. A. duodenaleduodenale

0.10 mm length, 0.4 mm 0.10 mm length, 0.4 mm diameter. Female > Malediameter. Female > Male

9000 eggs/day, eggs have 3-5 9000 eggs/day, eggs have 3-5 yrs survivalyrs survival

Buccal capsule set with two Buccal capsule set with two crescent-shaped cutting crescent-shaped cutting plates on ventral side plates on ventral side

Ingests 30 µl blood/dayIngests 30 µl blood/day

12 mm in length, 0.6 mm in diameter. Female > Male

20,000 eggs/day, eggs have 1 yr survival

Buccal capsule set with symmetric pair of sharp teeth on ventral side.

Ingests 260 µl blood/day

Gut InfectionGut Infection

Symptoms Associated Symptoms Associated with Infectionwith Infection

Skin InfectionSkin Infection stinging, burning, itching, pruritus, stinging, burning, itching, pruritus,

papulovesicular rash - can last up to 2 wkspapulovesicular rash - can last up to 2 wks Lung InfectionLung Infection

pneumonia, cough --rare and mild.pneumonia, cough --rare and mild. IngestionIngestion

throat soreness, hoarseness, nausea, vomitingthroat soreness, hoarseness, nausea, vomiting GI InfectionGI Infection

anemia, bloody stool (from former attachment anemia, bloody stool (from former attachment sites), abdominal painsites), abdominal pain

Associated MorbiditiesAssociated Morbidities

Anemia, iron deficiency.Anemia, iron deficiency. Hypoproteinemia, edema.Hypoproteinemia, edema. Mental, physical, growth Mental, physical, growth

retardation.retardation. Immunocompromised.Immunocompromised.

Complicates malaria, HIV, etc.Complicates malaria, HIV, etc.

Host Immune ResponseHost Immune Response

EosinophiliaEosinophilia InflammationInflammation IgG, IgM, IgE responsesIgG, IgM, IgE responses

IgG dominates; IgE minimal; IgA absentIgG dominates; IgE minimal; IgA absent NONE of the above immune NONE of the above immune

responses provide immune responses provide immune protection or reduction of worm protection or reduction of worm burdenburden

Infection with AgeInfection with Age

Worm burden Worm burden increase with increase with age.age.

Indicates no Indicates no natural immunity natural immunity is acquired with is acquired with age.age.

Global De-worming Global De-worming EffortsEfforts

2000, FRESH Partnership2000, FRESH Partnership Btw UNESCO, UNICEF, WHO, Education International, and Btw UNESCO, UNICEF, WHO, Education International, and

World BankWorld Bank Support the distribution of anti-helmintic drugs through Support the distribution of anti-helmintic drugs through

schools. schools. 20 projects targeting 45 million children in Africa.20 projects targeting 45 million children in Africa.

2001, Partnership for Parasite Control2001, Partnership for Parasite Control Btw WHO, World Food Program and World BankBtw WHO, World Food Program and World Bank 19 programs in Africa treating school-age children19 programs in Africa treating school-age children Train representatives of the ministries of health and Train representatives of the ministries of health and

education of 21 countrieseducation of 21 countries 2002, the Bill and Melinda Gates Foundation2002, the Bill and Melinda Gates Foundation

Will provide 35 million doses for young people, Will provide 35 million doses for young people, schoolchildren, women and all those at particular risk schoolchildren, women and all those at particular risk through their work. through their work.

Assist countries in making the transition to self sustained Assist countries in making the transition to self sustained programs. programs.

Global Deworming: Global Deworming: Obstacles Obstacles

High rates of re-infection.High rates of re-infection. As early as 3 mos.As early as 3 mos. To avoid drug resistance, can receive Rx no more To avoid drug resistance, can receive Rx no more

frequently than every 6 mos.frequently than every 6 mos. Public infrastructure, sanitation, agricultural Public infrastructure, sanitation, agricultural

methods must support medical efforts.methods must support medical efforts. Programs limited to young peoplePrograms limited to young people

To avoid drug resistance, Rx limited to high risk groups.To avoid drug resistance, Rx limited to high risk groups. Hookworm infection does not decrease with ageHookworm infection does not decrease with age Older populations left untreatedOlder populations left untreated

Danger of drug resistance.Danger of drug resistance. Safety during pregnancy not yet established.Safety during pregnancy not yet established.

Rationale for VaccineRationale for Vaccine

Anti-helminthic drugs failed to Anti-helminthic drugs failed to control hookworm.control hookworm.

Experience with hookworm does not Experience with hookworm does not confer immunity.confer immunity.

High rates of re-infection.High rates of re-infection. Current deworming efforts limited to Current deworming efforts limited to

school-age children.school-age children. Drug resistanceDrug resistance

Vaccine GoalsVaccine Goals

Prevent infectionPrevent infection Eliminate or reduce worm burden to Eliminate or reduce worm burden to

non-pathogenic levelsnon-pathogenic levels Eliminate or reduce fecundity of wormsEliminate or reduce fecundity of worms Provide sufficient duration of immunityProvide sufficient duration of immunity Will provide protection against Will provide protection against

infection of a genetic variety of infection of a genetic variety of hookwormshookworms

Affordable and convenientAffordable and convenient

Vaccine DevelopmentVaccine Development

Approach: Approach:

Find a hookworm antigen that is immunogenic but Find a hookworm antigen that is immunogenic but NOT pathogenic and that can confer protective NOT pathogenic and that can confer protective immunity to infectionimmunity to infection

    Molecular cloning and purification of Ac-TMP, a Molecular cloning and purification of Ac-TMP, a developmentally regulated putative tissue developmentally regulated putative tissue

inhibitor of metalloprotease released in relative inhibitor of metalloprotease released in relative abundance by adult Ancylostoma hookworms.abundance by adult Ancylostoma hookworms.

Zhan B.  Badamchian M.  Meihua B.  Ashcom J.  Feng J.  Hawdon J.  Shuhua Zhan B.  Badamchian M.  Meihua B.  Ashcom J.  Feng J.  Hawdon J.  Shuhua X.  Hotez PJ.X.  Hotez PJ.

  American Journal of Tropical Medicine & Hygiene.  66(3):238-44, 2002   American Journal of Tropical Medicine & Hygiene.  66(3):238-44, 2002 Mar.Mar.

Research OverviewResearch Overview

Used immuno-screening techniques to Used immuno-screening techniques to discover and characterize an adult discover and characterize an adult A. caninumA. caninum hookworm antigen that hookworm antigen that could possibly be used in a could possibly be used in a hookworm vaccinehookworm vaccine

Hookworm Ag for a Hookworm Ag for a VaccineVaccine

Interested in an Ag that is a secreted Interested in an Ag that is a secreted protein of adult hookwormprotein of adult hookworm Why?Why?

Proteins help worm evade host defenses Proteins help worm evade host defenses (ie. a suppressor of host immune agents_(ie. a suppressor of host immune agents_

Proteins allow worm growth and survival Proteins allow worm growth and survival of worm (ie. anticoagulant to keep of worm (ie. anticoagulant to keep continuous blood supply for worm)continuous blood supply for worm)

How?How? Use antibodies against secreted proteins Use antibodies against secreted proteins

to select for Agto select for Ag Sure of secreted proteins Sure of secreted proteins

immunogenicityimmunogenicity

Methods: Prepare Abs to Secreted Methods: Prepare Abs to Secreted ProteinsProteins

Grow worms in dogGrow worms in dog

Extract Extract wormsworms

Secreted Secreted proteinprotein

ssInject Inject rabbitrabbit

Anti-serum to Anti-serum to secreted proteins secreted proteins

bleedbleed

Develop Abs Develop Abs

Methods: Prepare cDNA Library of Methods: Prepare cDNA Library of Adult HookwormAdult Hookworm

TransformTransform PlatePlate

phage phage replicatreplicat

ee

PhagPhagee

E. E. ColiColi

rupturerupture

cDNA cDNA expressedexpressed

New New phage phage

releasedreleased

Hookworm Hookworm Ag Ag

releasedreleased

Methods: Immunoscreen for Methods: Immunoscreen for Secreted ProteinsSecreted Proteins

transfertransfer

membramembranene

Incubate Incubate with with

primary primary antibodyantibody

Incubate Incubate with with

secondarsecondary y

antibodyantibody

visualizevisualizeanti-anti-

secretory secretory protein protein

anti-anti-serumserum

anti-IgG anti-IgG antibodiantibodies with es with markermarker

Methods: cDNA and Peptide Methods: cDNA and Peptide SequencingSequencing

cDNA SequencecDNA Sequence

Peptide SequencePeptide Sequence

Identify Secreted ProteinIdentify Secreted Protein

Preliminary Preliminary Characterization StudiesCharacterization Studies

Why?Why? Rough evaluation of Ag promise in a Rough evaluation of Ag promise in a

vaccinevaccine Characteristics:Characteristics:

What class of Ab does Ag elicit? What class of Ab does Ag elicit? When Ag is expressed? When Ag is expressed? Which stages of development Ag Which stages of development Ag

important ? important ? How abundant/dominant is Ag?How abundant/dominant is Ag?

Methods: RT-PCRMethods: RT-PCREgEggg

AdultAdult

LarvaLarvaee

cDNAcDNA

Run Run gelgel

EgEggg

AdultAdult

LarvaLarvaee

ControControll

primeprimersrs

PCRPCR

EgEggg

AdultAdult LarvaLarvaee

cDNAcDNA

Methods: ChromatographyMethods: Chromatography

ChromatograpChromatographyhy

Secretory Secretory proteins proteins

separated separated based on based on relative relative

hydrophobicithydrophobicityy

Area under curve = abundance

Each peak corresponds to a

separated secretory protein

Fractions Fractions containing containing

unique unique proteinprotein

Methods: ChromatographyMethods: Chromatography

Amino acid sequenceAmino acid sequence

Fraction 51Fraction 51

Fraction 51

Compare to discovered secreted proteinCompare to discovered secreted protein

Secretory proteinSecretory protein

Results: Immuno-screeningResults: Immuno-screening

cDNA script for cDNA script for immunogenic immunogenic secretory protein secretory protein found!found!

Secretory protein Secretory protein is IgG reactiveis IgG reactive

Results: cDNA and Peptide Results: cDNA and Peptide SequencingSequencing

cDNA codes for predicted secretory protein, named Ac-TMPcDNA codes for predicted secretory protein, named Ac-TMP Ac-TMP shares 50% homology with Human Tissue Inhibitor of Ac-TMP shares 50% homology with Human Tissue Inhibitor of

Metalloprotease 2 (TIMP2)Metalloprotease 2 (TIMP2)

Results: RT-PCRResults: RT-PCR

Ac-TMP is only present in Ac-TMP is only present in adult hookwormadult hookworm

Results: Purification and Results: Purification and Identification of Antigen in Secretory Identification of Antigen in Secretory

ProductsProducts

Fraction 51 contained secretory Fraction 51 contained secretory protein that matched predicted protein that matched predicted amino acid sequence of Ac-TMPamino acid sequence of Ac-TMP

The corresponding RNA sequence of The corresponding RNA sequence of Fraction 51 secretory protein also Fraction 51 secretory protein also matched cDNA sequence of Ac-TMPmatched cDNA sequence of Ac-TMP

Ac-TMP approx. 6.3% of total Ac-TMP approx. 6.3% of total proteins secreted by adult hookwormproteins secreted by adult hookworm

DiscussionDiscussion

Ac-TMPAc-TMP Shares 50% homology to Human Shares 50% homology to Human

TIMP-2TIMP-2 Metalloprotease important in Metalloprotease important in

extracellular matrix remodellingextracellular matrix remodelling Matrilysin in intestinal tissue injury Matrilysin in intestinal tissue injury

repairrepair 6.3% of secretory products, one of 6.3% of secretory products, one of

most abundant secreted proteinsmost abundant secreted proteins Strong antibody responseStrong antibody response

ProblemsProblems

No evidence that it has anti-No evidence that it has anti-metalloprotease activitymetalloprotease activity

No evidence that the Ab to Ac-TMP No evidence that the Ab to Ac-TMP is important to immunityis important to immunity

Ac-TMP is IgG-reactiveAc-TMP is IgG-reactive

Future PlansFuture Plans

Test if Ac-TMP will work as a vaccineTest if Ac-TMP will work as a vaccine Immunize host with Ac-TMP, then challenge with Immunize host with Ac-TMP, then challenge with

hookworm, and observe if immune protection hookworm, and observe if immune protection acquiredacquired

Perform functional studies with Ac-TMPPerform functional studies with Ac-TMP What is its function?What is its function? What are its substrates?What are its substrates?

Investigate hookworm immune evasion Investigate hookworm immune evasion abilitiesabilities

Investigate hookworm properties essential to Investigate hookworm properties essential to its development and survivabilityits development and survivability

QuestionsQuestions

Quick and dirty way of finding a Quick and dirty way of finding a hookworm Ag?hookworm Ag?

Are vaccines the appropriate Are vaccines the appropriate solution to hookworm when solution to hookworm when hookworm is a public health/public hookworm is a public health/public sanitation problem?sanitation problem?