Victims, Vectors and
Vaccines: Battling
Malaria in the
21st Century
Anthony A. James, Ph.D.
Microbiology & Molecular Genetics
Molecular Biology & Biochemistry
University of California, Irvine
Plasmodium falciparum: merozoite stage (Photo:WHO/TDR)
Alexander the Great may have
died from malaria in 323BC
Children of Lugnano in Imperial
Rome died of malaria in 450AD
“Malaria disease leaves uncultivated two
million hectares of land in Italy …. it poisons
every year about two million inhabitants and
kills 15,000 of them .… There is no other
health problem so deeply linked to the
prosperity of our country.”
Giustino Fortunato and Leopoldo Franchetti
Members of the Italian Parliament
July 14, 1898
“Malaria is the single largest disease
in Africa and a primary cause of
poverty. Every day 3,000 children die
from malaria. Every year there are
500 million cases among children and
adults.”
Dr. Gro Harlem Brundtland
Director General
World Health Organization
May 13, 1998
“… the world has yet to commit the
resources needed to control this preventable
and treatable disease.
Today is Africa Malaria Day, but malaria is a
global problem, and the responsibility for
stopping it extends to the entire international
community.”
Dr. Regina Rabinovich
Director
Infectious Diseases Program
Bill & Melinda Gates Foundation
April 25, 2003
What is malaria?
Why is it still a problem?
What is being done to fight it?
What are the prospects for the future?
What is malaria?
Italian, from mala aria “bad air”:
1740
Plasmodium falciparum: merozoite stage (Photo:WHO/TDR)
Infectious disease
Intracellular protozoan parasite
Anopheles gambiae female. (WHO/TDR/Holt Studios)
Vector-borne (mosquitoes)
High degree of host specificity
Etiological agents in humans:
Plasmodium falciparum
Plasmodium vivax
C. Magowan, J.T. Brown, N. Mohandas, W. Meyer-Ilse
Plasmodium ovale
Plasmodium malariae
Plasmodium falciparum: merozoite stage (Photo:WHO/TDR)
Transmitted to humans by mosquitoes in the genus
Anopheles
442 described species
68 associated with
malaria transmission
40 as main vectors
Anopheles gambiae female. (WHO/TDR/Holt Studios)
An. gambiae, An. stephensi, An. dirus, An. fluviatilis, An. albimanus, An. aquasalis, An. darlingi
Humans are the only natural reservoirs of the
four species that cause disease
No free-living forms
Malaria parasites have a complex life cycle
Centers for Disease Control
Developmental stages in both hosts
Epidemiology
Malaria is endemic to the poorest countries
Epidemiology
300 to 500 million clinical cases
Greater than one million deaths each year
(>2,700 deaths/day; ~2 persons every minute)
More than 90% of malaria deaths occur in SubSaharan Africa
Epidemiology
Increasing
values
Despommier, Gwadz, Hotez, and Knirsch 2000
Age-related prevalence
Clinical aspects:
Children and pregnant women are affected
most severely
Symptoms of uncomplicated malaria:
Fever, malaise, fatigue, anemia,
headache, myalgias
Newton et al., 1998 Am J Trop Med Hyg 58: 673-683
Clinical aspects (cont.):
Severe and complicated malaria (often associated
with P. falciparum) characterized by:
Cerebral malaria (seizures, coma, convulsions)
Respiratory distress
Pulmonary edema
Acute renal failure
Metabolic acidosis
Newton et al., 1998 Am J Trop Med
Hyg 58: 673-683
Gary Baumbach, M.D., Department of Pathology, University of Iowa College of Medicine
Economic impact:
Sub-Saharan Africa:
slows economic growth
by as much as 1.3%/year
GDP/person average is
3X higher in malaria-free
countries
WHO/Harvard University/London School of
Hygiene and Tropical Medicine Report
Economic impact:
One healthy year of life
is gained for every $1 to
$8 spent on effectively
treating malaria cases
This makes malaria
treatment as costeffective a public health
investment as measles
vaccinations
WHO/Harvard University/London School of
Hygiene and Tropical Medicine Report
STANDARD ECONOMIC MODEL
ECONOMIC
GROWTH
HEALTH
NEW ECONOMIC MODEL
ECONOMIC
GROWTH
HEALTH
Why is malaria still a problem?
Anti-vector measures
Pyrethroid spraying in Gabon: (M.W. Service, WHO/TDR)
Indoor residual spraying (Andy Crump, WHO/TDR/Crump)
insecticide resistance
Prophylactic/therapeutic drugs
Dihydroartemisinin capsules (Andy Crump, WHO/TDR)
( Andy Crump, WHO/TDR)
Chloroquine, mefloquine,
fansidar, doxycycline, etc.
Chloroquine and panadol syrup (Andy Crump, WHO/TDR/Crump)
Drug resistance
RESISTANCE TO ANTIMALARIAL DRUGS
Chloroquine
16 years
Fansidar
6 years
Mefloquine
4 years
Atovaquone
6 months
1940
1950
1960
1970
1980
1990
Other contributing factors
Little private sector (commercial) interest
Decaying healthcare infrastructure
Political turmoil
What is being done to fight it?
Diagnostics
Therapeutics
Prevention
Three milestone efforts:
The sequence of the human
genome. (2001) Venter et al.,
Science 291:1304-1351.
The genome sequence of the
malaria mosquito Anopheles
gambiae. (2002) Holt et al., Science
298: 129-149.
Genome sequence of the human malaria
parasite Plasmodium falciparum. (2002)
Gardner et al., Nature 419: 498-511.
Diagnostics
Malaria symptoms overlap other febrile
diseases
Clinical diagnosis can be unreliable
Laboratory confirmation of clinical
diagnosis is desirable (laboratory
diagnosis can be confounded by the
prevalence of circulating blood
parasites in individuals without
symptoms)
Microscopic confirmation difficult in
peripheral health facilities and in the
community
Plasmodium falciparum: merozoite stage (Photo:WHO/TDR)
Diagnostics
Development of Rapid Diagnostic Tests (RDTs)
RDTs do not:
measure severity of infection
discriminate among all species
discriminate between sexual and
asexual parasite stages
Discriminate between malaria
antigens that persist in the blood
following treatment
“dipstick test”
Gene amplification tests
Therapeutics
It is estimated that a new drug must be available
every five years Dr. Robert Ridley,
Chief Scientific Officer
Medicines for Malaria Venture/WHO
Genomics has led to the discovery of hundreds of
new possible targets
Foth, B. J. et al. (2003). Dissecting apicoplast
targeting in the malaria parasite Plasmodium
falciparum. Science 299, 705-708.
Lell, B. et al. (2003). Fosmidomycin, a novel
hemotherapeutic agent for malaria. Antimicrob.
Agents Chemotherapy 47, 735-738.
Dihydroartemisinin capsules (Andy Crump, WHO/TDR)
Prevention
Vaccines
Antivector measures
POWER OF PREVENTION: IMPACT OF VACCINES
IN THE U.S.
Cases per 100,000
Poliomyelitis
Diphtheria
2.0
0.6
1.5
0.4
25
20
15
1.0
10
0.2
0.5
0
1960
1970
1980
1990
0
1960
Rubella
(German Measles)
Cases per 100,000
Measles
30
25
20
15
10
5
0
1960
1970
1980
1990
5
1970
1980
1990
0
1960
1980
1990
Pertussis
(Whooping Cough)
Mumps
60
50
40
30
20
10
0
1960
1970
10
8
6
4
2
1970
1980
1990
0
1960
1970
1980
1990
(Adapted from data presented by the Boston Consulting Group)
000322-1
Targets for vaccine development
1) Infection-blocking (sterile)
5) Anti-disease
6) Transmission-blocking
Insecticide-treated bed nets
Can reduce transmission of malaria by 17%, and
severe malaria by as much as 35-50% when used
properly
Phillips-Howard et al. (2003).
The efficacy of permethrintreated bed nets on child
mortality in Western Kenya.
Am. J. Trop. Med. Hygiene, in
press,
Research Program at UCI
Genetic Control Of
Vector-Borne Diseases
Identification of Malaria Antigens
for Vaccine Development
Vectors:
Aedes aegypti
Anopheles gambiae
Anopheles stephensi
Anopheles darlingi
Surface antigens on the
vertebrate-infectious forms of the
parasites, the sporozoites
Malaria parasites:
Plasmodium gallinaceum
Plasmodium falciparum
Dengue Viruses
Life cycle of the malaria parasite
Emphasis on the mosquito portion of the cycle
2
1
3
7
6
4
5
1)
Gametocytes are ingested and are carried in bloodmeal to midgut
2)
Gametes fuse to form zygote
3)
Zygote transforms into ookinete and penetrates midgut epithelium
4)
Ookinete develops into oocyst on basal surface of midgut epithelium wall
5)
Sporozoites develop in oocyst
6)
Sporozoites burst through oocyst wall and migrate through hemolymph to salivary glands
7)
Sporozoites invade salivary glands in preparation for transmission to a new vertebrate host
Target Organs
Midgut
Salivary glands
Hemolymph
Hypothesis
The introduction into a population of
vector insects of a gene that confers
resistance to a pathogen should lead to a
decrease in transmission of that pathogen
Implicit in this hypothesis is that less
transmission will result in less morbidity
and mortality
Major Areas of Research
Molecular genetic manipulation of
mosquitoes (laboratory)
Introduction of genes into
populations (laboratory and field)
Population characteristics of target
species (field)
Molecular genetic manipulation of
mosquitoes
Develop transgenesis
technology
Identify promoters for
expressing antiparasite
genes
Develop antiparasite
effector molecules
TRANSGENESIS
TECHNOLOGY
ITR------------------Transposase-----------------------------------------ITR
ITR
ITR-------------------Transposase
ITR
Chromosomal DNA
Target Site
ITR
Gene of Interest
Target Site
Gene of Interest
ITR
Chromosomal DNA
ITR
Mosquito
Element
Aedes aegypti
Hermes
Mos1 (mariner)
piggyBac1
Anopheles gambiae
piggyBac
Grossman et al., 2001
An. stephensi
Minos
piggyBac
Catteruccia et al., 2000
Nolan et al., 2002
An. albimanus
piggyBac
Perera et al., 2002
Culex quinquefasciatus
Hermes
Target Site
First Citation
Jasinskiene et al., 1998
Coates et al., 1998, 2000
Kokoza et al., 2001.
Allen et al., 2001
PROMOTERS
EXPRESSING
ANTIPATHOGEN
GENES
Mosquito Tissue-Specific Promoter
Midgut
Fat body
Salivary gland
Antipathogen Effector Gene
Tissue receptors
Parasite surface ligands
Insect immune response
Parasite gene expression
Antiparasite toxins
Midgut
Salivary glands
Hemolymph
3’UTR
MICROARRAY OF AN. GAMBIAE GENES AFTER MULTIPLE
BLOOD MEALS
24 HPBM
1st meal
24 HPBM
2nd meal
EFFECTOR MOLECULES
Malaria:
100
9
3
NoSin (108)
Sin4G2scFv (30)
SinEGFP (90)
SinN2scFv (102)
19
54
0
0
1
32
5
0
6
12
94
27
64
4
Control
80
scFv
Vg promoter
60
Number of mosquitoes
% Mosquitoes
Ub promoter
20
40
20
15
10
0
0-10
11-100
101-1000
>1000
5
Number of Parasites
Capurro et al., 2000
0-10
11-100
101-1000
Number of parasites
Dengue viruses:
3’UTR
Jasinskiene and James, unpublished
Control
MnpS
2prMAs
3’UTR
RNAi
Fold-back
Percent (%) of cells positive by IFA
for DEN-2 E antigen
100
90
80
70
60
50
Control
Fold-back
40
30
20
10
Adelman, Z.N. et al., J Virol 76: 12925-12933
>1000
Effector Molecule Strategies
Interfere with mosquito tissue-recognition
molecules (receptors)
Interfere with parasite surface ligands
Induce antiparasite insect immune response
Interfere with parasite gene expression
Secretion of antiparasite toxins
Blocking strategy:
prevent sporozoites from invading salivary glands
Animal Model System
Aedes aegypti
Plasmodium gallinaceum
Galliformes
Plasmodium gallinaceum Circumsporozoite proteins
Region I
Repeat region
Image courtesy of Nirmala Xavier
•Surface protein on immature and mature sporozoites
•64-76 kiloDalton polypeptide
•Single-copy gene
•Contains highly-immunogenic tandem repeat amino acid sequences
Region II
Blocking strategy cont.:
The mouse monoclonal antibody, N2H6D5, binds the
circumsporozoite protein of the avian parasite, P.
gallinaceum, and blocks sporozoite invasion of salivary
glands
Single-chain antibodies as anti-Plasmodium effector molecules
Fv
F(ab’)2
16 aa
linker
scFv
Linker
IgG
P
VH
VL
4 aa
linker
Fab
Dibody
Sindbis virus vectors for transient expression of
antipathogen scFvs in blocking experiments
Control virus
EGFP
Sindbis Promoter
Control single-chain virus
Sindbis Promoter
Mal-l
4G2scFV
E-tag
Experimental single-chain virus
Sindbis Promoter
Mal-l
N2scFV
E-tag
Capurro et al., 2000
Sindbis-expressed N2scFv binds sporozoites in vivo
N2scFv
EGFP
Phase Contrast
Fluorescence
Sporozoites isolated from the hemolymph of virus-infected mosquitoes and reacted with
anti-E-tag antibodies
Capurro et al., 2000
NoSin (108)
Sin4G2scFv (30)
SinEGFP (90)
SinN2scFv (102)
100
9 3 19 54
0
0 1 32
5
0 6 12
94 27 64 4
% Mosquitoes
80
60
40
20
0
0-10
11-100
101-1000
>1000
Number of Parasites
Capurro et al., 2000
Mosquitoes with salivary gland mean
intensities of infection as low as 10
sporozoites were able to infect
chickens
The target for salivary gland sporozoite
intensities is zero
Jasinskiene and James, unpublished
“Eleven patients requiring malaria therapy were inoculated
intradermally with … Plasmodium vivax. Estimated doses
of 10 sporozoites were given to four patients, of 100 to
three patients, of 1,000 to two patients, and of 10,000 to two
patients. Parasitaemia was detected in all patients 12 to 17
days after inoculation; fever began on the 14th to 19th
days.”
Ungureanu E, Killick-Kendrick R, Garnham PC, Branzei P, Romanescu C,
Shute PG. (1977) Prepatent periods of a tropical strain of Plasmodium vivax
after inoculations of tenfold dilutions of sporozoites. Trans R Soc Trop Med
Hyg 70: 482-483
Future directions
Move from animal models to human
pathogens
Develop mechanisms for driving genes
through populations
Study the transmission dynamics of
malaria in the field
Define target vector populations
“Roll Back Malaria” (RBM)
Goals to cut by half malaria
deaths in Africa by 2010
Insecticide treated bed nets must be made available to more people
more quickly (only 4% use them now). Taxes and tariffs on them
must be removed.
Pregnant women must get bed nets and anti-malarial medicines as
a priority.
Next-generation drugs must be made available to treat chloroquineresistant malaria. Even at $1-3 per treatment these drugs cost 100X
more then chloroquine.
Malaria Early Warning Systems, using weather forecasting and data
on malaria hotspots, must be created to help authorities mitigate
The Africa Malaria Report WHO/CDS/MAL/2003.1093 URL:
outbreaks.
http://mosquito.who.int/amd2003/ (available from 25.4.03)
“Big-picture” challenges:
Consider development of a live vaccine
Educate scientists and public on
genetically-engineered vectors
Design malaria control approaches with
eradication as the ultimate goal
Secure appropriate funding
Detail from the Ramakian (Thai version of the
Ramayana epic), Wat Phra Rattanasatsadaram
Image courtesy of Dr. C. Louis