Molecular Epidemiology and Molecular Epidemiology and SusceptibilitySusceptibility

to Malaria Infectionto Malaria Infection

Douglas Jay Perkins, Ph.D.Douglas Jay Perkins, Ph.D.

University of Pittsburgh Graduate School of Public HealthUniversity of Pittsburgh Graduate School of Public Health

Department of Infectious Diseases and MicrobiologyDepartment of Infectious Diseases and Microbiology

Centers for Disease Control and PreventionCenters for Disease Control and Prevention

Division of Parasitic Diseases-Immunology BranchDivision of Parasitic Diseases-Immunology Branch

Molecular Vaccine Section, Atlanta, GAMolecular Vaccine Section, Atlanta, GA

Malaria Transmission CycleMalaria Transmission CyclePre-erythrocytic

Erythrocytic

Sporozoites

Sporozoites Merozoites

Male and femalegametocytes

Clinical symptoms

Asymptomatic

Malaria in HumansMalaria in Humans

• Four species of genus Four species of genus PlasmodiumPlasmodium

infect humans: infect humans: P. falciparum, P. vivax, P. falciparum, P. vivax,

P. ovale, and P. malariaeP. ovale, and P. malariae

• Transmitted by female Transmitted by female AnophelineAnopheline

mosquitomosquito

• 300-500 million clinical cases per year300-500 million clinical cases per year

Populations at RiskPopulations at Risk• Infants, young children, and Infants, young children, and

pregnant women in malaria pregnant women in malaria endemic regionsendemic regions

– Greater than 3 million deaths (primarily Greater than 3 million deaths (primarily in children less than 5 y/o due to non-in children less than 5 y/o due to non-immune status)immune status)

• Non-immune individuals traveling Non-immune individuals traveling through and/or living in malaria through and/or living in malaria endemic regionsendemic regions

– 35 million non-immune individuals travel 35 million non-immune individuals travel through malaria endemic regions every through malaria endemic regions every

yearyear

Clinical Features of Clinical Features of P. falciparumP. falciparum

• P. falciparumP. falciparum can cause severe malaria: can cause severe malaria:-hyperparasitemia-hyperparasitemia

-severe anemia-severe anemia

-hypoglycemia-hypoglycemia

-respiratory distress-respiratory distress

-cerebral malaria-cerebral malaria

• Molecular determinants that regulate mild versus Molecular determinants that regulate mild versus

severe disease largely unknownsevere disease largely unknown

Current Situation: Major Current Situation: Major International Health ProblemInternational Health Problem

• Rapidly expanding number of clinical cases Rapidly expanding number of clinical cases

each year each year

• Growing problem of antimalarial drug Growing problem of antimalarial drug

resistance with few novel therapeutics resistance with few novel therapeutics

availableavailable

• Lack of an effective vaccineLack of an effective vaccine

Potential SolutionsPotential Solutions

• Gain an understand of the genetic and immunologic basis of Gain an understand of the genetic and immunologic basis of protective immunityprotective immunity

• Identify novel targets for therapeutic interventionIdentify novel targets for therapeutic intervention

• Determine reliable markers for measuring protection and Determine reliable markers for measuring protection and pathogenesis for use in pharmacologic and/or vaccine trials pathogenesis for use in pharmacologic and/or vaccine trials

Genetic SusceptibilityGenetic Susceptibilityto Malariato Malaria

• At least 10,000 years of “pressure” on the human genome from the malaria parasiteAt 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 In 1948 J.B.S. Haldane suggested that the high frequency of thalassemia in Mediterranean populations might confer a heterozygote advantage against malariapopulations might confer a heterozygote advantage against malaria

• Thalassemias are defects in synthesis of either Thalassemias are defects in synthesis of either or or globin chains of hemoglobin (hemoglobin globin chains of hemoglobin (hemoglobin adult = adult = 2222))

• Mechanism of protection may be related to increased binding of antibodies and/or increased Mechanism of protection may be related to increased binding of antibodies and/or increased retention of fetal hemoglobinretention of fetal hemoglobin

Sickle Cell Gene and ResistanceSickle Cell Gene and Resistanceto Malariato Malaria

• Over 400 abnormal hemoglobins but only three reach polymorphic frequencies (S, C, & E)Over 400 abnormal hemoglobins but only three reach polymorphic frequencies (S, C, & E)

• Homozygous state (SS) = sickle cell disease Homozygous state (SS) = sickle cell disease

• Heterozygous state (SC) = protection from malariaHeterozygous state (SC) = protection from malaria

• Mechanism unknown Mechanism unknown butbut red blood cells from (SC) individuals have reduced parasite growth red blood cells from (SC) individuals have reduced parasite growth and impaired invasion under low Oand impaired invasion under low O22 tension tension

• In addition to red cell abnormalities, there are many other genetic changes……..In addition to red cell abnormalities, there are many other genetic changes……..

Host Response Genes and Host Response Genes and Susceptibility to MalariaSusceptibility to Malaria

• In 1993 Murphy compared sequences of human and rodent genes and found greater variability among host In 1993 Murphy compared sequences of human and rodent genes and found greater variability among host defense genesdefense genes

• Polymorphisms in cytokines genes (e.g. TNF-Polymorphisms in cytokines genes (e.g. TNF-) and effector molecules (e.g. nitric oxide, NO) are now being ) and effector molecules (e.g. nitric oxide, NO) are now being investigatedinvestigated

• Study of genetic variation may utilize several types of DNA markers to analyze candidate susceptibility genesStudy of genetic variation may utilize several types of DNA markers to analyze candidate susceptibility genes

Single base pair variations = SNPsSingle base pair variations = SNPs

Microsatellite or variable number tandem repeats (VNTRs)Microsatellite or variable number tandem repeats (VNTRs)

OverviewOverview

Part 1. Part 1. NOS2 (G –954C) in Gabonese Children NOS2 (G –954C) in Gabonese Children with Severe Malarial Anemiawith Severe Malarial Anemia

Part 2.Part 2. NOS2 (G –954C) in Tanzanian Children NOS2 (G –954C) in Tanzanian Children with Cerebral Malariawith Cerebral Malaria

Part 3.Part 3. NOS2 (G –954C) in Kenyan Children NOS2 (G –954C) in Kenyan Children with Severe Malarial Anemiawith Severe Malarial Anemia

Nitric Oxide BiosynthesisNitric Oxide Biosynthesis

L-ArginineL-Arginine L-CitrullineL-Citrulline ++NONOL-NMMA

Aminoguanidine

NOSNOS

NONO22-- NONO33

--

Cellular LysateCellular Lysate

[[1414C]L-ArgC]L-Arg [[1414C]L-CitC]L-Cit

NOS Enzyme AssayNOS Enzyme Assay

Co-factorsCo-factors

[[1414C]L-Arg remainsC]L-Arg remains

[[1414C]L-Cit flows throughC]L-Cit flows through

Cation Exchange ColumnCation Exchange Column

Nitric Oxide SynthaseNitric Oxide Synthase

Constitutive ExpressionConstitutive Expression Inducible ExpressionInducible Expression- Ca- Ca2+2+- and Calmodulin- - and Calmodulin-

DependentDependent - - CaCa2+2+- and Calmodulin- - and Calmodulin- IndependentIndependent

NO Synthesis for Normal NO Synthesis for Normal Physiologic FunctionPhysiologic Function

NO Synthesis in the NO Synthesis in the Setting of InflammationSetting of Inflammation

eNOS & nNOSeNOS & nNOS NOS3 NOS1NOS3 NOS1

iNOSiNOSNOS2NOS2

Nitric Oxide: Previous Nitric Oxide: Previous Observations in MalariaObservations in Malaria

• Nitric oxide production is anti-plasmodialNitric oxide production is anti-plasmodial in vitro in vitro and and in in

vivovivo-(Oswald et al.,-(Oswald et al.,Comp Biochem Physiol Pharmacol Toxicol EndocrinoComp Biochem Physiol Pharmacol Toxicol Endocrino, ,

1994; 108:11-18)1994; 108:11-18)

• Elevated NO metabolites are associated with accelerated clinical Elevated NO metabolites are associated with accelerated clinical

cure and increased parasitologic clearance in Gabonese adults cure and increased parasitologic clearance in Gabonese adults

and childrenand children -(Kremsner et al., -(Kremsner et al., Trans R Soc Trop Med HygTrans R Soc Trop Med Hyg, 1996; 90: 44-47), 1996; 90: 44-47)

NO appears NO appears protectiveprotective against malaria against malaria

Model of NO Production in MalariaModel of NO Production in Malaria

NONONOS2NOS2

IL-10

TGF-1MonocyteLymphocyte TNF-

N = O

N = O

Monocyte/Macrophage

PRBC

Parasitic Products

IL-12

PRBC

IFN-

FeEnzSS

IFN-

HypothesisHypothesis

Increased capacity of the host to Increased capacity of the host to

generate nitric oxide is protective generate nitric oxide is protective

against severe malariaagainst severe malaria