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
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)
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.
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
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?