Ménard D, Barnadas C et al, Proc Natl Acad Sci U S A Mar 30;107(13):

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1 Plasmodium vivax malaria in Madagascar Pvivaxinfection P. infection & clinical malaria in Duffy-negative Malagasy people Ménard D, Barnadas C et al, Proc Natl Acad Sci U S A Mar 30;107(13): Insights into the possible molecular basis

2 P. vivax interaction with Duffy is required to enable its invasion of human erythrocytes Duffy/DARC : 10 4 copies/rbc SO 4 P. vivax Duffy Binding Protein Reticulocyte Binding Protein

3 Assaults on the paradigm: Major role of the Duffy antigen in P. vivax invasion mechanisms Ryan et al, 2006 : report of anopheles infected by P. vivax in Kenya (duffy negative populations) Worldwide distribution of P. vivax From Picot S, 2006 Cavasini et al, 2007 : report of P. vivax PCR positive patients, genotyped as Duffy negative (n=2) in Brazilian Amazon region

4 Madagascar: cross road RECENT (only 2300y before present) Peopled by succession of Asian and African migrations: Admixture Coexistence of 4 human malaria species falciparum, vivax, ovale, malariae

5 Study population 8 sentinel sites, Asymptomatic Schoolchildren (3-13 y old) (Razakandrainibe et al, 2009) Malaria Patients /Health Centers ( > 6 mo) (Barnadas et al, 2008) Malaria diagnosis: PCR SSU rdna & microscopy Duffy genotyping g Duffy -33, promoter +/- (FY ES ), codon 42, FY*A or FY*B; codon 89, FY*B or FY*X

6 Plasmodium infections and duffy genotype: asymptomatic ti population (Malagasy schoolchildren) hild 661/709 (93%) were successfully genotyped Duffy Phenotype Fya + /Fyb - Fya + /Fyb + Fya - /Fyb + Fya - /Fyb - Duffy Genotype FY*A/*A FY*A/*B ES FY*A/*B FY*B/*B FY*B/*B E S FY*B ES /*B ES Total population (3.8%) (17.7%) (1.7%) (0.1%) (4.7%) (72.0%) P. vivax infection (prevalence) (8.0%) (28.2%) (45.5%) (12.9%) (8.8%) Plasmodium sp Totals (13.0 %) Duffy infection genotyping was based on the SNPs Duffy -33, promoter +/-; codon 42, - FY*A or FY*B; codon 89, FY*B or FY*X) using (28.7 a post- PCR LDR-FMA (ligase detection (48.0%) reaction-fluorescent (38.5%) microsphere (54.5%) assay). (19.4%) (26.7%) (prevalence) 190 %) Duffy genotyping was based on the SNPs Duffy -33, promoter +/-; codon 42, FY*A or FY*B; codon 89, FY*B or FY*X) using a post-pcr LDR-FMA (ligase detection reaction-fluorescent microsphere assay). Plasmodium species diagnosis was based on species polymorphisms of the small-subunit ribosomal (SSU) rrna gene.

7 Andapa Maevatanana 7.8% 24.5% Tsiroanomandidy 2.0% 2.0% 71.4% 92.0% 14.8% 25.0% Moramanga 32.4% Miandrivazo 26.1% 5.7% 27.8% 58.0% 2.9% 34.8% Farafangana 14.0% 1.2% 62.3% Fy-Pv- Fy-Pv+ Fy+Pv+ Fy+Pv- 10.2% Ihosy 24.2% Ejeda 84.9% 75.8% 26.3% 73.7% A t ti hild l f it f th li? Asymptomatic children: release of merozoites from the liver? True intraerythrocytic infection?

Plasmodium infections and duffy genotype: clinical i l

vivax-positive iti infections in malaria patients:

vivax mono-infections 9 in Duffy-neg 33 P. vivax/p.

Tsiroanomandidy Miandrivazo i 1/18 7/98 7/52 Moramanga

8 Plasmodium infections and duffy genotype: clinical i l malaria 183 P. vivax-positive iti infections in malaria patients: Andapa 150 P. vivax mono-infections 9 in Duffy-neg 33 P. vivax/p. falciparum infections: 8 in Duffy-neg Maevatanana Tsiroanomandidy Miandrivazo i 1/18 7/98 7/52 Moramanga 1/4 Evidence of vivax clinical malaria 17/183 malaria patients with P. vivax are Duffy-neg Ejeda Ihosy 0/7 1/2 Farafangana Microscopy evidences?

9 P. vivax intra-erythrocytic infection in Duffy negative patients: microscopy (PCR confirmation and exclusion of P. ovale, P. malariae) - genotyped as Duffy-negative (FY*B ES /*B ES ), 4 year old female patient, Tsiroanomandidy, - microscopy: mixed infection P. vivax(3040 infected cells/µl) - P. falciparum (980 infected cells/µl). - PCR confirmation - genotyped as Duffy-negative (FY*BES/*BES), 3 year old female patient, Moramanga, - microscopy: P. vivax (3368 parasites infected cells/µl). - PCR confirmation Evidence for intraerythrocytic development :asexual and sexual stages

10 Panels A B: Patient ASA R genotyped as Duffy-negative (FY*B ES /*B ES ), - 4 year old female patient, Tsiroanomandidy, June, 26th, fever (37.8 C), headache and sweating, no previous antimalarial i l treatment - microscopy: mixed patients infection P. (5.9%) vivax (3040 infected cells/µl) - P. falciparum (980 infected cells/µl). - PCR confirmation 42 P. vivax infections (8.8%) in asymptomatic children genotyped Duffy negative 9 cases of P. vivax clinical malaria in Duffy negative PCR confirmation Evidence of intraerythrocytic th ti infections Genotype.. Phenotype??

11 Concordance between Duffy genotype and phenotype (serotyping) New collection of samples from 43 schoolchildren from Tsiroanomandidy - serology (DIAMED card): 100% concordant with genotyping results - flow cytometry (anti-fy6 antibody labeling) A Cell coun nts Isotype control Fy(a-b-) FY*B ES /*B ES Fy(a-b-) FY*B ES /*B ES (Malagasy blood sample) Fy(a-b+ weak ) FY*B ES /*X Fy(a-b+ weak ) FY*X/*X Fy(a+b-) FY*A/*B ES Fy(a+b+) FY*A/*B B N=4 MFI anti-f Fy N=30 N=2 N=6 MFI anti-fy Fy(a-b-) Fy(a-b+ weak ) Fy(a+b-) Fy(a+b+) Fy*B Pro ES Fyb/Fyb /*B ES Fy*B Pro+ Fya/Fyb ES /*X Fya/Fya Fy*A/*BPro /+ ES Fya/Fyb Fy*A/*B FyW Mean fluorescence intensities reflecting the Duffy antigen specific anti-fy6 antibody

12 Extended Duffy gene sequencing Extended genotyping g of Duffy blood group locus in 14 Duffy negative Malagasy patients : 1. single SNP (T > C) -33 in GATA-1 transcription factor binding site of the gene promoter that governs Duffy expression in erythroid cells 2. Intact, full length coding sequence % concordance with 3 Duffy negative West African samples. Primers used Primers used Primers used

13 Genetic diversity of P. vivax strains infecting Duffy- negative Malagasies Multilocus genotyping 1. Circumsporozoite protein (PvCSP) VK210, n=6; VK247, n=1; VK210 and VK247, n=9. 2. Microsatellites : 5 loci Heterogeneity for individual loci was > 75% (No alleles/locus = 6-13) >>> Multi-lineage background ; Duffy-neg =Duffy-pos multiple extant P vivaxstrains carry the capacity multiple extant P. vivax strains carry the capacity to infect Duffy negative erythrocytes

14 Diversity of P. vivax Duffy Binding Protein Duffy Binding domain Sequenced (N=243) 21 Alleles observed Skewed distribution, 8 alleles in Duffy negatives (n=27), 21 alleles in Duffy positives (n=216) (Chi2, p<0.01). In Tsiroanomandidy : 2 alleles in Duffy negatives (N=13) 8 alleles in Duffy positives (N=16) (Chi2, p=0.007): 007) Allele IX much higher frequency in Duffy-negatives (P<10-6)

15 PvDBPallele IX binds Duffy-negative RBC PvDBP allele IX: Baculovirus expression : N glycosylation sites mutated C-term His tag (5x10 7 RBC/assay) Neg & Pos Neg & Pos Neg & Pos Neg & Pos Neg & Pos Neg & Pos PvDBP (µg) with reduced efficacy of binding compared to Duffy-pos RBC

16 Hypothesis reservoir of Duffy positive infected carriers Duffy positive RBC Invasion of through DARC pathway Duffy negative RBC? Alternative invasion pathway (less efficient) but possibly involving mutant PvDBP.

17 Duffy binding site falciparum EBA sialic acid binding site Surface exposed contact residues required for recognition of DARC on human erythrocytes are in all P. vivax samples. Dual functionality?

18 Didier Ménard Céline Barnadas Vincent Thonier Jean-François Carod Olivier Domarle Ministère de la Santé, Madagascar Arsène Ratsimbasoa Unité d'immunologie Moléculaire des Parasites Didier Ménard Micheline Guillotte Odile Mercereau-Puijalon Pasteur Genopole Ile-de- France Christiane Bouchier BaculovirusProduction Platform Elodie Crublet Céline Barnadas Cara Henry-Halldin Laurie Gray Yves Colin Christopher L. King Olivier Bertrand Brian T. Grimberg Julien Picot Peter A. Zimmerman

Supporting Information

Supporting Information Ménard et al. 10.1073/pnas.0912496107 SI Appendix A: Malagasy Study Sites, Peopling History, and Ethnicities Location and Description of the Collection Sites, Madagascar, 2006 2007.