Post on 24-Dec-2015
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