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

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

The Malaria Parasite Lifecycle – human host

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 !

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

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

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

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

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

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

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…

Microsatellites 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 TA TA TA TA

3D7(GB) #61AATTAAATAG 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 GACTGATTTT TTAAGGtatg aataaaatga atataatata tatatatat: :::::::att W2 #121 GACTGATTTT TTAAGGtatg aataaaatga atataatata tatatat::: :::::::att Muz12 #121 GACTGATTTT TTAAGGtatg aataaaatga atataatata tatatatat: :::::::att Muz37 #121 GACTGATTTT TTAAGGtatg 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 Intron I Exon I 5’ Regulatory Domain Chromatin Binding Protein Intronic microsatellite polymorphism (TA) n

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

P. falciparum Erythrocyte Binding Antigen 175 (EBA175)

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? coding non-coding

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? Population biology of P. falciparum in PNG

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

“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

Microsatellite protocol bp = 116 bp PCR product = 8 repeat units bp = 120 bp PCR product = 10 repeat units 20 bp CACACACACACACACACACA GTGTGTGTGTGTGTGTGTGT 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

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

Microsatellite genotyping Locus 1 Isolates Sequencing gel Chromatogram 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 Locus 7 2 1

“In silico” analysis

Population biology of P. falciparum in PNG 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 Factors that may influence the distribution of parasites: -Malala boarding school -Mugil/Karkar Is. ferry -vector spp. -language groups -human genetics

Acknowledgements 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 Funding Harvard School of Public Health Caroline Buckee PNG Communities and Volunteers