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

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0.6

0.4

0.2

0

2520151050

1960 1970 1980 1990

3025201510

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1960 1970 1980 1990 1960 1970 1980 1990

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1086420

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0

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es p

er 1

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ases

per

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(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