Expression, Purification, Refolding and Characterization of Recombinant Domains 2X and 3X of var2CSA - PowerPoint PPT Presentation

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Expression, Purification, Refolding and Characterization of Recombinant Domains 2X and 3X of var2CSA

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Expression, Purification, Refolding and Characterization of Recombinant Domains ... 3 classic stages of paroxysm (6-10 hr): Abrupt onset cold stage with nasty shakes ... – PowerPoint PPT presentation

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Title: Expression, Purification, Refolding and Characterization of Recombinant Domains 2X and 3X of var2CSA


1
Expression, Purification, Refolding and
Characterization of Recombinant Domains 2X and 3X
of var2CSA from P. falciparum 3D7, which mediate
sequestration of infected erythrocytes in the
syncitiotrophoblast
  • Evaluation of Antigenicity and Cross-Reactivity

2
Malaria? I barely know her.
  • Just kidding

Or, why am I taking these damn pills?
3
Malaria Sucks
  • Arguably the most damaging disease in human
    history
  • 300-500 million clinical cases/year among 2
    billion people in endemic areas
  • 1.5 million-2.7 million deaths, mostly children
    in sub-Saharan Africa. 20 of all clinical
    presentations there are malaria.
  • Economically devastating, especially in the
    developing world, probably costs 2.2
    billion/year globally (treatment, prevention,
    productivity loss)

4
Its old
  • Splenomegaly and antigens found in Egyptian
    mummies from 3000 BC
  • Vedic texts (1500-800 BC) describe autumnal
    fevers as king of diseases and splenomegaly
  • Became prominent in Roman Republic round 200 BC.
    Association swamps led to name malaria, coined
    into English by Horace Walpole in 1740
  • Seemingly brought to Americas post-Columbian
  • Parasite identified in 1880 by Charles Laveran.
    Ronald Ross, working in India, proposed mosquito
    transmission.

5
So what should I look out for?
  • 3 classic stages of paroxysm (6-10 hr)
  • Abrupt onset cold stage with nasty shakes
  • Hot stage of 104 fever, restless, vomiting,
    convulsions
  • Sweating stage, defervesce, may sleep
  • Occurs in 4 3-day (severe) cycles
  • Headache, cough, flu-like, diarrhoea, jaundice,
    anaemia
  • Severe cases coma/convulsions, respiratory
    distress, circulatory collapse
  • Usually incubates 1-4 weeks some forms can hide
    for years before causing symptoms or relapses

6
How does it work?
animation
7
Sneaky expletive deleted Parasites
  • RBCs are a great place to hide, but
  • As blood circulates through the spleen, it clears
    abnormal RBCs.
  • P. falciparum avoids clearance by not
    circulating. A wide variety of gripper proteins,
    encoded by the var genes, enable the IEs to stick
    to endothelial tissue.

Passes through vessels normally
Cleared by spleen QC
  • X

Never reaches spleen, causes trouble where it
sticks
Sticks to vessel walls
8
What do we do?
  • Drugs stay tuned
  • Vector Control
  • Pesticide treatment
  • Habitat destruction
  • This is why theres no more malaria in the US and
    the CDC is in Atlanta.
  • Now that DDT is illegal ? bed nets, indoor
    spraying
  • Genetic Manipulation (sterile males, malaria
    lethal females, resistant strains)
  • Vaccines tricky because parasites hide so well
    in RBCs varying their surface proteins, but
    there are several under development EBA-175
    Duffy-binding

9
So what am I doing? The Problem
  • In endemic areas, children are at high risk.
    Adults develop immunity and generally have only
    mild symptoms when infected.
  • Except in pregnant women where the placenta
    presents a new hiding place for falciparum to
    sequester, very bad for mother and child (PAM).

10
So what am I doing? The Solution?
  • PAM is rarely a problem beyond 1st or 2nd
    pregnancy, so there must be some universal
    protein target for the immune system to attack.
  • That target might be pieces of var2CSA protein. I
    am making those pieces to see if they are good
    immune targets for preventing PAM.
  • Clone, generate and purify the protein pieces
  • See if they
  • Bind placental molecule, CSA
  • Elicit antibodies in mice which block CSA
    adhesion
  • Are bound specifically by antibodies from women
    whove been pregnant
  • If all those are true, these protein pieces are
    likely to make an effective vaccine.

11
The Curious Quinine Story
  • From cinchona bark, high alt evergreens. Used
    medicinally by Peruvian Indians.
  • Named for Countess of Chinchon, treated for
    malaria w/bark by Jesuits in early 17th century.
  • Oliver Cromwell refused to ingest the powder of
    the devil. He died of malaria.
  • Quinine isolated in 1820 by Peletier Caventou.
  • Forested almost to extinction till Dutch started
    plantations in Indonesia. Near monopoly till
    Japanese invade ? CQ.
  • British Navy studies of quinine/malaria 1st
    modern epidemiology
  • Made colonization of India Africa possible.
    Brits used 10,000 kg/yr in India 1860-1940.
    Union used 25,000 kg in American Civil War.

12
A Few Tips Prophylactic Drugs
  • Chloroquine 300mg (base)/wk, ok in 2 doses
  • Mefloquine 250mg/wk
  • Malarone atovaquone 250mg, proguanil 100mg/day
  • Maloprim pyrimethamine 12.5mg/dapsone 100mg/wk
  • Doxycycline 100mg/day, short term only
  • If you are taking quinine, Fansidar, clindamycin
    or halofantrine, stop.

13
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