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Victims, Vectors and Vaccines: Battling
Malaria in the 21st Century
Anthony A. James, Ph.D.
Microbiology & Molecular GeneticsMolecular 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 BrundtlandDirector General World Health OrganizationMay 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 RabinovichDirectorInfectious Diseases ProgramBill & Melinda Gates FoundationApril 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?
Plasmodium falciparum: merozoite stage (Photo:WHO/TDR)
Anopheles gambiae female. (WHO/TDR/Holt Studios)
Infectious disease
Intracellular protozoan parasite
Vector-borne (mosquitoes)
High degree of host specificity
Italian, from mala aria “bad air”: 1740
Plasmodium falciparumPlasmodium vivax
Plasmodium falciparum: merozoite stage (Photo:WHO/TDR)
Etiological agents in humans:
C. Magowan, J.T. Brown, N. Mohandas, W. Meyer-Ilse
Plasmodium ovalePlasmodium malariae
Anopheles gambiae female. (WHO/TDR/Holt Studios)
Transmitted to humans by mosquitoes in the genus Anopheles
442 described species
68 associated with malaria transmission
40 as main vectors
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
Centers for Disease Control
Malaria parasites have a complex life cycle
Developmental stages in both hosts
Epidemiology
Malaria is endemic to the poorest countries
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 Sub- Saharan Africa
Epidemiology
Age-related prevalence
Increasing values
Despommier, Gwadz, Hotez, and Knirsch 2000
Epidemiology
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:
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
Cerebral malaria (seizures, coma, convulsions)Respiratory distressPulmonary edemaAcute renal failureMetabolic acidosis
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 cost- effective a public health investment as measles vaccinations
WHO/Harvard University/London School of Hygiene and Tropical Medicine Report
STANDARD ECONOMIC MODEL
ECONOMICGROWTH HEALTH
ECONOMICGROWTH HEALTH
NEW ECONOMIC MODEL
Why is malaria still a problem?
Anti-vector measures
insecticide resistance
Indoor residual spraying (Andy Crump, WHO/TDR/Crump)Indoor residual spraying (Andy Crump, WHO/TDR/Crump)Pyrethroid spraying in Gabon: (M.W. Service, WHO/TDR)
Prophylactic/therapeutic drugs
( Andy Crump, WHO/TDR)
Dihydroartemisinin capsules (Andy Crump, WHO/TDR)
Chloroquine and panadol syrup (Andy Crump, WHO/TDR/Crump)
Chloroquine, mefloquine, fansidar, doxycycline, etc.
Drug resistance
RESISTANCE TO ANTIMALARIAL DRUGS
1940
Chloroquine 16 years
Fansidar 6 years
Mefloquine 4 years
Atovaquone 6 months
1950 1960 1970 1980 1990
Little private sector (commercial) interest
Other contributing factors
Political turmoil
Decaying healthcare infrastructure
What is being done to fight it?
Diagnostics
Therapeutics
Prevention
Genome sequence of the human malaria parasite Plasmodium falciparum. (2002) Gardner et al., Nature 419: 498-511.
The genome sequence of the malaria mosquito Anopheles gambiae. (2002) Holt et al., Science 298: 129-149.
The sequence of the human genome. (2001) Venter et al., Science 291:1304-1351.
Three milestone efforts:
DiagnosticsMalaria 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)
Development of Rapid Diagnostic Tests (RDTs)Diagnostics
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
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.
Genomics has led to the discovery of hundreds of new possible targets
Dihydroartemisinin capsules (Andy Crump, WHO/TDR)
Vaccines
Antivector measures
Prevention
POWER OF PREVENTION: IMPACT OF VACCINES IN THE U.S.
000322-1
Poliomyelitis Diphtheria Measles
Pertussis(Whooping Cough)Mumps
Rubella(German Measles)
2.0
1.5
1.0
0.5
0
0.6
0.4
0.2
0
2520151050
1960 1970 1980 1990
3025201510
50
1960 1970 1980 1990 1960 1970 1980 1990
1960 1970 1980 1990 1960 1970 1980 1990 1960 1970 1980 1990
1086420
605040302010
0
Cas
es p
er 1
00,0
00C
ases
per
100
,000
(Adapted from data presented by the Boston Consulting Group)
1) Infection-blocking (sterile)
5) Anti-disease
6) Transmission-blocking
Targets for vaccine development
Insecticide-treated bed netsCan 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 permethrin- treated 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 aegyptiAnopheles gambiaeAnopheles stephensiAnopheles darlingi
Malaria parasites:Plasmodium gallinaceumPlasmodium falciparum
Dengue Viruses
Surface antigens on the vertebrate-infectious forms of the parasites, the sporozoites
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
1
2
3
54
67
Life cycle of the malaria parasiteEmphasis on the mosquito portion of the cycle
Salivary glands
Midgut
Hemolymph
Target Organs
HypothesisThe 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 ResearchMolecular genetic manipulation of mosquitoes (laboratory)Introduction of genes into populations (laboratory and field)Population characteristics of target species (field)
Molecular genetic manipulation of mosquitoesDevelop transgenesis technology
Identify promoters for expressing antiparasite genes
Develop antiparasite effector molecules
TRANSGENESIS TECHNOLOGY
Chromosomal DNA Chromosomal DNATarget Site
Gene of InterestITR ITR
Target Site Target SiteGene of InterestITR ITR
ITRITR--------------------------------------TransposaseTransposase------------------------------------------ITRITR
Mosquito Element First Citation
Aedes aegypti HermesMos1 (mariner)
piggyBac1
Jasinskiene et al., 1998Coates et al., 1998, 2000Kokoza et al., 2001.
Anopheles gambiae piggyBac Grossman et al., 2001
An. stephensi MinospiggyBac
Catteruccia et al., 2000Nolan et al., 2002
An. albimanus piggyBac Perera et al., 2002
Culex quinquefasciatus Hermes Allen et al., 2001
PROMOTERS EXPRESSING ANTIPATHOGEN GENES Salivary glands
Midgut
Hemolymph
Antipathogen Effector GeneMosquito Tissue-Specific Promoter 3’UTR
MidgutFat body
Salivary gland
Tissue receptorsParasite surface ligands Insect immune responseParasite gene expression
Antiparasite toxins
24 HPBM1st meal
24 HPBM2nd meal
MICROARRAY OF AN. GAMBIAE GENES AFTER MULTIPLE BLOOD MEALS
EFFECTOR MOLECULES
0
20
40
60
80
100
0-10 11-100 101-1000 >1000
Number of Parasites
% M
osqu
itoes
NoSin (108) Sin4G2scFv (30)SinEGFP (90) SinN2scFv (102)
9 3 19 54 0 0 1 32 5 0 6 12 94 27 64 4
Capurro et al., 2000
Num
ber
of m
osqu
itoes
0-10
Control
Ub promoter
Vg promoter
11-100 101-1000 >1000
Number of parasites
5
10
15
20
Jasinskiene and James, unpublished
scFv
Malaria:
Dengue viruses:3’UTR
2prMAsMnpS 3’UTR
RNAi
Control
Fold-back
Perc
ent (
%) o
f cel
ls p
ositi
ve b
y IF
A
for
DE
N-2
E a
ntig
en
10
90
70
100
80
60
50
40
30
20
Control Fold-back
Adelman, Z.N. et al., J Virol 76: 12925-12933
Effector Molecule StrategiesInterfere 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
Plasmodium gallinaceum GalliformesAedes aegypti
Plasmodium gallinaceum Circumsporozoite proteins
•Surface protein on immature and mature sporozoites
•64-76 kiloDalton polypeptide
•Single-copy gene
•Contains highly-immunogenic tandem repeat amino acid sequences
Region I Region IIRepeat region
Image courtesy of Nirmala Xavier
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
P VH VL
4 aalinker
16 aalinker
LinkerIgG
F(ab’)2Fv
Fab
scFv
Dibody
Single-chain antibodies as anti-Plasmodium effector molecules
Sindbis virus vectors for transient expression of antipathogen scFvs in blocking experiments
Sindbis Promoter EGFP
Sindbis Promoter N2scFVMal-l E-tag
Sindbis Promoter 4G2scFVMal-l E-tag
Control virus
Control single-chain virus
Experimental single-chain virus
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
0
20
40
60
80
100
0-10 11-100 101-1000 >1000
Number of Parasites
% M
osqu
itoes
NoSin (108) Sin4G2scFv (30)SinEGFP (90) SinN2scFv (102)
9 3 19 54 0 0 1 32 5 0 6 12 94 27 64 4
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
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
Future directions
The Africa Malaria Report WHO/CDS/MAL/2003.1093 URL:http://mosquito.who.int/amd2003/ (available from 25.4.03)
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 chloroquine- resistant 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 outbreaks.
“Roll Back Malaria” (RBM) Goals to cut by half malaria
deaths in Africa by 2010
“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
Image courtesy of Dr. C. LouisDetail from the Ramakian (Thai version of the Ramayana epic), Wat Phra Rattanasatsadaram