Transcript 041118-SusanWong

Hookworm Infection
Prevalence
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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
Worldwide Infection
Prevalence
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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
 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
Offending Pathogens
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)
Global Distribution
A. duodenale and N.
americanus
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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Infection
Hookworm Egg and Larvae
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N. americanus and A.
duodenale
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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
Gut Infection
Symptoms Associated with
Infection
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Skin Infection
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Lung Infection
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pneumonia, cough --rare and mild.
Ingestion
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stinging, burning, itching, pruritus, papulovesicular
rash - can last up to 2 wks
throat soreness, hoarseness, nausea, vomiting
GI Infection
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anemia, bloody stool (from former attachment sites),
abdominal pain
Associated Morbidities
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Anemia, iron deficiency.
Hypoproteinemia, edema.
Mental, physical, growth retardation.
Immunocompromised.
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Complicates malaria, HIV, etc.
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Host Immune Response
Eosinophilia
 Inflammation
 IgG, IgM, IgE responses
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 IgG
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dominates; IgE minimal; IgA absent
NONE of the above immune
responses provide immune protection
or reduction of worm burden
Infection with Age
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Worm burden
increase with age.
Indicates no natural
immunity is
acquired with age.
Global De-worming Efforts
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2000, FRESH Partnership
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2001, Partnership for Parasite Control
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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.
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.
Global Deworming: Obstacles
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High rates of re-infection.
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Public infrastructure, sanitation, agricultural
methods must support medical efforts.
Programs limited to young people
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As early as 3 mos.
To avoid drug resistance, can receive Rx no more
frequently than every 6 mos.
To avoid drug resistance, Rx limited to high risk groups.
Hookworm infection does not decrease with age
Older populations left untreated
Danger of drug resistance.
Safety during pregnancy not yet established.
Rationale for Vaccine
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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
Vaccine Goals
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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
Vaccine Development
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.
Research Overview
Used immuno-screening techniques to
discover and characterize an adult A.
caninum hookworm antigen that could
possibly be used in a hookworm vaccine
Hookworm Ag for a Vaccine
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Interested in an Ag that is a secreted protein
of adult hookworm
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Why?
 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
bleed
Anti-serum to
secreted proteins
Develop Abs
Methods: Prepare cDNA Library of Adult
Hookworm
Phage
Transform
Plate
E. Coli
cDNA
expressed
New
phage
released
Hookworm
Ag released
rupture
phage
replicate
Methods: Immunoscreen for Secreted
Proteins
membrane
transfer
anti-IgG
antibodies
with
marker
visualize
Incubate
with
secondary
antibody
Incubate
with
primary
antibody
antisecretory
protein
anti-serum
Methods: cDNA and Peptide Sequencing
cDNA Sequence
Peptide Sequence
Identify Secreted Protein
Preliminary Characterization
Studies
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Why?
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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?
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Methods: RT-PCR
Eg
g
Adult
Larvae
cDNA
cDNA
primers
PCR
Eg
g
Adult
Egg Adult Larvae Control
Larvae
Run
gel
Methods: Chromatography
Each peak
corresponds to a
separated secretory
protein
Chromatography
Secretory
proteins
separated based
on relative
hydrophobicity
Fractions
containing
unique protein
Area under curve
= abundance
Methods: Chromatography
Fraction 51
Fraction 51
Amino acid
sequence
Secretory protein
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
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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
Discussion
Ac-TMP
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Shares 50% homology to Human TIMP-2
Metalloprotease important in extracellular
matrix remodelling
 Matrilysin in intestinal tissue injury repair
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6.3% of secretory products, one of most
abundant secreted proteins
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Strong antibody response
Problems
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No evidence that it has antimetalloprotease activity
No evidence that the Ab to Ac-TMP is
important to immunity
Ac-TMP is IgG-reactive
Future Plans
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Test if Ac-TMP will work as a vaccine
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Perform functional studies with Ac-TMP
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Immunize host with Ac-TMP, then challenge with
hookworm, and observe if immune protection
acquired
What is its function?
What are its substrates?
Investigate hookworm immune evasion abilities
Investigate hookworm properties essential to its
development and survivability
Questions
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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?