I never found the order I searched for But always a sinister And well-planned disorder That...

I never found the order I searched for

But always a sinister

And well-planned disorder

That increases in the hands

Of those who hold power

While the others who clamor for

A more kindly world

A world with less hunger and more hopefulness

Die of torture in the prisons

Claribel Alegria, Nicaraguan poet

Leading causes of death in Sub-Saharan Africa, South Asia, and Southeast Asia for persons age 0-44 (World Health Organization)

How can we defeat this disease?

First, how does your body strike back?

Can you say antibodies and T cells?

Antibodies work at all stages

but T cells are only effective

when the parasite is in the liver

Why?

Red blood cells do NOT express MHC proteins

and thus infected cells cannot

generate a T cell response!

In adults the immune system does

a pretty good job,

but in kids it’s a different story

www.columbia.edu/itc/hs/medical/pathophys/parasitology/2006/PAR-05Color .pdf

Young kids are hit much harder by malaria

Because of differences in their immune response


How does the malaria parasite respond to

the natural selection produced by immune attack?



generate a T cell response

One clue comes from the fact that malaria makes

Red blood cells “sticky” so they lodge in capillaries




This allows them to prevent their host cells from

Going through the spleen where they can be killed




Infected red blood cells can be recognized by a change

In shape--the cells get “knobs”

www3.niaid.nih.gov/.../ malariaGeneticsSection/

parasite




The “knobs” and the stickiness result from

Plasmodium proteins that are put on the plasma membrane

of the red blood cell including the PfEMP proteins





PfEMP-1 proteins are encoded by the var (for variable) genes.

individual parasite genomes contain 50-150 var genes

but only one is expressed at any one time.

PfEMP-1 proteins are very antigenic and thus we mount

a strong and effective response




Parasites fight back: they switch on different var genes

during asexual reproduction allowing them

to evade the initial immune response!

Nature 415: 673 (2002)

Variation also allows the parasite to re-infect previously exposed hosts

Nature 415: 673 (2002)


Despite this, as we have seen, most adults

fight off the infection over time


How can we speed up this process, protect children

and the elderly, and reduce the death rate?


How can we speed up this process, protect children

and the elderly, and reduce the death rate?

We need drugs

We’ll focus on the deadliest parasite

http://drnelson.utmem.edu/Woods.Hole/slide5.png

But there is a problem that takes us back to the family tree

You and memushrooms

plants

Plasmodium

http://drnelson.utmem.edu/Woods.Hole/slide5.png

We seem like distant relatives

but this is only part of the tree of life

You and memushrooms

plants

Plasmodium

Here’s the bigger picture--we are actuallyrelatively closely related to Plasmodium

E. coli

Staph. aureus

TB bug

Genome Research 12, 1080-1090 (2002)

Plasmodium and me!

We share much more of our cellular machineryThan we do with bacteria.

E. coli

Staph. aureus

TB bug

Genome Research 12, 1080-1090 (2002)

Plasmodium and me!

We need to find drug targets

that Plasmodium have

And people do not share

The first insight goes back >400 years

Following their arrival in the New World, Spanish Jesuit missionaries in Peru learned of a medicinal bark That the native Quechuaused to treat “fever”.

web1.caryacademy.org/.../ Quinine/history.htm

Legend suggests that the Countess of Chinchón, wife of the Viceroy of Peru, was cured of her fever with the bark.The tree was named Cinchona after the countess.

The Quechua called itQuinquina = “bark of barks”

In 1820 J.B. Caventou and P.J. Pelletier isolated the active chemical and named it quinine

A synthesis approach wasdiscovered in the 1940sBut most is still purified From Cinchona bark

Quinine remains an effective drugand was dominant through 1950.But chemists soughtmore effective derivativesthat had less side effects Hans Andersag at Bayer discovered chloroquine in 1934and by the 1950’s itbecame the drug of choice

How do quinine And its derivativeskill Plasmodium?

Most current drugs target the red blood cell stage

Plasmodium eats blood! (actually really hemoglobin)

Plasmodium eats blood!

When living in red blood cellsIt survives by digesting Hemoglobin in its food vacuole(the parasite lysosome).

However, the leftover“heme”, the metal complexthat actually carries oxygen,Is quite toxic.

Wikipedia.com

Plasmodium eats blood!

The parasite’s

heme polymerase converts

the toxic heme into

non-toxic hemazoin

Which then crystalizes

Chloroquine accumulates in the food vacoule

Where it inhibits conversion of heme to hemazoin

Either by directly binding heme and/or

By inhibiting heme polymerase

http://www.tulane.edu/~wiser/protozoology/notes/drugs.html

Chloroquine accumulates in the food vacoule

Where it inhibits conversion of heme to hemazoin

Either by directly binding heme and/or

By inhibiting heme polymerase


No, not chloroquine!

Ahhhhhhhhh…..

Chloroquine allowed major progress against malaria--

What do you think happened next?


No, not chloroquine!

Ahhhhhhhhh…..



What cellular changes underline resistance?




Chloroquine resistance is linked to a mutation in a membrane-located food vacuolar drug-metabolite transporter protein Mutant PfCRT is thought to move Chloroquine out of the food vacuole


www.uhhg.org/mcrh/resources/video/malariappt.pdf

Choroquine resistance set back

the fight against malaria

www.uhhg.org/mcrh/resources/video/malariappt.pdf


Option 1: make more derivatives of quinine or use quinine again


Option 2: new drug targets

Pyrimethamine (DHFR) + sulfadoxine or dapsone (DHPS)

attack enzymes involved in making nucleotides

(we can take in folate from our diet- they cannot)


One of the newest medicines has one of the longest histories

Artemisia has been used by Chinese herbalistsfor >1000years to treat many illnesses including malaria

Med Trop (Mars). 1998;58(3 Suppl):13-7.Int J Parasitol. 2002 Dec 4;32(13):1655-60.

The active compound was found to be Artemisinin


Artemisinin is not toxic until it is cleavedinside the parasite by exposure to heme-iron

A resulting free radical intermediate may kill the parasite by poisoning one or more essential malarial protein(s).


What’s another rule to prevent resistancewe learned from treating TB?

When you use new drugs, use them in combination!

Current CDC guidelines are:

If malaria was acquired in areas without If malaria was acquired in areas without chloroquine resistancechloroquine resistance

Then Treat with chloroquineThen Treat with chloroquine


If from area with resistance If from area with resistance :: 1. quinine plus doxycycline or tetracycline1. quinine plus doxycycline or tetracycline

oror

2. Atovaquone + proguanil (Malarone)2. Atovaquone + proguanil (Malarone)


If P. vivax P. vivax or or P. Ovale P. Ovale Chloroquine plusChloroquine plus PRIMAQUINE to hit liver stage


Plasmodium is also evolvingresistance to other drugs

Research continues to identify new drugs

Nature 415: 686 (2002)

Scientists also sequenced the genome of P. falciparum

In 2002 looking for new drug targets

Nature 415: 686 (2002)

23 million base pairs

5,300 proteins encoded

60% are not similar

to known proteins!

The genome of P. vivax was completed this past month

(October 2008)

More similar to P. falciparum than expected

Only 150 unique proteins

pathmicro.med.sc.edu/ppt-vir/vaccine.ppt

Hey wait a minute, aren’t youforgetting something?

pathmicro.med.sc.edu/ppt-vir/vaccine.ppt

What about a vaccineagainst malaria?

(Program for Appropriate Technology in Health)

Genetically attenuated P. falciparum sporozoites.

Developmentally arrested at an early stage

following liver cell invasion,

or

Radiation attenuated sporozoite

Of all malaria vaccines, GlaxoSmithKline’s

RTS,S/AS02A is the furthest along

in clinical testing, "at least by four to five years,"

Zarifah Reed at the WHO Initiative for Vaccine Research

RTS,S/AS02A is generated against CSP, the most abundant cell surface protein

during the malaria parasite's infectious sporozoite stage

The RTS,S/AS02A antigen is a fusion of CSP with a surface protein from hepatitis B,

to stimulate a more effective immune response

CS is attached to the plasma membrane

and contains a large immunodominant domain

that consists of repeats of a short, species-specific peptide.

It is important for invasion of liver cells

Membrane anchor

Since RTS,S incorporates a hepatitis B antigen,

it "will also be a hepatitis B vaccine," she adds.

An estimated one million people die annually worldwide

from hepatitis B and ensuing liver complications.

After Phase I trials demonstrated safety,

the vaccine entered Phase II trials in children in Africa

Here’s the data--did it work?

It continues to look good after a longer follow up

The Lancet 366 10 December 2005, pp. 2012-2018

Other vaccines are following this

though trials are less far along

That’s high tech—how about low tech

Reduce Contact Between Humans And Mosquitoes

Personal protective measures– DEET– PERMETHRIN– Bed nets

Insecticide treated bednetscan reduce infection by 63%

UNICEF Ethiopia

For example, 20 million Bednets

have been distributed in Ethiopia

Legend: PSI - Population Services International;

WB -World Bank.

GFATM 2, 5 - Global Fund for AIDS, TB and Malaria

Cum nets: Cumulative number of nets distributed.

WHO, UNICEF, the World Bank and the UN Development Program formed a

new partnership in 1998

It’s goal: eliminate malaria as a majordisease by 2015

Accessible drugs

Nets and Insecticide

Rapid diagnostic tests

More Artemesia

A U.S. government initiative designed to cut malaria deaths in half in sub-Saharan Africa.

Announced by President Bush on June 30, 2005.

Pledges to increase U.S. funding of malaria prevention and treatment in sub-Saharan Africa by > $1.2 billion over 5 years.

Before we end, let’s consider one other cool aspect of malaria

J. Mol. Biol., 235, 657

This brings us back to blood and how it carries oxygen

J. Mol. Biol., 235, 657

Hemoglobin is a finely tuned machine crafted by natural selection

to deliver oxygen and remove CO2

A single amino acid change alters hemoglobin structure

And allows the protein to form long “rods”

A single amino acid change alters hemoglobin structure

And allows the protein to form long “rods”

These protein “rods” change the shape of the Red blood cell

and this change occurs when hemoglobin is not bound to oxygen

Sickled cell block capillaries and trigger

lower oxygen and more “sickling”

The disease is treatable in the developed world

But still shortens life expectancy

Untreated, as in much of the developing world,

the disease is fatal in childhood

The mutation responsible for sickle cell anemia

is surprisingly common in parts of the world

anthro.palomar.edu/synthetic/synth_4.htm

Why hasn’t natural selection eliminated this allele?


Does this map look familiar?


Does this map look familiar?

Malaria distribution

Heterozygotes have an advantage!

While red blood cells from heterozygotes are usually normal,

they are prone to sickle and be destroyed if infected by Plasmodium,

eliminating the blood stage of the parasite before it can reproduce

And this is not the only such example!

Protects red blood cells

Against oxidative stress

Glucose-6-phosphate dehydrogenase plays a key role

in glucose metabolism but also generates glutathione



200 million people are deficient in this process



Complete loss of function can

lead to episodes of anemia



Milder reductions can provide partial resistance

to malaria as malaria-infected red cells

are susceptible to more rapid death



However, these individuals are also

hypersensitive to certain antimalarials

like primaquine that affect oxidative stress

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