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In 2002, WHO estimated 1.3 billion infected.
65,000 deaths from its associated anemia.
Predominates in tropic and subtropic regions
Disease of developing and under-developed world, disease of the poorest of the poor
Most vulnerable:
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Children
Pregnant women
 Persons without shoes or adequate protective clothing
 Agriculturalists

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US infection rates are very low
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 very low prevalence in Southeastern US
Zoonotic transmission of hookworm species or acquired tropical dermatitis more common
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Ancylostoma caninum – Abdominal pain, eosinophilic enteritis
Ancylostoma brazilinese – Cutaneous larva migrans
Was widespread in Southeastern US in early 20th C.
The Rockefeller Sanitary Commission (1905-1919) was founded in response to eradicate hookworm.
Hookworm infection is not major a public health concern, research support limited

Majority of hookworm infections in humans caused by
1) Ancylostoma duodenale
(an-cy-CLO-sto-ma doe-AH-den-al)
2) Necator americanus
(ne-KAY-tor am-er-i-CON-us)

Soil-transmitted helminthic nematodes
 infection through skin contact with soil laden with hookworm larvae.
 helminth a.k.a. worm
 nematode a.k.a. roundworm --> nonsegmented, having cylindrical bodies that narrow at each end, with a simple gut tube.

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0.10 mm length, 0.4 mm diameter. Female > Male
9000 eggs/day, eggs have 3-5 yrs survival
Buccal capsule set with two crescent-shaped cutting plates on ventral side
Ingests 30 µl blood/day
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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

 Skin Infection
 stinging, burning, itching, pruritus, papulovesicular rash - can last up to 2 wks
 Lung Infection
 pneumonia, cough --rare and mild.
 Ingestion
 throat soreness, hoarseness, nausea, vomiting
 GI Infection
 anemia, bloody stool (from former attachment sites), abdominal pain

 Anemia, iron deficiency.
 Hypoproteinemia, edema.
 Mental, physical, growth retardation.
 Immunocompromised.
 Complicates malaria, HIV, etc.

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 IgG dominates; IgE minimal; IgA absent
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 Worm burden increase with age.
 Indicates no natural immunity is acquired with age.

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2000, FRESH Partnership
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Btw UNESCO, UNICEF, WHO, Education International, and
World Bank
Support the distribution of anti-helmintic drugs through schools.
20 projects targeting 45 million children in Africa.
2001, Partnership for Parasite Control
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Btw WHO, World Food Program and World Bank
19 programs in Africa treating school-age children
Train representatives of the ministries of health and education of
21 countries
2002, the Bill and Melinda Gates Foundation
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Will provide 35 million doses for young people, schoolchildren, women and all those at particular risk through their work.
Assist countries in making the transition to self sustained programs.

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High rates of re-infection.
 As early as 3 mos.
 To avoid drug resistance, can receive Rx no more frequently than every 6 mos.
Public infrastructure, sanitation, agricultural methods must support medical efforts.
Programs limited to young people
 To avoid drug resistance, Rx limited to high risk groups.
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Hookworm infection does not decrease with age
Older populations left untreated
Danger of drug resistance.
Safety during pregnancy not yet established.

 Anti-helminthic drugs failed to control hookworm.
 Experience with hookworm does not confer immunity.
 High rates of re-infection.
 Current deworming efforts limited to school-age children.
 Drug resistance

 Prevent infection
 Eliminate or reduce worm burden to nonpathogenic levels
 Eliminate or reduce fecundity of worms
 Provide sufficient duration of immunity
 Will provide protection against infection of a genetic variety of hookworms
 Affordable and convenient

Approach:
Find a hookworm antigen that is immunogenic but
NOT pathogenic and that can confer protective immunity to infection
Molecular cloning and purification of Ac-TMP, a developmentally regulated putative tissue inhibitor of metalloprotease released in relative abundance by adult Ancylostoma hookworms.
Zhan B. Badamchian M. Meihua B. Ashcom J. Feng J. Hawdon J. Shuhua
X. Hotez PJ.
American Journal of Tropical Medicine & Hygiene. 66(3):238-44, 2002 Mar.

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

 Interested in an Ag that is a secreted protein of adult hookworm
 Why?
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 Proteins help worm evade host defenses (ie. a suppressor of host immune agents_
 Proteins allow worm growth and survival of worm (ie. anticoagulant to keep continuous blood supply for worm)
How?
 Use antibodies against secreted proteins to select for Ag
 Sure of secreted proteins immunogenicity

Methods: Prepare Abs to Secreted Proteins
Secreted proteins
Inject rabbit
Extract worms
Grow worms in dog
Anti-serum to secreted proteins bleed
Develop Abs

Phage
Methods: Prepare cDNA Library of Adult
Hookworm
Transform
Plate
E. Coli cDNA expressed phage replicate
New phage released
Hookworm
Ag released rupture

visualize
Methods: Immunoscreen for Secreted
Proteins membrane transfer anti-IgG antibodies with marker
Incubate with primary antibody
Incubate with secondary antibody antisecretory protein anti-serum

Methods: cDNA and Peptide Sequencing cDNA Sequence
Peptide Sequence
Identify Secreted Protein

 Why?
 Rough evaluation of Ag promise in a vaccine
 Characteristics:
 What class of Ab does Ag elicit?
 When Ag is expressed?
 Which stages of development Ag important ?
 How abundant/dominant is Ag?

Eg g
Adult Larvae cDNA
Methods: RT-PCR cDNA primers
PCR
Eg g
Adult Larvae
Egg Adult Larvae Control
Run gel

Methods: Chromatography
Each peak corresponds to a separated secretory protein
Area under curve
= abundance
Chromatography
Secretory proteins separated based on relative hydrophobicity
Fractions containing unique protein

Fraction 51
Fraction 51
Secretory protein
Amino acid sequence
Compare to discovere d secreted protein

Results: Immuno-screening
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 cDNA script for immunogenic secretory protein found!
Secretory protein is
IgG reactive

Results: cDNA and Peptide Sequencing
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 cDNA codes for predicted secretory protein, named Ac-TMP
Ac-TMP shares 50% homology with Human Tissue Inhibitor of
Metalloprotease 2 (TIMP2)

Results: RT-PCR
 Ac-TMP is only present in adult hookworm

Results: Purification and Identification of
Antigen in Secretory Products
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Fraction 51 contained secretory protein that matched predicted amino acid sequence of Ac-TMP
The corresponding RNA sequence of
Fraction 51 secretory protein also matched cDNA sequence of Ac-TMP
Ac-TMP approx. 6.3% of total proteins secreted by adult hookworm

Ac-TMP
 Shares 50% homology to Human TIMP-2
 Metalloprotease important in extracellular matrix remodelling
 Matrilysin in intestinal tissue injury repair
 6.3% of secretory products, one of most abundant secreted proteins
 Strong antibody response

 No evidence that it has antimetalloprotease activity
 No evidence that the Ab to Ac-TMP is important to immunity
 Ac-TMP is IgG-reactive

 Test if Ac-TMP will work as a vaccine
 Immunize host with Ac-TMP, then challenge with hookworm, and observe if immune protection acquired
 Perform functional studies with Ac-TMP
 What is its function?
 What are its substrates?
 Investigate hookworm immune evasion abilities
 Investigate hookworm properties essential to its development and survivability

 Quick and dirty way of finding a hookworm
Ag?
 Are vaccines the appropriate solution to hookworm when hookworm is a public health/public sanitation problem?