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Hookworm Infection

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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Hookworm Infection

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  1. Hookworm Infection

  2. 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

  3. Worldwide Infection

  4. 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

  5. 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)

  6. Global Distribution

  7. 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.

  8. Infection

  9. Hookworm Egg and Larvae

  10. 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

  11. Gut Infection

  12. 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

  13. Associated Morbidities • Anemia, iron deficiency. • Hypoproteinemia, edema. • Mental, physical, growth retardation. • Immunocompromised. • Complicates malaria, HIV, etc.

  14. 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

  15. Infection with Age • Worm burden increase with age. • Indicates no natural immunity is acquired with age.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  21. 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

  22. 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

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

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

  25. 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

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

  27. 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?

  28. Methods: RT-PCR Egg Adult Larvae cDNA cDNA primers PCR Egg Adult Control Larvae Egg Adult Larvae Run gel

  29. 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

  30. Methods: Chromatography Fraction 51 Fraction 51 Amino acid sequence Secretory protein Compare to discovered secreted protein

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

  32. 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)

  33. Results: RT-PCR • Ac-TMP is only present in adult hookworm

  34. 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

  35. 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

  36. 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

  37. 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

  38. 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?

  39. j0nks!

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