YAA Reuben Mangi (I56/ 81536/ 2012) Bsc. University of Nairobi (2012) Identification of Plasmodium Falciparum MSP-1 Variants in Malaria sequential infections YAA Reuben Mangi (I56/ 81536/ 2012) Bsc. University of Nairobi (2012) Supervisors Dr. Isabella Oyier, Wellcome- Trust, Kenya Medical Research Institute (KEMRI), Kilifi Dr. George Obiero, Centre for Bioinformatics and Biotechnology (CEBIB) University of Nairobi

Introduction Malaria is an intermittent and remittent fever disease caused by a species in the genus Plasmodium. Malaria thrives in the tropical areas of Asia, Africa, and Central & South America, where it infects millions of people. Figure 1. Malaria Victim( National Geographic) There are approximately 219 million malaria cases and mortality rates of approximately 1 million cases a year in the world. 28 million Kenyans living in malaria risk regions and majority being children.

Introduction Cont.. The erythrocytic stages of the parasite life cycle are responsible for all symptoms and pathologies of the disease. The symptoms are; fever, headache, malaise, fatigue, muscular pains, and occasionally nausea, vomiting and diarrhoea. Sequestered RBCs adhere to the blood vessels resulting to destruction and blockage of vessels leading to hypoxia and toxemia in vital tissues and organs. Figure 2 Malaria Parasites Amid Red Blood Cells (National Geographic)

Life cycle of Plasmodium falciparum The parasite undergoes a complex life cycle that requires both a human host and a female Anopheles mosquito. Figure 3. Life cycle of P. falciparum (Nature)

Molecular basis of invasion The surface is comprised of Glycosyl Phosphatidyl Inositol (GPI) anchored membrane proteins(Ligands) and their associated partners. These include Merozoite Surface proteins(MSP), Apical Membrane Antigen-1(AMA-1), The Reticulocyte Binding Homologue proteins (RBLs), Erythrocyte Binding like proteins (EBLs). Other peripheral proteins involved in primary contact also exist. Well characterized EBLs are, EBA-175, EBA-140 and their receptors are glycophorin A, Glycophorin C respectively. Figure 4: Fine structure of Plasmodium falciparum Merozoite (Cowman and Crabb 2006)

Invasion of Erythrocytes by Plasmodium falciparum Merozoite Figure 5: Diagram of RBC invasion -(Cowman and Crabb 2006) A process that involves is multi-step sequence of initial recognition, re-orientation and entry. The contact requires interactions of merozoite surface proteins (MSP) and specific receptors on the host cell.

Immunity to Invasion by Merozoites Clinical immunity to malaria is slow to develop and short lived. One reason for this is the extensive diversity found in Plasmodium antigens. Merozoites are among the parasite stages targeted by immune system. The merozoite stage represents an attractive target for controlling malaria due to its uniqueness towards its association with the disease clinical symptoms. The merozoite surface proteins(MSP) are the major targets of immune attack by antibodies. The well characterized MSP is MSP-1. Other MSP gene families exist. Clinical immunity to malaria is slow to develop and short lived. One reason for this is the extensive diversity found in Plasmodium antigens, which facilitate parasite escape from host immune detection But still single infections are however important in developing initial low levels of immunity to severe malaria and death With increasing age, there is a shift from severe malaria to clinical malaria and eventually to asymptomatic parasitemia. MSP abundantly expressed. and have been shown to elicit both humoral and cell mediated immune responses during exposure to natural infections and in experimental immunization.

Plasmodium falciparum MSP-1 gene Located at the middle of chromosome 9 Organized into blocks Its polymorphic and genetically diverse The product of this is MSP-1, a high-molecular-weight precursor which is then processed into several fragments . At the time of red cell invasion, only the 19-kDa C-terminal fragment remains on the surface. and is carried into the invaded erythrocytes. Since MSP-1, can elicit immune responses in natural malaria infections, and is genetically diverse containing multiple polymorphisms in P. falciparum it will be considered in sequential infections in this study.

Research Question Hypothesis Are there Plasmodium falciparum MSP-1 polymorphic differences in sequential clinical malaria infections There are no Plasmodium falciparum MSP-1 polymorphic variants in sequential cases of clinical malaria infections. Hypothesis

General Objective Specific Objectives Determine MSP-1 19kDa genetic variants in parasite obtained from individuals with multiple malaria sequential infections. Identify MSP-119kDa gene polymorphisms in sequential clinical malaria infections. Determine allelic changes in individuals across the sequential infections. Determine the population proportion of allele frequencies between infections. Specific Objectives

Justification Malaria parasite resistant to drugs. Vector resistant to insecticides. Developing a vaccine towards malaria would be the most practical way to arrest this quagmire. However, efforts that are underway have been unsuccessful. This work will determine the frequency of MSP-119kDa gene polymorphisms and allele prevalence in individuals with multiple sequential infections and contribute to the knowledge of MSP-1 diversity which is useful towards the development of malaria vaccine merozoite candidates.

Methodology Study design: This will be Longitudinal study where MSP-1 variable will be measured in all subjects in the cohort Study site The lab work will be conducted at Centre for Biotechnology and Bioinformatics (CEBIB) molecular labs in Nairobi and at ILRI. Study population The target group will be composed of 33 children aged between 0 and 5 years who had been followed up weekly with over 9 malaria infection episodes after visiting a dispensary in Junju, Kilifi County. Ethical clearance Ethical clearance for this study has already been obtained from the KEMRI ethics review board for integrated studies of natural immunity to malaria. .

Amplification and sequencing of MSP-119 kDa genes PCR Gel electrophoresis Optimization Gene specific primers for MSP-1 19KDa, High Fidelity PCR Taq (Roche) and Controls (3D7) Samples run in batches Gel preparation using Cyber Safe® and view by Gel Doc Amplicons will be purified using ethanol precipitation. Cycle sequencing PCR. BigDye PCR Data Analysis Sequencing Analysis by DNA analyser machine (Applied Biosystems) Quality checking and contig assembly using BioEdit, DNASTAR, CLC bio softwares. Alignment to identify Nucleotide polymorphisms Gene polymorphisms frequencies by R

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Acknowledgements: Parwos Abraham: CEBIB admin Colleagues

YAA Reuben Mangi (I56/ 81536/ 2012) Bsc. University of Nairobi (2012) Identification of Plasmodium Falciparum MSP-1 Variants in Malaria sequential infection YAA Reuben Mangi (I56/ 81536/ 2012) Bsc. University of Nairobi (2012) THANK YOU