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Molecular Epidemiology and
Susceptibility
to Malaria Infection
Douglas Jay Perkins, Ph.D.
University of Pittsburgh Graduate School of Public Health
Department of Infectious Diseases and Microbiology
Centers for Disease Control and Prevention
Division of Parasitic Diseases-Immunology Branch
Molecular Vaccine Section, Atlanta, GA
Malaria Transmission Cycle
Pre-erythrocytic
Asymptomatic
Merozoites
Sporozoites
Erythrocytic
Clinical symptoms
Sporozoites
Male and female
gametocytes
Malaria in Humans
• Four species of genus Plasmodium
infect humans: P. falciparum, P.
vivax, P. ovale, and P. malariae
• Transmitted by female Anopheline
mosquito
• 300-500 million clinical cases per year
Populations at Risk
• Infants, young children, and
pregnant women in malaria
endemic regions
–
Greater than 3 million deaths
(primarily in children less than 5 y/o
due to non-immune status)
• Non-immune individuals
traveling through and/or living
in malaria endemic regions
–
35 million non-immune individuals
travel through malaria endemic
regions every year
Clinical Features of
P. falciparum
• P. falciparum can cause severe malaria:
-hyperparasitemia
-severe anemia
-hypoglycemia
-respiratory distress
-cerebral malaria
• Molecular determinants that regulate mild
versus severe disease largely unknown
Current Situation: Major
International Health Problem
• Rapidly expanding number of clinical cases
each year
• Growing problem of antimalarial drug
resistance with few novel therapeutics
available
• Lack of an effective vaccine
Potential Solutions
• Gain an understand of the genetic and
immunologic basis of protective immunity
• Identify novel targets for therapeutic
intervention
• Determine reliable markers for measuring
protection and pathogenesis for use in
pharmacologic and/or vaccine trials
Genetic Susceptibility
to Malaria
• At least 10,000 years of “pressure” on the human
genome from the malaria parasite
• In 1948 J.B.S. Haldane suggested that the high
frequency of thalassemia in Mediterranean populations
might confer a heterozygote advantage against malaria
• Thalassemias are defects in synthesis of either a- or bglobin chains of hemoglobin (hemoglobin adult = a2b2)
• Mechanism of protection may be related to increased
binding of antibodies and/or increased retention of fetal
hemoglobin
Sickle Cell Gene and Resistance
to Malaria
• Over 400 abnormal hemoglobins but only three reach
polymorphic frequencies (S, C, & E)
• Homozygous state (SS) = sickle cell disease
• Heterozygous state (SC) = protection from malaria
• Mechanism unknown but red blood cells from (SC)
individuals have reduced parasite growth and
impaired invasion under low O2 tension
• In addition to red cell abnormalities, there are many
other genetic changes……..
Host Response Genes and
Susceptibility to Malaria
• In 1993 Murphy compared sequences of human and
rodent genes and found greater variability among host
defense genes
• Polymorphisms in cytokines genes (e.g. TNF-a) and
effector molecules (e.g. nitric oxide, NO) are now being
investigated
• Study of genetic variation may utilize several types of
DNA markers to analyze candidate susceptibility genes
Single base pair variations = SNPs
Microsatellite or variable number tandem repeats (VNTRs)
Overview
Part 1.
NOS2 (G –954C) in Gabonese Children
with Severe Malarial Anemia
Part 2.
NOS2 (G –954C) in Tanzanian Children
with Cerebral Malaria
Part 3.
NOS2 (G –954C) in Kenyan Children
with Severe Malarial Anemia
Nitric Oxide Biosynthesis
L-Arginine
NOS
L-Citrulline + NO
L-NMMA
Aminoguanidine
NO2-
NO3-
NOS Enzyme Assay
Cellular Lysate
[14C]L-Arg remains
[14C]L-Arg Co-factors [14C]L-Cit
[14C]L-Cit flows through
Cation Exchange Column
Nitric Oxide Synthase
eNOS & nNOS
iNOS
NOS3
NOS2
NOS1
Constitutive Expression Inducible Expression
- Ca2+- and CalmodulinDependent
- Ca2+- and Calmodulin-
NO Synthesis for Normal
Physiologic Function
NO Synthesis in the
Setting of Inflammation
Independent
Nitric Oxide: Previous
Observations in Malaria
• Nitric oxide production is anti-plasmodial in vitro
and in vivo
-(Oswald et al.,Comp Biochem Physiol Pharmacol
Toxicol Endocrino, 1994; 108:11-18)
• Elevated NO metabolites are associated with
accelerated clinical cure and increased parasitologic
clearance in Gabonese adults and children
-(Kremsner et al., Trans R Soc Trop Med Hyg, 1996; 90: 44-47)
 NO appears protective against malaria
Model of NO Production in Malaria
IFN-g
IL-12
Lymphocyte
IL-10
Monocyte
TNF-a
TGF-b1
IFN-a
Parasitic
Products
PRBC
NOS2
NO
S
Enz
Fe
S
PRBC
Monocyte/Macrophage
N=O
N=O
Hypothesis
Increased capacity of the host to
generate nitric oxide is protective
against severe malaria