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 Erythrocytic Sporozoites Merozoites Male and female gametocytes Clinical symptoms Asymptomatic

Malaria in Humans Four species of genus Plasmodium infect humans: P. falciparum, P. vivax, P. ovale, and P. malariaeFour species of genus Plasmodium infect humans: P. falciparum, P. vivax, P. ovale, and P. malariae Transmitted by female Anopheline mosquitoTransmitted by female Anopheline mosquito million clinical cases per year million clinical cases per year

Populations at Risk Infants, young children, and pregnant women in malaria endemic regionsInfants, 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 regionsNon-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 malariaP. falciparum can cause severe malaria: -hyperparasitemia -severe anemia -hypoglycemia -respiratory distress -cerebral malaria Molecular determinants that regulate mild versus severe disease largely unknownMolecular determinants that regulate mild versus severe disease largely unknown

Current Situation: Major International Health Problem Rapidly expanding number of clinical cases each yearRapidly expanding number of clinical cases each year Growing problem of antimalarial drug resistance with few novel therapeutics availableGrowing problem of antimalarial drug resistance with few novel therapeutics available Lack of an effective vaccineLack of an effective vaccine

Potential Solutions Gain an understand of the genetic and immunologic basis of protective immunityGain an understand of the genetic and immunologic basis of protective immunity Identify novel targets for therapeutic interventionIdentify novel targets for therapeutic intervention Determine reliable markers for measuring protection and pathogenesis for use in pharmacologic and/or vaccine trialsDetermine 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 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 populations might confer a heterozygote advantage against malariaIn 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  or  globin chains of hemoglobin (hemoglobin adult =  2  2 )Thalassemias are defects in synthesis of either  or  globin chains of hemoglobin (hemoglobin adult =  2  2 ) Mechanism of protection may be related to increased binding of antibodies and/or increased retention of fetal hemoglobinMechanism 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)Over 400 abnormal hemoglobins but only three reach polymorphic frequencies (S, C, & E) Homozygous state (SS) = sickle cell diseaseHomozygous state (SS) = sickle cell disease Heterozygous state (SC) = protection from malariaHeterozygous state (SC) = protection from malaria Mechanism unknown but red blood cells from (SC) individuals have reduced parasite growth and impaired invasion under low O 2 tensionMechanism unknown but red blood cells from (SC) individuals have reduced parasite growth and impaired invasion under low O 2 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 Susceptibility to Malaria In 1993 Murphy compared sequences of human and rodent genes and found greater variability among host defense genesIn 1993 Murphy compared sequences of human and rodent genes and found greater variability among host defense genes Polymorphisms in cytokines genes (e.g. TNF-  ) and effector molecules (e.g. nitric oxide, NO) are now being investigatedPolymorphisms in cytokines genes (e.g. TNF-  ) 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 genesStudy 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-ArginineL-Citrulline + NO L-NMMA Aminoguanidine NOS NO 2 - NO 3 - Cellular Lysate [ 14 C]L-Arg [ 14 C]L-Cit NOS Enzyme Assay Co-factors [ 14 C]L-Arg remains [ 14 C]L-Cit flows through Cation Exchange Column

Nitric Oxide Synthase Constitutive Expression Inducible Expression - Ca 2+ - and Calmodulin- Dependent Dependent - Ca 2+ - and Calmodulin- Independent Independent NO Synthesis for Normal Physiologic Function NO Synthesis in the Setting of Inflammation eNOS & nNOS NOS3 NOS1 NOS3 NOS1 iNOSNOS2

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)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)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 NONOS2 IL-10 TGF-  1 Monocyte Lymphocyte TNF-  N = O Monocyte/Macrophage PRBC Parasitic Products IL-12 PRBC IFN-  Fe Enz S S IFN- 

Hypothesis Increased capacity of the host to generate nitric oxide is protective against severe malaria