Malaria genomic epidemiology research Dr. Alyssa Barry Malaria Genomic Epidemiology Lab., Centre for Population Health

1. Malaria genomic epidemiology researchDr. Alyssa BarryMalaria Genomic Epidemiology Lab., Centre for Population Health

2. What is Malaria? • A disease caused by infection with Plasmodium spp. parasites • Carried from person to person by anopheline mosquitoes • Six species of Plasmodium cause malaria • P. vivax, P.falciparum, P. malariae P. ovale curtisii, P. ovale wallikeri, P. knowlesii • P.falciparum causes most morbidity and mortality • Symptoms include fever, nausea, vomiting, diarrhoea, tissue damage, multiple organ failure, severe anaemia, coma (cerebral Malaria), death

3. The Malaria Parasite Lifecycle – human host

4. The Burden of Malaria • ~ 50% of the global population at risk of malaria • Half a billion clinical attacks each year • At least 1 million deaths each year Two or three people die of malaria every minute !

5. Who are Most at Risk? • Children under 5yrs old • In the top 5 causes of death • Pregnant women • 400million births/yr in malaria affected areas • Other non-immunes • natural disaster • war • environmental change • climate change

6. Effects of Malaria Besides direct morbidity and mortality: • Reduced school attendance • Lower productivity • Impaired intellectual development • Developmental abnormalities • 2% less GDP growth in malarious countries • Costs Africa about US$12 billion a year

7. Malaria Genomic Epidemiology… • Genomic epidemiology (Def’n): The systematic investigation of how variation in the human genome, and in the genomes of human pathogens, affect the occurrence and clinical outcome of disease • We are investigating patterns of genomic diversity within natural malaria parasite populations to: • Monitor patterns and routes of transmission (molecular epidemiology, population genetics, ecology) • Design malaria vaccines (what strains circulate?) • Understand parasite evolution (changes over time, immune selection, interactions with host molecules) • Understand how humans naturally acquire immunity to diverse malaria parasites

8. Malaria parasite diversity Variant specific antibodies A diverse parasite population will be more resilient to interventions Vaccine Rapid evolution of drug resistance and other advantageous traits Partial efficacy of single strain vaccines. A malaria vaccine may need to contain multiple variants.

9. Malaria parasite population structure C Gene flow Variability in allele frequencies A B Unique alleles D Movement of different strains between populations Population specific approaches to malaria control (e.g. tailored vaccines, efforts targeted to specific foci) • Speed and direction of the dissemination of advantageous traits

10. Polymorphism 101 • Derived from the Greek language • Poly = many πολύ • Morph = form μορφή • The occurrence in a population (or among populations) of several phenotypic forms associated with alleles (variants, types) of one gene • Genetic polymorphism: the occurrence together in the same population of one or more allele or genetic marker (e.g. nucleotide or string of nucleotides) at the same locus (position in the genome) • Therefore: Genetic variation results in the occurrence of several different forms or types of individuals among the members of a single species (diversity) • e.g. Humans: blood group, hair colour, eye colour, disease status in • e.g. microorganisms: drug sensitivity/resistance, growth characteristics, antigenic diversity (strains) • Caused by mutation

11. Types of polymorphism Fragment size/pattern analysis (electrophoresis): • AFLP: Amplified Fragment Length Polymorphism • RFLP: Restriction Fragment Length Polymorphism • SSLP: Short Sequence Length Polymorphism • Microsatellites: tandem repeats (2-3 bp) • Minisatellites:tandem repeats (>3 bp) Sequence analysis (sequencing, but also RFLP, SSLP): • SNP : single nucleotide polymorphism • Indel: Insert or Deletion • Simple sequence repeats: polynucleotides (AAAA), microsatellites (TATATA) etc…

12. Microsatellites TA TA TA TA TA TA TA TA • Arrays of short tandem repeats 1-4 bp long • A class of variable number tandem repeat (VNTR) used in DNA fingerprinting • Also known as simple sequence repeats (SSR) • Abundant and rapidly evolving • Highly polymorphic • Detected by size variation • Fairly evenly spaced through the genome • Cheap to analyze

13. Chromatin Binding Protein Intronic microsatellite polymorphism (TA)n 3D7(GB) #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAAAT 3D7 #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAAAT HB3 #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAAAT W2 #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAAAT Muz12 #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAAAT Muz37 #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAAAT Muz51 #61 AATTAAATAG GATTAAAATA ATTGTCATAA AAAAAATTAT ATATACTTGA AAAAGCAAAT 3D7(GB) #121 GACTGATTTT TTAAGgtatg aataaaatga atataatata tatatatat: :::::::att 3D7 #121 GACTGATTTT TTAAGGtatg aataaaatga atataatata tatatatat: :::::::att HB3 #121 GACTGATTTTTTAAGGtatg aataaaatga atataatata tatatatat: :::::::att W2 #121 GACTGATTTTTTAAGGtatg aataaaatga atataatata tatatat::: :::::::att Muz12 #121 GACTGATTTTTTAAGGtatg aataaaatga atataatata tatatatat: :::::::att Muz37 #121 GACTGATTTTTTAAGGtatg aataaaatga atataatata tatatatat: :::::::att Muz51 #121 GACTGATTTT TTAAGGtatg aataaaatga atataatata tatatatata tatat::att 3D7(GB) #181 taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt 3D7 #181 taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt HB3 #181 taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt W2 #181 taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt Muz12 #181 taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt Muz37 #181 taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt Muz51 #181 taacctaaga tatatatgtt ttttcatata atagttaata taatataaac aaaatatatt 5’ Regulatory Domain Exon I Intron I Intron I

14. Single Nucleotide Polymorphisms (SNPs) • Point mutation, variation at a single nucleotide position • e.g. A/C, G/A etc… • Clustered in rapidly evolving genes e.g. human MHC genes, P. falciparum var, HIV env, • A good SNP map is useful for population genetics and linkage analysis • Rapid, high throughput detection possible but can be expensive

15. P. falciparum Erythrocyte Binding Antigen 175 (EBA175)

16. coding non-coding Population genetic markers for P. falciparum • Different markers show different patterns • The P. falciparum genome : SNP “islands” • Selected markers • Vaccine candidate antigens • inform vaccine design • Novel vaccine candidates - immune selection? • Drug resistance genes and their genetic background • Is it spreading (how fast, which direction) or multiple independent origins? • Neutral markers • Genome wide microsatellites and SNPs • Population biology e.g. how diverse (fit) is the parasite population? gene flow? i.e. how difficult will the parasite population be to control?

17. Population biology of P. falciparumin PNG • Intense year round transmission of P. falciparum in the lowlands (50-60%), epidemics in the highlands • Any spp. (~80%), P. vivax (~50%), P. malariae (~20%), P. ovale (~5%) • Diverse micro-epidemiology- Spatially variable transmission, host genetics, vector species, malaria control (bednets) • Complex population genetics?

18. Volunteers Isolate Blood Extract genomic DNA Analysis Collecting samples Pic of Ivo here

19. “Wet” lab. methods gDNA n ~ 3000 Screen for P.falciparum infection(msp2 PCR, multiplex) n ~1500 Count the number of msp2 bands Mean MOI = 1.7 (1-13) Whole genome amplification of single infections n ~ 700 Microsatellite genotyping Antigen gene PCR and sequencing Data Analysis

20. Microsatellite protocol 50 + 50 + 16 bp = 116 bp PCR product = 8 repeat units 50 + 50 + 20 bp = 120 bp PCR product = 10 repeat units CACACACACACACACACACA GTGTGTGTGTGTGTGTGTGT 20 bp • Small size difference (4bp) – cannot be detected by agarose gel electrophoresis • Solution: Sequencing, or for cheaper high throughput run PCR products on an ABI Sequencer • The latter solution requires products to be fluorescently labeled

21. Approach:Fragment analysis on an ABI Sequencer Dye attached to the 5’ end of primer TA TA TA TA TA TA TA TA TA TA TA TA TA Fluorescent dye is incorporated into PCR product TA TA TA TA TA TA TA TA TA TA TA TA TA

22. Microsatellite genotyping Multilocus genotyping: Different sizes and differentcoloureddyes increase the number of loci that can be analyzed in a single run and the sensitivity of the assay Chromatogram Sequencing gel Isolates Locus 1 Locus 7 2 1 The haplotype, a string of alleles (e.g. the number of repeats per loci 15_12_6_8_10_6) is then determined for each isolate

23. “In silico” analysis

24. Population biology of P. falciparum in PNG Factors that may influence the distribution of parasites: -Mugil/Karkar Is. ferry -Malala boarding school -vector spp. -language groups -human genetics High but variable diversity and population structure Implications for control, elimination and the spread of vaccine and drug resistance (mapping routes of transmission) Currently sequencing several vaccine candidate antigens in two of these populations to inform vaccine design

25. Acknowledgements PNG Communities and Volunteers PNGIMR Peter Siba Ivo Mueller Nicholas Senn Livingstone Tavul Ore Toporua Benson Kiniboro Joe Nale Thomas Adiguma Elias Namosha Burnet Institute Lee Schultz Pilate Ntsuke Johanna Wapling John Reeder Harvard School of Public Health Caroline Buckee Funding