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Hookworm Infection. Prevalence. 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: Children Pregnant women

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prevalence
Prevalence
  • 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:
    • Children
    • Pregnant women
    • Persons without shoes or adequate protective clothing
    • Agriculturalists
prevalence1
Prevalence
  • US infection rates are very low
    • 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
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)

a duodenale and n americanus
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 --> non-segmented, having cylindrical bodies that narrow at each end, with a simple gut tube.
n americanus and a duodenale
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

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

N. americanus and A. duodenale
symptoms associated with infection
Symptoms Associated with Infection
  • 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
associated morbidities
Associated Morbidities
  • Anemia, iron deficiency.
  • Hypoproteinemia, edema.
  • Mental, physical, growth retardation.
  • Immunocompromised.
  • Complicates malaria, HIV, etc.
host immune response
Host Immune Response
  • Eosinophilia
  • Inflammation
  • IgG, IgM, IgE responses
    • IgG dominates; IgE minimal; IgA absent
  • NONE of the above immune responses provide immune protection or reduction of worm burden
infection with age
Infection with Age
  • Worm burden increase with age.
  • Indicates no natural immunity is acquired with age.
global de worming efforts
Global De-worming Efforts
  • 2000, FRESH Partnership
    • 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
    • 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
    • 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
Global Deworming: Obstacles
  • 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.
    • Hookworm infection does not decrease with age
    • Older populations left untreated
  • Danger of drug resistance.
  • Safety during pregnancy not yet established.
rationale for vaccine
Rationale for Vaccine
  • 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
Vaccine Goals
  • Prevent infection
  • Eliminate or reduce worm burden to non-pathogenic 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
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
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
Hookworm Ag for a Vaccine
  • Interested in an Ag that is a secreted protein of adult hookworm
    • 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
Methods: Prepare Abs to Secreted Proteins

Grow worms in dog

Secreted proteins

Inject rabbit

Extract worms

bleed

Anti-serum to

secreted proteins

Develop Abs

methods prepare cdna library of adult hookworm
Methods: Prepare cDNA Library of Adult Hookworm

Phage

Plate

Transform

E. Coli

cDNA expressed

phage replicate

New phage released

rupture

Hookworm Ag released

methods immunoscreen for secreted proteins
Methods: Immunoscreen for Secreted Proteins

membrane

transfer

Incubate with primary antibody

anti-IgG antibodies with marker

visualize

Incubate with secondary antibody

anti-secretory protein anti-serum

methods cdna and peptide sequencing
Methods: cDNA and Peptide Sequencing

cDNA Sequence

Peptide Sequence

Identify Secreted Protein

preliminary characterization studies
Preliminary Characterization Studies
  • 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?
methods rt pcr
Methods: RT-PCR

Egg

Adult

Larvae

cDNA

cDNA

primers

PCR

Egg

Adult

Control

Larvae

Egg

Adult

Larvae

Run gel

methods chromatography
Methods: Chromatography

Each peak corresponds to a separated secretory protein

Chromatography

Area under curve = abundance

Secretory proteins separated based on relative hydrophobicity

Fractions containing unique protein

methods chromatography1
Methods: Chromatography

Fraction 51

Fraction 51

Amino acid sequence

Secretory protein

Compare to discovered secreted protein

results immuno screening
Results: Immuno-screening
  • cDNA script for immunogenic secretory protein found!
  • Secretory protein is IgG reactive
results cdna and peptide sequencing
Results: cDNA and Peptide Sequencing
  • 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
Results: RT-PCR
  • Ac-TMP is only present in adult hookworm
results purification and identification of antigen in secretory products
Results: Purification and Identification of Antigen in Secretory Products
  • 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
Discussion

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
problems
Problems
  • No evidence that it has anti-metalloprotease activity
  • No evidence that the Ab to Ac-TMP is important to immunity
  • Ac-TMP is IgG-reactive
future plans
Future Plans
  • 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
questions
Questions
  • 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?