Title: Expression, Purification, Refolding and Characterization of Recombinant Domains 2X and 3X of var2CSA
1Expression, 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
2Malaria? I barely know her.
Or, why am I taking these damn pills?
3Malaria 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)
4Its 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.
5So 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
6How does it work?
animation
7Sneaky 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
Never reaches spleen, causes trouble where it
sticks
Sticks to vessel walls
8What 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
9So 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).
10So 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.
11The 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.
12A 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.
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